I SCIENTIFIC RESULTS IBB 2017 - bornbunge.be · may be related to the aetiology of suicidal ideation in older adults (Rymo et al., 2017, Acta ... We provide consensus guidelines for

Scientific Report Institute Born-Bunge 2017. Page 4

ADDENDUM I

SCIENTIFIC RESULTS IBB 2017

I. INTRODUCTION

This report summarizes the scientific results of research in the fields of neurology, neurobiology and cardiology performed in 2016 within the framework of an agreement between the Research Fund – Flanders, the University of Antwerp and the Institute Born Bunge (IBB). The IBB encompasses the following laboratories and or Central Facilities : 1) Biobank (Coordinator Prof. Dr. P.P. De Deyn) 2) Laboratory of Neurobiology (Prof. P. Cras) focussing on the study of prion diseases; 3) Laboratory of Neurogenetics (Prof. C. Van Broeckhoven) focussing on the study of central nervous system disorders; 4) Laboratory of Peripheral nerve systems (PNS) diseases (Prof. P. De Jonghe, Prof. V. Timmerman); 5) Laboratory of Neuromuscular Pathology (Prof. J. Baets); 6) Laboratory of Theoretical Neurobiology and Neuroengineering (Prof. M. Giugliano) focussing on cellular and network neurobiology; 7) Laboratory of Neurochemistry and Behaviour (Prof. P.P. De Deyn). Since 2005, Prof. P.P. De Deyn is Scientific director of the IBB, and in 2006, a new organigram was implemented to further support the expansion of the Biobank Central Core Facility; the Laboratory for Ultrastructural Neuropathology was established and three task forces were defined: 1) Neuromuscular Diseases; 2) Prion Diseases, and 3) Central Nervous system Neurodegenerative Disorders.

II. STUDY OF DEMENTIA AND THE ESTABLISHMENT OF A BRAIN BANK

II.1 From the Laboratory of Neurochemistry and Behaviour (P.P. De Deyn, S. Engelborghs, D.

Van Dam, Y. Vermeiren and co-workers)

A. Improved characterization of the MCI and Dementia study population

The possible relationships between suicidal ideation and cerebrospinal fluid (CSF) levels of glial markers YKL-40 (also known as chitinase-3-like protein 1), growth-associated protein-43 (GAP-43) and myelin basic protein (MBP) was investigated. Therefore, samples were obtained from the Prospective Population Study of Women and 86 women without dementia who underwent both psychiatric examinations and lumbar puncture (LP) were included. Eight of these women reported past-month suicidal ideation. The results showed significantly higher CSF levels of both YKL-40 and GAP-43 in women with past-month suicidal ideation. Associations with suicidal ideation remained for both YKL-40 and GAP-43 in regression models adjusted for smoking status, BMI and age. CSF levels of YKL-40, GAP-43 and MBP did not differ by depression status. Higher levels of CSF GAP-43 were associated with feelings of worthlessness; a strong relationship was demonstrated in the fully adjusted model (OR 5.95 CI [1.52-23.20], P = 0.01). These findings of elevated CSF concentrations of both YKL-40 and GAP-43 in women with suicidal ideation, compared to those without, suggest that a disrupted synaptic glial functioning and inflammation may be related to the aetiology of suicidal ideation in older adults (Rymo et al., 2017, Acta psychiatrica Scandinavica).

In a somewhat unrelated paper, a review of literature and consensus guidelines from dementia experts and the Belgian road safety institute (endorsed by the Belgian Medical Association) regarding AD and driving is provided (Versijpt et al., 2017, Acta Neurologica Belgica).

In the quest for prevention or treatment, there is a need to find early markers for preclinical dementia. Therefore, memory clinic patients with subjective cognitive impairment (SCI) and


normal cognitive function at baseline were observed. The primary aim was to address SCI as a potential risk factor for cognitive decline. The secondary aim was to address a potential relation between (1) baseline cerebrospinal fluid biomarkers and (2) a decline in memory performance over the first 2 years of follow-up, with a possible cognitive decline after 6 years. Eighty-one patients (mean age 61 years) were recruited from university memory clinics and followed up for 6 years. Of those patients, eighty-six percent remained cognitively stable or improved, 9% developed mild cognitive impairment, and only 5% (n = 4) developed dementia. Regression analysis revealed that low levels of Aβ42 at baseline and memory decline during the first 2 years predicted dementia. When combined, these variables were associated with a 50% risk of developing dementia. In conclusion, cognitive stability for 86% of the cohort suggests that SCI is predominantly a benign condition with regard to neuropathology. The low number of individuals who developed dementia limits the generalizability of the results and discussion of progression factors. So, subjective cognitive impairment is a predominantly benign condition in memory clinic patients followed for 6 years: The Gothenburg-Oslo MCI Study (Hessen et al., 2017, Dementia and geriatric cognitive disorders extra).

B. Validation, standardisation and harmonisation of AD CSF biomarkers

A review was published focusing on the validation and standardization of Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers, as well as on the current clinical indications and rational use of CSF biomarkers in daily clinical practice. The validated AD CSF biomarkers, Aβ1-42, T-tau, and P-tau181, have an added value in the (differential) diagnosis of AD and related disorders, including mixed pathologies, atypical presentations, and in case of ambiguous clinical dementia diagnosis. CSF biomarkers should not be routinely used in the diagnostic work-up of dementia and cannot be used to diagnose non-AD dementias. In cognitively healthy subjects, CSF biomarkers can only be applied for research purposes, e.g., to identify pre-clinical AD in the context of clinical trials with potentially disease-modifying drugs. Therefore, biomarker-based early diagnosis of AD offers great opportunities for preventive treatment development in the near future (Niemantsverdriet et al., 2017, Acta Neurologica Belgica).

Standardisation and harmonization of (pre-)analytical aspects of CSF biomarkers

A paper was published on the topic of multicenter analytical validation of Aβ40 immunoassays. This paper concludes that all validated Aβ40 appeared to be of good technical quality and performed generally well accoriding to predefined criteria. A novel version of the validation SOP is developed based on these findings, to further facilitate implementation of novel immunassays in clinical practice (van Waalwijk van Doorn et al., 2017, Frontiers in Neurology).

Harmonisation of lumbar puncture (LP) for CSF sampling

Cerebrospinal fluid collection by lumbar puncture (LP) is performed in the diagnostic workup of several neurological brain diseases. Reluctance to perform the procedure is among others due to a lack of standards and guidelines to minimize the risk of complications, such as post-LP headache or back pain. We provide consensus guidelines for the LP procedure to minimize the risk of complications. The recommendations are based on (1) data from a large multicenter LP feasibility study (evidence level II-2), (2) systematic literature review on LP needle characteristics and post-LP complications (evidence level II-2), (3) discussion of best practice within the Joint Programme Neurodegenerative Disease Research Biomarkers for Alzheimer’s disease and Parkinson’s Disease and Biomarkers for Multiple Sclerosis consortia (evidence level III). Our consensus guidelines address contraindications, as well as patient-related and procedure-related risk factors that can influence the development of post-LP complications. Discussion: When an LP is performed correctly, the procedure is well tolerated and accepted with a low complication rate (Engelborghs et al., 2017, Alzheimers Dement: DADM).


C. AD CSF biomarkers in clinical practice and clinical research

A decade of AD CSF biomarker analyses at BIODEM, IBB/UAntwerp

Although the core cerebrospinal fluid Alzheimer's disease (AD) biomarkers amyloid-β (Aβ1-42) and tau show a high diagnostic accuracy, there are still limitations due to overlap in the biomarker levels with other neurodegenerative and dementia disorders. During Aβ1-42 production and clearance in the brain, several other Aβ peptides and amyloid precursor protein fragments are formed that could potentially serve as biomarkers for this ongoing disease process. Therefore, a review was written to describe the current status of the findings for amyloid precursor protein and Aβ peptide isoforms in AD and clinically related disorders. This review concludes that adding new Aβ isoforms to the AD biomarker panel may improve early differential diagnostic accuracy and increase the cerebrospinal fluid biomarker concordance with AD neuropathological findings in the brain (Somers et al., 2017, Biomarkers in Medicine).

An article was published that presents recommendations, based on the Grading of Recommendations, Assessment, Development, and Evaluation method, for the clinical application of cerebrospinal fluid (CSF) Amyloid-β1–42, tau, and phosphorylated tau in the diagnostic evaluation of patients with mild cognitive impairment (MCI). The recommendations were developed by a multidisciplinary working group and based on the available evidence and consensus from focused group discussions for 1) prediction of clinical progression to Alzheimer’s disease (AD) dementia, 2) cost-effectiveness, 3) interpretation of results, and 4) patient counseling. The working group recommended using CSFAD biomarkers in the diagnostic workup of MCI patients, after prebiomarker counseling, as an add-on to clinical evaluation to predict functional decline or conversion to AD dementia and to guide disease management. Because of insufficient evidence, it was uncertain whether CSF AD biomarkers outperform imaging biomarkers. Furthermore, the working group provided recommendations for interpretation of ambiguous CSF biomarker results and for pre- and post-biomarker counseling (Herukka et al., 2017, Alzheimers Dement).

Another published article discusses the recommendations for CSF AD biomarkers in the diagnostic evaluation of dementia (Simonsen et al., 2017, Alzheimers Dement).

Early AD diagnosis: from timely diagnosis to population screening

We have been invited to write an opinion paper with regard to early (biomarker-based) AD diagnosis (De Roeck et al., in press in J Am Med Dir Assoc).

Recent advances in neuroimaging, CSF assays, and other biomarkers now provide the ability to detect evidence of the AD pathophysiological process in vivo. This stirs the debate about how and when AD is best detected. It is important to bear in mind that (1) nowadays disease-modifying drugs are still not available and (2) there might be differences between the interests and needs for individual patients, for wider society and for research purposes.

Alzheimer's disease biomarkers are important for early diagnosis in routine clinical practice and research. Three core CSF biomarkers for the diagnosis of Alzheimer's disease (Aβ42, T-tau, and P-tau) have been assessed in numerous studies, and several other Alzheimer's disease markers are emerging in the literature. However, there was a lack of a comprehensive meta-analyses of their diagnostic performance. We systematically reviewed the literature for 15 biomarkers in both CSF and blood to assess which of these were most altered in Alzheimer's disease (Olsson et al., 2016, Lancet Neurology).

We are convinced that in the future, when disease modifying medication are available, one should seek to diagnose AD in its pre-symptomatic phase. Therefore, minimally invasive (or preferably: non-invasive) and affordable techniques to detect AD in the pre-symptomatic phase should be optimized through (clinical) research. In other words, at this moment, research should focus on the development/optimization of cost efficient screening tools to identify people in the asymptomatic


phase. Besides, it is important to underline that drug development can profit from the improvement of detection techniques. Partly due to the advance in detection techniques, research for potential disease-modifying treatments has changed its focus from the dementia phase to the MCI phase and currently also to the pre-symptomatic phase of AD. Both PET scans with Aβ ligands and analysis of Aβ in the CSF are used as an enrichment strategy for clinical trials in the field of AD.

Next to the improvement of screening techniques, more attention is needed for population based research. There is a lack of population based studies for cognitive screening instruments as well as for biomarkers. Little is known about how cognitive functions change with age. Population based research could result in more insight in how cognitive functions and biomarkers change with age.

D. CSF and imaging biomarkers for improved differential dementia diagnosis

Biomarkers for AD versus non-AD dementia differential diagnosis

Virtually nothing is known about a potential diagnostic role of non-phospho-epitopes of Tau (Non-P-Tau) in cerebrospinal fluid (CSF). Therefore, the objective was to establish and analytically and clinically characterize the first assay capable to measure concentrations of Non-P-Tau in human CSF. An antibody (1G2) was developed that selectively binds to the Tau molecule non-phosphorylated at the positions T175 and T181, and was used in establishing a sandwich ELISA capable to measure Non-P-Tau in human CSF, following analytical and clinical validation of the method. The results indicated that the 1G2 antibody shows decreasing reactivity to tau peptides containing phosphorylation mainly at positions T175 and T181. Detection limit of the assay is 25 pg/ml; the coefficients of variation (CVs) of the optical densities of the repeated standard curves were between 3.6–15.9%. Median intra-assay imprecision of double measurements was 4.8%; inter-assay imprecision was in the range of 11.2%–15.3%. Non-P-Tau concentrations are stable in the CSF samples sent to distinct laboratories under ambient temperature; inter-laboratory variation was approximately 30%. The Non-P-Tau CSF concentrations were highly significantly increased in patients with Alzheimer’s disease in stage of mild cognitive impairment or dementia (AD/MCI, n = 58, 109.2±32.0 pg/mL) compared to the non-demented Controls (n = 42, 62.1±9.3 pg/mL, p < 0.001). At the cut-off of 78.3 pg/mL, the sensitivity and the specificity were 94.8% and 97.6%, respectively. So, for the first time, an assay is reported to reliably measure concentrations of non-phosphorylated Tau in human CSF (Lewczuk et al., 2017, JAD).

Biomarkers for AD versus dementia with Lewy bodies (DLB) differential diagnosis

In one study, we evaluated the diagnostic performance of five CSF biomarkers across a well-characterized cohort of patients diagnosed with AD, DLB, PDD, and Parkinson’s disease (PD). A total of 208 patients were enrolled in 3 European centers. The diagnostic groups (AD, n = 48; DLB, n = 40; PDD, n = 20; PD, n = 54) were compared with cognitively healthy neurological control subjects (patients with other neurological diseases [OND], n = 46). CSF levels of fatty acid binding protein 3, heart type (FABP3), α-synuclein (α-syn), amyloid-β peptide 1–42, total tau (t-tau), and phosphorylated tau 181 (p-tau) were assessed with immunoassays. Univariate and multivariate statistical analyses were applied to calculate the diagnostic value of the biomarkers as well as their association with clinical scores. The results showed that FABP3 levels were significantly increased in patients with AD and DLB compared with those with PD and OND (p < 0.001). CSF t-tau, p-tau, and α-syn were significantly higher in patients with AD than in patients with PDD, DLB, PD, and OND. Combination of FABP3 with p-tau showed high accuracy for the differential diagnosis between AD and DLB (AUC 0.92), whereas patients with AD were separated from those with PDD using a combination of p-tau, FABP3, and α-syn (AUC 0.96). CSF FABP3 was inversely associated with Mini Mental State Examination score in the whole cohort (r = −0.42, p < 0.001). In conclusion, the combination of CSF biomarkers linked to different aspects of neurodegeneration, such as FABP3, α-syn, and AD biomarkers, improves the biochemical characterization of AD and Lewy body disorders (Chiasserini et al., 2017, Alzheimers Res Ther).


The Alzheimer’s disease (AD) cerebrospinal fluid (CSF) biomarkers Aβ1–42, t-tau, and p-tau181 overlap with other diseases. New tau modifications or epitopes, such as the non-phosphorylated tau fraction (p-taurel), may improve differential dementia diagnosis. The goal of this study is to investigate if p-taurel can improve the diagnostic performance of the AD CSF biomarker panel for differential dementia diagnosis. The study population consisted of 45 AD, 45 frontotemporal lobar degeneration (FTLD), 45 dementia with Lewy bodies (DLB), and 21 Creutzfeldt-Jakob disease (CJD) patients, and 20 cognitively healthy controls. A substantial subset of the patients was pathology-confirmed. CSF levels of Aβ1–42, t-tau, p-tau181, and p-taurel were determined with commercially available single-analyte enzyme-linked immunosorbent assay (ELISA) kits. Diagnostic performance was evaluated by receiver operating characteristic (ROC) curve analyses, and area under the curve (AUC) values were compared using DeLong tests. The diagnostic performance of single markers as well as biomarker ratios was determined for each pairwise comparison of different dementia groups and controls. The addition of p-taurel to the AD biomarker panel decreased its diagnostic performance when discriminating non-AD, FTLD, and DLB from AD. As a single marker, p-taurel increased the diagnostic performance for CJD. No significant difference was found in AUC values with the addition of p-taurel when differentiating between AD or non-AD dementias and controls. In conclusion, the addition of p-taurel to the AD CSF biomarker panel failed to improve differentiation between AD and non-AD dementias (Goossens et al., 2017, Alzheimers Res Ther).

Biomarkers for AD versus FTLD differential diagnosis

See also previous paper by Goossens et al., 2017, Alzheimers Res Ther.

We explored the diagnostic performance of CSF biomarkers allowing differentiation between FTLD and AD, as well as between FTLD pathological subtypes. CSF levels of routine AD biomarkers (p-tau181, t-tau, Aβ1-42) and neurofilament proteins, as well as progranulin levels in both CSF and serum were quantified in definite FTLD (n=46), clinical AD (n=45), and cognitively healthy controls (n=20). FTLD subgroups were defined by genetic carrier status and/or postmortem neuropathological confirmation (FTLD-TDP: n=34, including FTLD-C9orf72: n=19 and FTLD-GRN: n=9; FTLD-tau: n=10). GRN mutation carriers had significantly lower progranulin levels compared to other FTLD patients, AD and controls. Both t-tau and p-tau181 were normal in FTLD patients, even in FTLD-tau. Aβ1-42 levels were very variable in FTLD. Neurofilament light chain (Nf-L) was significantly higher in FTLD compared to AD and controls. The reference logistic regression model based on the established AD biomarkers could be improved by the inclusion of CSF Nf-L, which was also important for the differentiation between FTLD and controls. Within the FTLD cohort, no significant differences were found between FTLD-TDP and FTLD-tau, but GRN mutation carriers had higher t-tau and Nf-L levels than C9orf72 mutation carriers and FTLD-tau patients. In conclusion, there is an added value for Nf-L in the differential diagnosis of FTLD. Progranulin levels in CSF depend on mutation status, and GRN mutation carriers seem to be affected by more severe neurodegeneration (Goossens et al., 2018, Alzheimers Res Ther).

EEG as a biomarker for AD versus FTLD differential diagnosis

We investigated the power of EEG as biomarker in differential diagnosis of Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD). EEG was recorded from 106 patients with AD or FTLD, of which 37 had a definite diagnosis, and 40 controls. Dominant frequency peaks were extracted for all 19 channels, for each subject. The average frequency of the largest dominant frequency peaks (maxpeak) was significantly lower in AD than FTLD patients and controls. Based on ROC analysis, classification could be made with diagnostic accuracy of 78.9%. Our findings show that quantitative analysis of EEG maxpeak frequency is an easy and useful measure for differential dementia diagnosis (Goossens et al., 2017, JAD).

Amyloid-PET in combination with CSF biomarkers to improve diagnosis


Evidence suggests that the concordance between amyloid-PET and cerebrospinal fluid (CSF) amyloid-β (Aβ) increases when the CSF Aβ 1–42/Aβ 1–40 ratio is used as compared to CSF Aβ 1–42 levels alone. In order to test this hypothesis, we set up a prospective longitudinal study comparing the concordance between different amyloid biomarkers for Alzheimer’s disease (AD) in a clinical setting. Seventy-eight subjects (AD dementia (n = 17), mild cognitive impairment (MCI, n = 48), and cognitively healthy controls (n = 13)) underwent a [18F]Florbetapir ([18F]AV45) PET scan, [18F]FDG PET scan, MRI scan, and an extensive neuropsychological examination. In a large subset (n = 67), a lumbar puncture was performed and AD biomarkers were analyzed (Aβ 1–42, Aβ 1–40, T-tau, P-tau181). We detected an increased concordance in the visual and quantitative (standardized uptake value ratio (SUVR) and total volume of distribution (VT)) [18F]AV45 PET measures when the CSF Aβ 1–42/Aβ 1–40 was applied compared to Aβ 1–42 alone. CSF biomarkers were stronger associated to [18F]AV45 PET for SUVR values when considering the total brain white matter as reference region instead of cerebellar grey matter. Conclusion: The concordance between CSF Aβ and [18F]AV45 PET increases when the CSF Aβ 1–42/Aβ 1–40 ratio is applied. This finding is of most importance for the biomarker-based diagnosis of AD as well as for selection of subjects for clinical trials with potential disease-modifying therapies for AD (Niemantsverdriet et al., 2017, JAD).

Increased brain uptake of 18F-AV45 visualized by PET is a key biomarker for Alzheimer disease (AD). The SUV ratio (SUVR) is widely used for quantification, but is subject to variability based on choice of reference region and changes in cerebral blood flow. Here we validate the SUVR method against the gold standard volume of distribution (VT) to assess cross-sectional differences in plaque load. Methods: Dynamic 60-min 18F-AV45 (291 6 67 MBq) and 1-min 15O-H2O (370 MBq) scans were obtained in 35 age-matched elderly subjects, including 10 probable AD, 15 amnestic mild cognitive impairment (aMCI), and 10 cognitively healthy controls (HCs). 18F-AV45 VT was determined from 2-tissue-compartment modeling using a metabolite-corrected plasma input function. Static SUVR was calculated at 50–60 min after injection, using either cerebellar gray matter (SUVRCB) or whole subcortical white matter (SUVRWM) as the reference. Additionally, whole cerebellum, pons, centrum semiovale, and a composite region were examined as alternative references. Blood flow was quantified by 15O-H2O SUV. Data are presented as mean 6 SEM. Results: There was rapid metabolization of 18F-AV45, with only 35% of unchanged parent remaining at 10 min. Compared with VT, differences in cortical Ab load between aMCI and AD were overestimated by SUVRWM (14% 6 2%) and underestimated by SUVRCB (210% 6 2%). VT correlated better with SUVRWM (Pearson r: from 0.63 for posterior cingulate to 0.89 for precuneus, P , 0.0001) than with SUVRCB (Pearson r: from 0.51 for temporal lobe [P 5 0.002] to 0.82 for precuneus [P , 0.0001]) in all tested regions. Correlation results for the alternative references were in between those for CB and WM. 15O-H2O data showed that blood flow was decreased in AD compared with aMCI in cortical regions (25% 6 1%) and in the reference regions (CB, 29% 6 8%; WM, 28% 6 8%). Conclusion: Increased brain uptake of 18F-AV45 assessed by the simplified static SUVR protocol does not truly reflect Ab load. However, SUVRWM is better correlated with VT and more closely reflects VT differences between aMCI and AD than SUVRCB (Ottoy et al., 2017, J Nucl Med).

In a related study, we evaluated how SUVR of a cortical target region is affected by blood flow changes in the target and two frequently used reference regions. Regional baseline time-activity curves (TACs) were simulated based on metabolite-corrected plasma input functions and pharmacokinetic parameters obtained from our previously acquired data in healthy control (HC; n = 10), amnestic mild cognitive impairment (aMCI; n = 15) and AD cohorts (n = 9). Blood flow changes were simulated by altering the regional tracer delivery rate K1 (and clearance rate k2) between -40% and +40% from its regional baseline value in the target region and/or cerebellar grey


(CB) or subcortical white matter (WM) reference regions. The corresponding change in SUVR was calculated at 50± 60 min post-injection. The results showed a -40% blood flow reduction in the target resulted in an increased SUVRtarget (e.g. SUVRprecuneus: +10.0±5% in HC, +2.5±2% in AD), irrespective of the used reference region. A -40% blood flow reduction in theWMreference region increased SUVRWM (+11.5±4% in HC, +13.5±3% in AD) while a blood flow reduction in CB decreased SUVRCB (-9.5±6% in HC, -5.5±2% in AD), irrespective of the used target region. A -40% flow reduction in both the precuneus and referenceWM (i.e., global flow change) induced an increased SUVR (+22.5±8% in HC, +16.0±4% in AD). When considering reference CB instead, SUVR was decreased by less than -5% (both in HC and AD). So, blood flow changes introduce alterations in [18F]AV45 PET SUVR. Flow reductions in the CB andWM reference regions resulted in a decreased and increased SUVR of the target, respectively. SUVR was more affected by global blood flow changes when considering WM instead of CB normalization (Ottoy et al., 2017, PloS one).

E. The monoaminergic assessment of Down syndrome revisited: a (pre)clinical approach

Down syndrome (DS) mouse models

Altered concentrations of monoamine neurotransmitters and metabolites have been repeatedly found in people with Down syndrome (DS, trisomy 21). Because of the limited availability of human post-mortem tissue, DS mouse models are of great interest to study these changes and the underlying neurobiological mechanisms. Although previous studies have shown the potential of Ts65Dn mice – the most widely used mouse model of DS – to model noradrenergic changes, a comprehensive monoaminergic characterization in multiple brain regions has not been performed so far. Here, we used RP-HPLC with electrochemical detection to quantify (nor)adrenergic (NA, adrenaline and MHPG), dopaminergic (DA, HVA and DOPAC), and serotonergic compounds (tryptophan, 5-HT and 5-HIAA) in ten regionally dissected brain regions of Ts65Dn mice, as well as in Dp1Tyb mice – a novel DS mouse model. Comparing young adult aneuploid mice (2.5–5.5 months) with their euploid WT littermates did not reveal generalized monoaminergic dysregulation, indicating that the genetic overload in these mice barely affected the absolute concentrations at this age. Moreover, we studied the effect of aging in Ts65Dn mice: comparing aged animals (12–13 months) with their younger counterparts revealed a large number of significant changes. In general, the (nor)adrenergic system appeared to be reduced, while serotonergic compounds were increased with aging. Dopaminergic alterations were less consistent. These overall patterns appeared to be relatively similar for Ts65Dn and WT mice, though more observed changes were regarded significant for WT mice. Similar human post-mortem studies are necessary to validate the monoaminergic construct validity of the Ts65Dn and Dp1Typ mouse models (Dekker et al., 2017, Neurobiol Dis.).

Down syndrome (DS) human brain tissue and biofluids

Review - Down syndrome (DS), present in nearly six million people, is associated with an extremely high risk to develop Alzheimer's disease (AD). Amyloid-β and tau pathology are omnipresent from age 40 years onward, but clinical symptoms do not appear in all DS individuals. Dementia diagnostics is complex in this population, illustrating the great need for predictive biomarkers. Although blood biomarkers have not yet proven useful, cerebrospinal fluid (CSF) biomarkers (low amyloid-β42, high t-tau, and high p-tau) effectively contribute to AD diagnoses in the general population and are increasingly used in clinical practice. Surprisingly, CSF biomarkers have been barely evaluated in DS. Breaking the taboo on CSF analyses would finally allow for the elucidation of its utility in (differential) diagnoses and staging of disease severity. A sensitive and specific biomarker profile for AD in DS would be of paramount importance to daily care, adaptive caregiving, and specific therapeutic interventions (Dekker et al. 2017, Alz Dem).


People with Down syndrome (DS) are at high risk for Alzheimer’s disease (AD). Defects in monoamine neurotransmitter systems are implicated in DS and AD but have not been comprehensively studied in DS. Therefore, noradrenaline, adrenaline, and their metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG); dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid; and serotonin and its metabolite 5-hydroxyindoleacetic acid were quantified in 15 brain regions of DS without AD (DS, n=4), DS with AD (DS+AD, n=17), early-onset AD (EOAD, n=11) patients, and healthy non-DS controls (n=10) in the general population. Moreover, monoaminergic concentrations were determined in cerebrospinal fluid (CSF)/plasma samples of DS (n=37/149), DS with prodromal AD (DS+pAD, n=13/36), and DS+AD (n=18/40). In brain, noradrenergic and serotonergic compounds were overall reduced in DS+AD versus EOAD, while the dopaminergic system showed a bidirectional change. For DS versus non-DS controls, significantly decreased MHPG levels were noted in various brain regions, though to a lesser extent than for DS+AD versus EOAD. Apart from DOPAC, CSF/plasma concentrations were not altered between groups. In conclusion, monoamine neurotransmitters and metabolites were evidently impacted in DS, DS+AD, and EOAD. DS and DS+AD presented a remarkably similar monoaminergic profile, possibly related to early deposition of amyloid pathology in DS. To confirm whether monoaminergic alterations are indeed due to early amyloid beta accumulation, future avenues include positron emission tomography studies of monoaminergic neurotransmission in relation to amyloid deposition, as well as relating monoaminergic concentrations to CSF/plasma levels of amyloid beta and tau within individuals (Dekker et al., 2018, Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring). F. Cerebrospinal fluid (CSF) and serum 3-methoxy-4-hydroxyphenylglycol (MHPG) as a potential biomarker for differential dementia diagnosis

Among parkinsonian disorders

Lewy body disorders, including Parkinson’s disease (PD), Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB), are characterized by profound central and peripheral monoaminergic dysfunction. Consequently, we wanted to investigate whether these alterations depend on dementia status, and thus measured cerebrospinal fluid (CSF) and serum monoamine and metabolite levels across subgroups of the cognitive spectrum, and evaluated their marker potential afterwards. In total, 153 subjects were included, of which 43 healthy controls (HC), 28 PD patients with normal cognition (PD-NC), 26 patients with PD and mild cognitive impairment (PD-MCI), 18 PDD patients, and 38 DLB patients. The levels of monoamines and metabolites in paired CSF and serum samples were analyzed applying reversed-phase high-performance liquid chromatography with electrochemical detection. Firstly, when comparing subgroups, CSF 3-methoxy-4-hydroxyphenylglycol (MHPG) levels were found lowest in HC and PD-NC groups and significantly higher in PDD/DLB patients. In addition, CSF 5-hydroxyindoleacetic acid (5-HIAA) levels differed significantly between HC and PD-MCI/PDD, and DLB patients (P≤.001), but not between HC and PD-NC patients. Secondly, when performing logistic regression, it was shown that particularly CSF/serum MHPG levels and the serum MHPG to noradrenaline (NA) ratio effectively differentiated between HC and (non-)pooled PD subgroups (AUC=.914-.956), and PDD and DLB patients (AUC=.822), respectively. Furthermore, CSF 5-HIAA was the most discriminative parameter to differentiate between PD-NC and PD-MCI (AUC=.808), and, PD-NC and PDD subgroups (AUC=.916). To conclude, our data revealed that especially alterations of the noradrenergic neurotransmitter system could distinguish between Lewy body disorder subtypes, pinpointing CSF/serum MHPG and NA as potential stage markers across the cognitive spectrum (van der Zee et al., 2018, J Parkinsons Dis.).

Alzheimer’s disease (AD) vs. dementia with Lewy bodies (DLB)


Given the challenges concerning the differential diagnosis of dementia, we investigated the possible added value of monoaminergic compounds to the standard cerebrospinal fluid (CSF) Alzheimer’s disease (AD) biomarkers. Particularly, regarding the AD versus dementia with Lewy bodies (DLB) comparison, monoamines or their metabolites might have added discriminative value as there is a more severe neuropathological burden in the locus coeruleus of DLB patients, the principal site of noradrenaline synthesis. We applied enzyme-linked immunosorbent assay (ELISA) to analyze CSF amyloid b peptide of 42 amino acids, total tau, and tau phosphorylated at threonine 181, in patients with AD, frontotemporal dementia, DLB/Parkinson’s disease dementia, and controls. Reversed-phase highperformance liquid chromatography with electrochemical detection was implemented to study monoamine and metabolite levels in CSF and serum. Stepwise forward conditional logistic regression and receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic accuracy of these newly fitted models containing the most discriminative indicators of disease status. As for our results, most significant differences in CSF and serum were confined to the noradrenergic system. More specifically, CSF 3-methoxy-4-hydroxyphenylglycol (MHPG) levels were higher, whereas serum MHPG levels were lower, in DLB patients compared with all other groups. Addition of CSF and serum MHPG levels to the CSFAD biomarker panel significantly increased diagnostic accuracy between DLB/Parkinson’s disease dementia and AD. Interestingly, a model only including CSF and serum MHPG without the classic AD biomarker panel reached similar area under the curve values. In short, we hypothesize that varying degrees of neuronal loss in the locus coeruleus of DLB/Parkinson’s disease dementia versus AD patients result in differentially altered MHPG levels, making this metabolite a valuable biomarker (Janssens et al., 2018, Alzheimers Dement (Amst).). G. Exploring the neurochemical continuum between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) Behavioral variant frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are early-onset debilitating diseases with genetic and clinicopathological commonalities, and are hypothesized to be part of a disease continuum alongside FTD-ALS. Although serotonergic and dopaminergic disturbances were recently identified in brain and spinal cord of ALS patients, information about neurochemical status of these disorders in body fluids remains scarce. Furthermore, few studies have compared neurochemical parameters directly between FTD and ALS. No FDA-approved disease-modifying drug therapies are available for FTD, nor ALS, with the sole exception of riluzole. An unmet medical need thus exists for both conditions, necessitating the identification of new pharmacological targets or the establishment of biomarkers for early disease detection. We, therefore, retrospectively analyzed paired cerebrospinal fluid (CSF) – serum samples from 40 FTD, 26 ALS, 4 FTD-ALS and 40 age-matched control subjects using reversed-phase ultra-high-performance liquid chromatography with electrochemical detection. Levels of serotonergic, dopaminergic and (nor)adrenergic compounds were measured for all samples. As for our results, statistically significant differences in monoamine concentrations were predominantly present between FTD and age-matched control subjects, with significantly increased CSF (P<0.001) and serum (P<0.001) dopamine (DA) concentrations in FTD subjects. In contrast, levels of 3,4-dihydroxyphenylacetic acid (DOPAC) (P=0.008 for CSF; P=0.003 for serum) and DOPAC/DA ratios (P<0.001 for CSF; P<0.001 for serum), indicative for dopaminergic turnover, were lowered in latter patients. A similar pattern was observed between the ALS and age-matched control group, despite insignificant differences for DOPAC/DA ratios and DOPAC levels in CSF and serum, respectively. Concerning the (nor)adrenergic system, a significant increase in CSF MHPG concentrations was identified in FTD compared to age-matched control subjects (P=0.006). No dissimilarities could be detected in any of the monoaminergic compounds between FTD and ALS patients. Given the dopaminergic differences in CSF and serum samples in the age-matched control group compared to FTD and ALS groups, we hypothesize that both may be characterized by a


dopaminergic disturbance. Furthermore, since no monoaminergic differences could be identified between patients, such neurodegenerative disorders do not only seem to belong to a genetic and clinicopathological continuum, but are likely to share a similar neurochemical pathophysiology as well (poster presented during AAIC London, 2017; Janssens et al., 2017; Alzheimers Dement 13(7): supplement page P1514).

H. Novel biochemical biomarkers for dementia

The cerebrospinal fluid neurogranin/BACE1 ratio is a potential correlate of cognitive decline in

AD

In diagnosing AD, ratios of CSF biomarkers, such as CSF Aβ1-42/tau, have an improved diagnostic performance compared to the single analytes, yet, still a limited value to predict cognitive decline. Since synaptic dysfunction/loss is closely linked to cognitive impairment, synaptic proteins are investigated as candidate CSF AD progression markers. We studied CSF levels of the postsynaptic protein neurogranin and protein BACE1, predominantly localized presynaptically, and their relation to CSF total-tau, Aβ1-42, Aβ1-40, and Aβ1-38. All six analytes were considered as single parameters as well as ratios. Every ELISA involved was based on monoclonal antibodies, including the BACE1 and neurogranin immunoassay. The latter specifically targets neurogranin C-terminally truncated at P75, a more abundant species of the protein in CSF. We studied patients with MCI due to AD (n = 38) and 50 dementia due to AD patients, as well as age-matched cognitively healthy elderly (n = 20). A significant subset of the patients was followed up by clinical and neuropsychological (MMSE) examinations for at least one year. The single analytes showed statistically significant differences between the clinical groups, but the ratios of analytes indeed had a higher diagnostic performance. Furthermore, only the ratio of CSF neurogranin trunc P75/BACE1 was significantly correlated with the yearly decline in MMSE scores in patients with MCI and dementia due to AD, pointing toward the prognostic value of the ratio. This is the first study demonstrating that the CSF neurogranin trunc P75/BACE1 ratio, reflecting postsynaptic/presynaptic integrity, is related to cognitive decline (De Vos et al, Journal of Alzheimer’s Disease).

While neurogranin has no value as plasma biomarker for Alzheimer’s disease, it may be a potential blood biomarker for traumatic brain injury. This evokes the question whether there are changes in neurogranin levels in blood in other conditions of brain injury, such as acute ischemic stroke (AIS). We therefore explored neurogranin in paired cerebrospinal fluid (CSF)/plasma samples of AIS patients (n = 50) from a well-described prospective study. In parallel, we investigated another neuronal protein, i.e. tau, which has already been suggested as potential AIS biomarker in CSF and blood. ELISA as well as Single Molecule Array (Simoa) technology were used for the biochemical analyses. Statistical analyses included Shapiro-Wilk testing, Mann-Whitney analyses and Pearson’s correlation analysis. The results showed that, in contrast to tau, of which high levels in both CSF and plasma were related to stroke characteristics like severity and long-term outcome, plasma neurogranin levels were only correlated with infarct volume. Likewise, CSF neurogranin levels were significantly higher in patients with an infarct volume > 5 mL than in patients with smaller infarct volumes. Finally, neurogranin and tau were significantly correlated in CSF, whereas a weaker relationship was observed in plasma. These findings indicate that although plasma and CSF neurogranin may reflect the volume of acute cerebral ischemia, this synaptic protein is less likely to be a potential AIS biomarker. Levels of tau correlated with severity and outcome of stroke in both plasma and CSF, in the present study as well as previous reports, confirming the potential of tau as an AIS biomarker (De Vos et al., 2017; BMC Neurology).

The β-site amyloid-β protein precursor (AβPP) cleaving enzyme-1 (BACE1) is the rate limiting enzyme in the generation of amyloid-β peptide (Aβ) from AβPP, one of the major pathways in Alzheimer’s disease (AD) pathology. Increased BACE1 levels and activity have been reported in the brain of patients with sporadic AD. Therefore, changes of BACE1 levels in the cerebrospinal


fluid (CSF) have also been investigated as a possible biomarker of the disease. We analyzed BACE1 levels in CSF of elderly healthy participants before and after chronic treatment with a BACE inhibitor (BACEi) and evaluated the correlation between BACE1 levels and downstream AD markers. Overall, BACE1 CSF levels showed strong correlations to all downstream AD markers investigated. This is the first reported finding that shows BACE1 levels in CSF were well correlated to its end product Aβ1-42. As previously described, BACE1 levels were strongly correlated to total-tau and phosphorylated tau levels in CSF. Generally, chronic BACE inhibition did not influence BACE1 CSF protein levels. Follow-up studies including early-stage AD pathophysiology and prodromal AD patients will help to understand the importance of measuring BACE1 routinely in daily clinical practice and AD clinical trials (Timmers et al., 2017, JAD).

I. Generation of new AD-related hypotheses

Sleep and Alzheimer's disease: A pivotal role for the suprachiasmatic nucleus.

Alzheimer's disease (AD), which accounts for most of the dementia cases, is, aside from cognitive deterioration, often characterized by the presence of non-cognitive symptoms. Society is desperately in need for interventions that alleviate the economic and social burden related to AD. Circadian dysrhythmia, one of these symptoms in particular, immensely decreases the self-care ability of AD patients and is one of the main reasons of caregiver exhaustion. Studies suggest that these circadian disturbances form the root of sleep-wake problems, diagnosed in more than half of AD patients. Sleep abnormalities have generally been considered merely a consequence of AD pathology. Recent evidence suggests that a bidirectional relationship exists between sleep and AD, and that poor sleep might negatively impact amyloid burden, as well as cognition. The suprachiasmatic nucleus (SCN), the main circadian pacemaker, is subjected to several alterations during the course of the disease. Its functional deterioration might fulfill a crucial role in the relation between AD pathophysiology and the development of sleep abnormalities. This review aims to give a concise overview of the anatomy and physiology of the SCN, address how AD pathology precisely impacts the SCN and to what degree these alterations can contribute to the progression of the disease (Van Erum et al. 2017, Sleep Med Rev).

Targeting the norepinephrinergic system in Parkinson's disease and related disorders: The locus

coeruleus story.

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD) are related, progressive and debilitating neurodegenerative disorders with hallmark features that include a variety of motor and non-motor symptoms (behavioral, autonomic and cognitive dysfunction). For almost half a century, the motor aspects have been attributed to Lewy pathology (LP) predominantly in the substantia nigra (SN), causing a major loss of dopaminergic neurons. However, the relative success of dopaminergic replacement therapies for alleviation of solely the parkinsonian features has prompted researchers to further explore other monoaminergic strategies which may tackle all PD-related aspects. In this regard, recent evidence suggests that LP in the locus coeruleus (LC), the brain's main source of norepinephrine (NE), precedes that of the SN, and, may be one of the very first etiological events in PD. Interestingly, oxidized NE has neuroprotective properties and may even prevent the formation of toxic and higher molecular weight α-synuclein oligomers associated with PD. Moreover, norepinephrinergic neurons directly innervate the SN, and, LC lesioning causes more severe dopaminergic cell loss and supplementary motor manifestations, as shown in preclinical research. In fact, the LC may be considered one of the main orchestrators that controls the other major monoaminergic nuclei, such as the SN and raphe nuclei. Apart from its regulating function, disruption of such a sustainable but vulnerable LC-NE system has been linked to the cognitive pathophysiology of dementia as well. Consequently, LC neuronal loss and the accompanying norepinephrinergic deficiency constitute an important pharmacological target for the (symptomatic) treatment of PD/DLB/PDD. This review, therefore, summarizes and


discusses all relevant neurochemical research, including the intriguing link with (prodromal) dementia, several biomarker opportunities, the latest therapeutic strategies to enhance NE signaling, and, finally, some overarching comments and perspectives for future research (Vermeiren and De Deyn, 2017, Neurochem Int.)

Link between glaucoma and AD

A growing body of evidence suggests a link between Alzheimer’s disease (AD) and glaucoma. A prospective case study including AD patients with glaucoma and AD patients without glaucoma , as well as non-AD dementia patients and control subjects, is set up to confirm the high rate of comorbidity between AD and glaucoma. The planned yearly follow-up will allow estimates of glaucoma incidence in this patient population. All patients undergo routine physical examination with recording of medical history, neurologic examination with measurement of lumbar CSF pressure, blood sampling for future genetic association studies, ophthalmological examination, measurement of CSF biomarkers, magnetic resonance imaging, and profound neuropsychological examination. This study will establish whether reduced CSFP and a high trans-lamina cribrosa pressure difference play an important role in the pathogenesis of glaucoma in AD. Various hypothesis for the involvement of e.g. CSF flow, intracranial pressure fluctuations, and the glymphatic system in the development of glaucoma were discussed (Wostyn et al. 2017, Invest Ophthalmol Vis Sci.; Wostyn et al. 2017, Ann Neurol. 2017; Wostyn et al. 2017, Clin Exp Optom. 2017; Wostyn et al. 2017, Acta Ophthalmol.; Wostyn et al. 2017, Acta Neurochir Wien; Wostyn et al. 2017, Biomed Res Int; Wostyn et al., 2017, J Neuroophthalmol; Wostyn & De Deyn, 2017, Biomark Med; Wostyn & De Deyn, 2017, Invest Ophthalmol Vis Sci; Wostyn & De Deyn, 2018 JAMA Ophthalmol.). J. Study of Cerebrovascular Disease

Stroke

Dipeptidyl peptidase IV (DPPIV) inhibition may be a promising therapeutic strategy for acute stroke treatment, given its potential to prolong the biological half-life of neuroprotective substrates. A related protease, fibroblast activation protein (FAP), was recently shown to inactivate the same substrates. Therefore, it should also be investigated as a potential target in stroke. The study aimed to investigate whether stroke severity and outcome correlate with DPPIVand FAP activities and their kinetics shortly after acute ischemic stroke. DPPIV and FAP activities were analyzed in the serum of 50 hyperacute stroke patients at admission, 1 day, 3 days, and 7 days after stroke onset and in 50 age-matched healthy controls. This was done as part of the Middelheim’s Interdisciplinary Stroke Study. DPPIV activity tended to increase shortly after stroke compared to the control population. DPPIV and FAP activities steadily decreased in the first week after stroke onset. Higher infarct volumes (≥5 ml) and a more severe stroke (NIHSS >7) at admission were correlated with a stronger decrease in the activities of both enzymes. Moreover, these patients more often developed a progressive stroke, were more often institutionalized. Patients with a stronger increase in DPPIV activity at admission and decrease in the activity of both DPPIVand FAP during the first week after stroke onset had a more severe stroke and worse short-term outcomes (Baerts et al, 2017, Transl Stroke Res). Little is known of procarboxypeptidase U (proCPU) in cerebrospinal fluid (CSF) of stroke patients. Therefore, ProCPU levels were studied in CSF of controls and non-thrombolyzed acute ischemic stroke patients. The results showed that ProCPU is elevated in CSF of stroke patients compared with controls. In conclusion, ProCPU in CSF correlates with stroke progression, outcome, and blood-brain barrier dysfunction (Mertens et al., 2017, Journal of Thrombosis and Haemastasis).

Subcortical small vessel disease


Vascular cognitive impairment (VCI) is a heterogeneous entity with multiple aetiologies, all linked to underlying vascular disease. Among these, VCI related to subcortical small vessel disease (SSVD) is emerging as a major homogeneous subtype. Its progressive course raises the need for biomarker identification and/or development for adequate therapeutic interventions to be tested. In order to shed light in the current status on biochemical markers for VCI-SSVD, experts in field reviewed the recent evidence and literature data. The group conducted a comprehensive search on Medline, PubMed and Embase databases for studies published until 15.01.2017. The proposal on current status of biochemical markers in VCI-SSVD was reviewed by all co-authors and the draft was repeatedly circulated and discussed before it was finalized. This review identifies a large number of biochemical markers derived from CSF and blood. There is a considerable overlap of VCI-SSVD clinical symptoms with those of Alzheimer’s disease (AD). Although most of the published studies are small and their findings remain to be replicated in larger cohorts, several biomarkers have shown promise in separating VCI-SSVD from AD. These promising biomarkers are closely linked to underlying SSVD pathophysiology, namely disruption of blood-CSF and blood–brain barriers (BCB-BBB) and breakdown of white matter myelinated fibres and extracellular matrix, as well as blood and brain inflammation. The leading biomarker candidates are: elevated CSF/blood albumin ratio, which reflects BCB/BBB disruption; altered CSF matrix metalloproteinases, reflecting extracellular matrix breakdown; CSF neurofilment as a marker of axonal damage, and possibly blood inflammatory cytokines and adhesion molecules. The suggested SSVD biomarker deviations contrasts the characteristic CSF profile in AD, i.e. depletion of amyloid beta peptide and increased phosphorylated and total tau. Conclusions: Combining SSVD and AD biomarkers may provide a powerful tool to identify with greater precision appropriate patients for clinical trials of more homogeneous dementia populations. Thereby, biomarkers might promote therapeutic progress not only in VCI-SSVD, but also in AD (Wallin et al. 2017, BMC Neurol.).

K. Study of Neuroinflammation Neutrophil gelatinase-associated lipocalin (NGAL) is an inflammatory protein with gaining increasing interest for its use as marker in blood and cerebrospinal fluid (CSF) for several chronic diseases. Its biochemical properties make it an attractive marker. However, changes in blood and CSF NGAL concentrations during the diurnal rhythm in the elderly are unknown. This information is important for its optimal use as marker in studies with older people. Serial paired plasma and CSF samples were obtained from 8 healthy elderly males over a 30-hour period. NGAL and cortisol were quantified with ELISA. No significant changes in plasma and CSF NGAL concentrations over time were found, whereas cortisol (included as internal control) concentrations displayed significant changes over time. Significant circadian patterns were found for plasma NGAL and for cortisol in both plasma and CSF. However, CSF NGAL concentrations did not follow a diurnal pattern in elderly males. This study illustrates the temporal regulation of NGAL in plasma and CSF, which potentially is a useful reference for studies measuring NGAL as biomarker in older individuals (Naudé et al., 2017 Eur J Clin Invest.). Co-existing depression worsens Alzheimer's disease (AD) pathology. Neutrophil gelatinase-associated lipocalin (NGAL) is a newly identified (neuro)inflammatory mediator in the pathophysiologies of both AD and depression. This study aimed to compare NGAL levels in healthy controls, AD without depression (AD-D), and AD with co-existing depression (AD+D) patients. Protein levels of NGAL and its receptors, 24p3R and megalin, were assessed in nine brain regions from healthy controls (n = 19), AD-D (n = 19), and AD+D (n = 21) patients. NGAL levels in AD-D patients were significantly increased in brain regions commonly associated with AD. In the hippocampus, NGAL levels were even further increased in AD+D subjects. Unexpectedly, NGAL levels in the prefrontal cortex of AD+D patients were comparable to those in controls. Megalin levels were increased in BA11 and amygdala of AD+D patients, while no changes in 24p3R were


detected. These findings indicate significant differences in neuroimmunological regulation between AD patients with and without co-existing depression. Considering its known effects, elevated NGAL levels might actively promote neuropathological processes in AD with and without depression (Dekens et al. 2017, J Alzheimers Dis.). Chronic neuroinflammation, which is primarily mediated by microglia, plays an essential role in aging and neurodegeneration. It is still unclear whether this microglia-induced neuroinflammation occurs globally or is confined to distinct brain regions. In this study, we investigated microglia activity in various brain regions upon healthy aging and Alzheimer's disease (AD)-related pathology in both human and mouse samples. In purified microglia isolated from aging mouse brains, we found a profound gene expression pattern related to pro-inflammatory processes, phagocytosis, and lipid homeostasis. Particularly in white matter microglia of 24-month-old mice, abundant expression of phagocytic markers including Mac-2, Axl, CD16/32, Dectin1, CD11c, and CD36 was detected. Interestingly, in white matter of human brain tissue the first signs of inflammatory activity were already detected during middle age. Thus quantification of microglial proteins, such as CD68 (commonly associated with phagocytosis) and HLA-DR (associated with antigen presentation), in postmortem human white matter brain tissue showed an age-dependent increase in immunoreactivity already in middle-aged people (53.2 ± 2.0 years). This early inflammation was also detectable by non-invasive positron emission tomography imaging using [11C]-(R)-PK11195, a ligand that binds to activated microglia. Increased microglia activity was also prominently present in the white matter of human postmortem early-onset AD (EOAD) brain tissue. Interestingly, microglia activity in the white matter of late-onset AD (LOAD) CNS was similar to that of the aged clinically silent AD cases. These data indicate that microglia-induced neuroinflammation is predominant in the white matter of aging mice and humans as well as in EOAD brains. This white matter inflammation may contribute to the progression of neurodegeneration, and have prognostic value for detecting the onset and progression of aging and neurodegeneration (Raj et al., 2017, Front Mol Neurosci.) Alzheimer's disease (AD) is strongly associated with microglia-induced neuroinflammation. Particularly, Aβ plaque-associated microglia take on an "activated" morphology. However, the function and phenotype of these Aβ plaque-associated microglia are not well understood. We show hyperreactivity of Aβ plaque-associated microglia upon systemic inflammation in transgenic AD mouse models (i.e., 5XFAD and APP23). Gene expression profiling of Aβ plaque-associated microglia (major histocompatibility complex II+ microglia) isolated from 5XFAD mice revealed a proinflammatory phenotype. The upregulated genes involved in the biological processes (gene ontology terms) included: "immune response to external stimulus" such as Axl, Cd63, Egr2, and Lgals3, "cell motility", such as Ccl3, Ccl4, Cxcr4, and Sdc3, "cell differentiation", and "system development", such as St14, Trpm1, and Spp1. In human AD tissue with similar Braak stages, expression of phagocytic markers and AD-associated genes, including HLA-DRA, APOE, AXL, TREM2, and TYROBP, was higher in laser-captured early-onset AD (EOAD) plaques than in late-onset AD plaques. Interestingly, the nonplaque parenchyma of both EOAD and late-onset AD brains, the expression of above-mentioned markers were similarly low. Here, we provide evidence that Aβ plaque-associated microglia are hyperreactive in their immune response and phagocytosis in the transgenic AD mice as well as in EOAD brain tissue. We suggest that Aβ plaque-associated microglia are the primary source of neuroinflammation related to AD pathology (Yin et al., 2017, Neurobiol aging). II.2 From the IBB Biobank (P.P. De Deyn, J-J. Martin and co-workers) and Laboratory of

Neuromuscular Pathology and TF Neuromuscular disorders (J. Baets with key input

from M. Lammens, dept. head Department of Pathology UZA)


A. Neurodegenerative disorders – routine practice

The diagnosis of a series of conditions characterized by dementia depends on the neuropathological and immunohistochemical study of representative areas of the central nervous system. The accurate description of the structure and of the topography of the histological lesions is a prerequisite for further research. As a result of this morphological approach and taking into account the recommendations of international collaborative groups it is possible to give a correct neuropathological diagnosis of a series of neurodegenerative disorders affecting the nervous system. These results are then used as a golden standard for research in the field of neurochemistry and molecular genetics. Conversely the results of this research can be used to improve the histopathological classification of the disorders, eg in the field of the fronto-temporal lobe dementias (FTLD).

The brains of patients who have been adequately examined during their life are removed within 2-3 hours after death. The right half of the brain, brainstem and cerebellum is fixed in formalin, while the left part of the brain is immediately deep-frozen. After a period of 2-4 weeks, the formalin-fixed part of the brain is photographed and examined macroscopically. Representative parts of the brain (Brodmann’s areas 6, 7, 8, 9, 10, 11, 12, 17, 18, 22, 24, 46, neostriatum, putamen and pallidum, thalamus at the level of the centrum medianum, corpus subthalamicum, mesencephalon, pons at the level of the locus coeruleus, medulla oblongata and cerebellum) are embedded in paraffin. Classical staining techniques like cresyl violet and HE are used for the cytology, the method of Gallyas for the neurofibrillary tangles, the method of Klüver-Barrera for the myelin. Immunohistochemistry is made with antibodies against hyperphosphorylated tau (AT8), amyloid Aß+ (4G8), ubiquitine, alpha-synucleïne and against glial fibrillary acidic protein according to standard methods.

The subsequent table summarizes the Biobank Brain inclusions from Jan 2015 till December 2017 Number of cases

Neuropathological diagnosis 2015 2016 2017

Adult onset leucoencephalopathy with axonal spheroids and pigmented cells 1 - -

Alzheimer disease neuropathological changes 13 13 26

Anti-Hu encephalopathy - - 1

Amyotrophic lateral sclerosis 2 5 5

Brain tumor - 2 -

Cerebral amyloid angiopathy (CAA) 2 2 2

Controls - - 1

Corticobasal degeneration - - -

Creutzfeldt-Jakob disease (sporadic/familial) 10 11 9

Diffuse Lewy body disease (various forms) 7 5 2

FTLD-MND (TDP types 1-4, UPS, tau, C9orf72 repeat expansion, Pick) 3 - 7

Globular glial tauopathy - - 1

Huntington chorea 1 1 1

Liver encephalopathy - 2

Leukodystrophy - 1 1

Mixed dementia (vascular +SDAT) - 3 -

Multiple system atrophy 2 2 2

Multiple vascular brain injury 1 1 -

PART (primary age related tauopathy) 1 3 3

Pick disease - 1 -

Progressive supranuclear palsy - - 4

Small vessel disease (cerebrovascular disease) 2 4 3

Spinocerebellar ataxias 1 1 -

Total 46 57 68

B. Neurodegenerative disorders – contribution to research projects


Neuropathological evaluation of a large FTLD-TDP family carrying a GRN null mutation

We describe the clinical and neuropathological findings of nine members of the Belgian progranulin

gene (GRN) founder family. In this family, the loss-of-function mutation IVS1 + 5G > C was

identified in 2006. In 2007, a clinical description of the mutation carriers was published that

revealed the clinical heterogeneity among IVS1 + 5G > C carriers. We report our comparison of our

data with the published clinical and neuropathological characteristics of other GRN mutations as

well as other frontotemporal lobar degeneration (FTLD) syndromes, and we present a review of the

literature. For each case, standardized sampling and staining were performed to identify

proteinopathies, cerebrovascular disease, and hippocampal sclerosis. The neuropathological

substrate in the studied family was compatible in all cases with transactive response DNA-binding

protein (TDP) proteinopathy type A, as expected. Additionally, most of the cases presented also

with primary age-related tauopathy (PART) or mild Alzheimer's disease (AD) neuropathological

changes, and one case had extensive Lewy body pathology. An additional finding was the presence

of cerebral small vessel changes in every patient in this family. Our data show not only that the

IVS1 + 5G > C mutation has an exclusive association with FTLD-TDP type A proteinopathy but

also that other proteinopathies can occur and should be looked for. Because the penetrance rate of

the clinical phenotype of carriers of GRN mutations is age-dependent, further research is required to

investigate the role of co-occurring age-related pathologies such as AD, PART, and cerebral small

vessel disease. (Sieben et al. 2018, Alzheimer's Research & Therapy).

Ongoing project (2016-2018): Hippocampal sclerosis in FTLD: the link with underlying

proteinopathies and cerebrovascular disease. (research project dr. Anne Sieben)

Neuropathological contributions to other studies:

- Teenage-onset progressive myoclonic epilepsy due to a familial C9orf72 repeat expansion (van den Ameele et al. 2018, Neurology).

- Neuropathological evaluation of all IBB cases with Down syndrome (Dekker et al., 2018, Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring).

- Neuropathology of C9orf72 mutation carriers (Van Mossevelde et al. 2017, JAMA Neurol) - Neuropathological re-examination of all FTLD cases in IBB (Goossens et al. 2017,

Alzheimers Res Ther; Goossens et al. 2017, J Alzheimers Dis.) - Neuropathology of VPS13C mutation carriers (research project Stephanie Philtjens) - Neuropathology of ABC17 mutation carriers (PhD research project Tobi Van Den Bossche) - Neuropathology of DPP6 mutation carriers (PhD research project Rita Cacace) - Neuropathological re-evaluation of 100 AD cases using Montines criteria (PhD research

project Clarisse Somers)

C. Routine histopathology including electron microscopy (EM) of tissues for diagnostic purposes in a wide variety of neurological disorders

The context of the Laboratory of Neuromuscular Pathology has changed in the recent year, first of all logistically due to the new and modernized facility at T5 in 2016 and more recently due to the retirement of the former head of the Laboratory for Ultrastructural Neuropathology, Chantal Ceuterick and most recently of Edith Peeters senior technician. Also, Karen Sterck will leave us in April 2018. Two new technicians have been appointed in the meantime, both with MLT-certificates (Medical Laboratory Technician) as required for the new regulations in terms of accreditation. Training of these people is largely complete concerning light microscopy techniques and still ongoing for electron microscopy techniques. Preparatory steps are being taken in order to fulfill the accreditation requirements for diagnostic histopathology in order to be able to keep performing these assays with RIZIV reimbursement. This will be rolled out with help and close supervision of


prof. M. Lammens of the department of pathology at the UZA given his expertise and also taking into account that our diagnostic histology unit will de facto function as a sub-branch of the pathology department at UZA. For the year 2017 the following numbers of diagnostic test have been performed, as a reference overall numbers are listed for the two preceding years:

Light microscopy Immunohistochemistry EM RIZIV nomenclature

588276/588240 588070/588081 588114/588125

Numbers 2015 105 39 121

2016 106 50 97

2017 98 61 113

D. Laboratory of Neuromuscular Pathology – reseach activities - Integration with Neurogenetics Laboratory and P. De Jonghe

Close and largely integrated work with the activities of P. De Jonghe within the Neurogenetics Laboratory, with J. Baets as a staff scientist. Scientific activities on molecular genetics as described above under the headings “Cerebellar ataxias and Hereditary Spastic Paraplegias, Inherited Peripheral Neuropathies and Muscle disorders”. Detailed description not repeated to avoid redundancy. Additional key experimental input from LNP concerning muscle biobanking and diagnostic workup of myopathy patients. These include a large cohort of patients with genetic muscle disease as a preparatory step leading up to further genetic studies (MYO-SEQ in collaboration with the University of Newcastle, UK). Additionally, systematic histopathologic workup is necessary in a cohort of patients with inflammatory myopathies most particularly inclusion body myositis (sIBM). Using the biosamples of a large cohort of sIBM patients we have started a collaborative study with prof. S. Maudsley at the VIB Centre for Molecular Neurology in using state-of-the-art preoteomics techniques in order to pinpoint the key mechanism driving this poorly understood degenerative muscle disease.

II.3 Laboratory of Neurogenetics Neurodegenerative Brain Diseases (C. Van Broeckhoven, M. Cruts, K. Sleegers, J. Theuns, Julie

van der Zee and co-workers)

A. Genetic follow-up of novel dementia genes

Investigating the role of ALS genes CHCHD10 and TUBA4A in Belgian FTD-ALS spectrum

patients

Mutation screen and phenotypic profiling of two ALS and FTD associated genes, CHCHD10 and TUBA4A, was performed in a Belgian cohort of 459 FTD, 28 FTD-ALS and 429 ALS patients. In CHCHD10, we identified a novel nonsense mutation (p.Gln108*) in a patient with atypical clinical FTD and pathology-confirmed Parkinson’s disease leading to loss of transcript. In TUBA4A, we detected a novel frameshift mutation (p.Arg64Glyfs*90) leading to a truncated protein in one FTD patient with family history of Parkinson’s disease and cognitive impairment, and a novel missense mutation (p.Thr381Met) in two sibs with familial ALS and memory problems in whom we previously identified a pathogenic C9orf72 repeat expansion mutation. This study confirmed the role of CHCHD10 and TUBA4A in the FTD-ALS spectrum, although genetic variations in these two genes are extremely rare in the Belgian population and often associated with symptomatology


of related neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease (Perrone, Nguyen et al., Neurobiology of Aging 2017).

TBK1 mutation spectrum in an extended European patient cohort with frontotemporal dementia

and amyotrophic lateral sclerosis.

We investigated the mutation spectrum of the TANK-Binding Kinase 1 (TBK1) gene and its associated phenotypic spectrum by exonic resequencing of TBK1 in a cohort of 2538 patients with FTD, ALS or FTD plus ALS, ascertained within the European Early-Onset Dementia Consortium. We assessed pathogenicity of predicted protein truncating mutations by measuring loss of RNA expression. Functional effect of in-frame amino acid deletions and missense mutations was further explored in vivo on protein level and in vitro by an NFκB-induced luciferase reporter assay and measuring phosphorylated TBK1. The protein truncating mutations led to loss of transcript through nonsense-mediated mRNA decay. For the in-frame amino acid deletions, we demonstrated loss of TBK1 or phosphorylated TBK1 protein. An important fraction of the missense mutations compromised NFκB activation indicating that at least some functions of the TBK1 protein are diminished or lost, implying that also missense mutations might be disease-causing or contributing to increased risk for disease (van der Zee, Gijselinck et al., Human Mutation 2017).

B. Rare variants in AD Deleterious ABCA7 mutations and transcript rescue mechanisms in early-onset Alzheimer’s disease

Premature termination codon (PTC) mutations in ABCA7 have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer’s disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age; indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early-onset AD (EOAD) - control cohort, and examined the effect on transcript level with comprehensive third generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5–41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD (De Roeck, Van den Bossche et al., Acta Neuropathologica 2017).

Identification and description of three families with familial Alzheimer disease that segregate

variants in the SORL1 gene.

The majority of AD cases are sporadic, while up to 5% are families with EOAD. Mutations in one of the three genes: amyloid beta precursor protein (APP), presenilin 1 (PSEN1) or presenilin 2 (PSEN2) can be disease causing. However, most EOAD families do not carry mutations in any of these three genes, and candidate genes, such as the sortilin-related receptor 1 (SORL1), have been


suggested to be potentially causative. To identify AD causative variants, we performed whole-exome sequencing on five individuals from a family with EOAD and a missense variant, p.Arg1303Cys (c.3907C > T) was identified in SORL1 which segregated with disease and was further characterized with immunohistochemistry on two post mortem autopsy cases from the same family. In a targeted re-sequencing effort on independent index patients from 35 EOAD-families, a second SORL1 variant, c.3050-2A > G, was found which segregated with the disease in 3 affected and was absent in one unaffected family member. The c.3050-2A > G variant is located two nucleotides upstream of exon 22 and was shown to cause exon 22 skipping, resulting in a deletion of amino acids Gly1017- Glu1074 of SORL1. Furthermore, a third SORL1 variant, c.5195G > C, recently identified in a Swedish case control cohort included in the European Early-Onset Dementia consortium study, was detected in two affected siblings in a third family with familial EOAD. The finding of three SORL1-variants that segregate with disease in three separate families with EOAD supports the involvement of SORL1 in AD pathology. The cause of these rare monogenic forms of EOAD has proven difficult to find and the use of exome and genome sequencing may be a successful route to target them (Thonberg et al., Acta Neuropathologica Communications 2017).

C. Genotype-phenotype correlations in neurodegenerative dementia

Clinical evidence of disease anticipation in families segregating a C9orf72 repeat expansion.

Patients carrying a C9orf72 repeat expansion leading to frontotemporal dementia and/or amyotrophic lateral sclerosis have highly variable ages at onset of disease, suggesting the presence of modifying factors. We aimed to provide clinical-based evidence for disease anticipation in families carrying a C9orf72 repeat expansion by analyzing age at onset, disease duration, and age at death in successive generations. This cohort study was performed from June 16, 2000, to June 1, 2016, in 36 extended Belgian families in which a C9orf72 repeat expansion was segregating. The generational effect on age at onset, disease duration, and age at death was estimated using a mixed effects Cox proportional hazards regression model, including random-effects terms for within-family correlation and kinship. Time until disease onset or last examination, time from disease onset until death or last examination, or age at death was collected for for 244 individuals (132 proven or obligate C9orf72 carriers), of whom 147 were clinically affected (89 proven or obligate C9orf72 carriers). Among the 111 individuals with age at onset available (66 men and 45 women; mean [SD] age, 57.2 [9.1] years), the mean (SD) age at onset per generation (from earliest-born to latest-born generation) was 62.5 (8.3), 57.1 (8.2), 54.6 (10.2), and 49.3 (7.5) years. Censored regression analysis on all affected and unaffected at-risk relatives confirmed a decrease in age at onset in successive generations (P < .001). No generational effect was observed for disease duration or age at death.The clinical data provide supportive evidence for the occurrence of disease anticipation in families carrying a C9orf72 repeat expansion by means of a decrease in age at onset across successive generations. This finding may help clinicians decide from which age onward it may be relevant to clinically follow presymptomatic individuals who carry a C9orf72 repeat expansion (Van Mossevelde et al., JAMA Neurology 2017). Genetic Alzheimer Disease and Sporadic Dementia With Lewy Bodies: A Comorbidity Presenting

as Primary Progressive Aphasia.

We report a 44-year-old woman, with a family history of early-onset dementia, presenting with primary progressive aphasia. This clinically variable syndrome has multiple underlying pathologies, and correlations between clinical manifestations and postmortem neuropathologic findings are controversial. Our patient suffered worsening language impairment with major word-finding difficulties but preserved comprehension. She also developed episodic memory impairment. Her condition progressed to dementia with behavioral changes. Magnetic resonance imaging showed


early left perisylvian and bitemporal atrophy. The patient died shortly afterward from colon cancer. Neuropathologic examination revealed advanced early-onset Alzheimer and Lewy body disease, plus a clinically nonrelevant metastasis of her colon cancer in her left parietal lobe. Genetic examination revealed a p.Glu184Asp mutation in the presenilin1 gene. Our findings confirm the importance of a thorough appreciation for the clinical and neuropathologic correlations in patients with atypical neurodegenerative dementias (Picková et al., Cognitive and Behavioral Neurology 2017).

Familial primary lateral sclerosis or dementia associated with Arg573Gly TBK1 mutation.

Primary lateral sclerosis (PLS) is a rare variant of motor neuron disease (MND) characterised by selective upper motor neuron features whose causes and pathogenic mechanisms remain largely unknown. While some familial cases of childhood to young–adult onset with recessive transmission have been reported in association with mutations in the Alsin, SPG11 and SPG7 genes, most adult cases occur sporadically. Recently, TBK1 mutations have been identified in 1.9% of FTD and/or ALS cohorts. However, pathogenicity of many TBK1 missense mutations is difficult to establish in the absence of cosegregation data. A screening for TBK1 mutations carried out by the European Early-Onset Dementia Consortium in a large series of FTD/ALS cases found one carrier of the Arg573Gly mutation.3 The index case belonged to a Spanish family with autosomal dominant disease manifesting in the sixth decade as either dementia or PLS, and in whom we were able to demonstrate cosegregation (Gómez-Tortosa et al., JNNP 2017).

D. Consortium participations Through our collaborations within the European Alzheimer’s Disease Initiative (EADI) consortium we contributed to identification of novel risk loci for AD harboring rare genetic variants in a rare variant analysis of the AD GWAS data gathered by the International Genomics of Alzheimer’s Project (IGAP) (Sims et al., Nature Genetics 2017).

Through our collaboration within the International FTD-Genomics Consortium (IFGC), we contributed to identification of risk variants for clinical subtypes of FTD (Mishra et al., Brain 2017) and analysis of overlap between FTD genetic architecture and AD and PD (Ferrari et al., JNNP 2017).

We contributed to the meta-analysis of a large-scale investigation of ATXN2 trinucleotide repeat length in risk of ALS (Sproviero et al., Neurobiol Aging 2017).

E. Reviews in leading journals and books

We published three review papers and one book chapter highlighting different aspects of FTD genetics. Modifiers of GRN-associated frontotemporal lobar degeneration.

Heterozygous loss-of-function (LOF) mutations in GRN cause FTLD by a mechanism of haploinsufficiency. Patients present most frequently with frontotemporal dementia, which is the second most common neurodegenerative dementia at young age. Currently, no disease-modifying therapies are available for these patients. Stimulating GRN protein expression or inhibiting its breakdown is an obvious therapeutic strategy, and is indeed the focus of current preclinical research and clinical trials. Multiple studies have demonstrated the heterogeneity in clinical presentation and wide variability in age of onset in patients carrying a GRN LOF mutation. Recently, this heterogeneity became an opportunity to identify disease modifiers, considering that these might


constitute suitable targets for developing disease-modifying or disease-delaying therapies (Wauters et al., Trends Mol Med 2017).

The Genetics of C9orf72 Expansions.

Repeat expansions in the promoter region of C9orf72 are the most common genetic cause of ALS and related disorders of the ALS/ FTLD spectrum. Remarkable clinical heterogeneity among patients with a repeat expansion has been observed, and genetic anticipation over different generations has been suggested. Genetic factors modifying the clinical phenotype have been proposed, including genetic variation in other known disease genes, the genomic context of the C9orf72 repeat, and expanded repeat size, which has been estimated between 45 and several thousand units. The role of variability in normal and expanded repeat sizes for disease risk and clinical phenotype is under debate. Different pathogenic mechanisms have been proposed, including loss of function, RNA toxicity, and dipeptide repeat (DPR) protein toxicity resulting from abnormal translation of the expanded repeat, but the major mechanism is yet unclear (Gijselinck et al., Cold Spring Harbor Perspectives in Medicine 2017).

Relationship between C9orf72 repeat size and clinical phenotype

Patient carriers of a C9orf72 repeat expansion exhibit remarkable heterogeneous clinical and pathological characteristics suggesting the presence of modifying factors. In accordance with other repeat expansion diseases, repeat length is the prime candidate as a genetic modifier. Observations of earlier onset ages in younger generations of large families suggested a mechanism of disease anticipation. Yet, studies of repeat size and onset age have led to conflicting results. Also, the correlation between repeat size and diagnosis is poorly understood. We review what has been published regarding C9orf72 repeat size as modifier for phenotypic characteristics. Conclusive evidence is lacking, partly due to the difficulties in accurately defining the exact repeat size and the presence of repeat variability due to somatic mosaicism (Van Mossevelde et al., Current Opinion in Genetics & Development 2017).

Frontotemporal dementia

Frontotemporal dementia is a collective term for devastating dementias characterized by behavioral and personality changes or language disturbances due to neurodegeneration of the frontal and temporal areas of the brain. Currently no disease-modifying therapies are available. Research into the genetics and disease mechanisms of FTD has pointed towards novel putative drug targets in the different pathogenetic subtypes of FTD, including FTLD-tau caused by mutations in the microtubule-associated protein tau, and FTLD-TDP, caused by mutations in C9orf72, or GRN. Some targets are currently taken into pre-clinical research and early stages of clinical trials. In this chapter we review current symptomatic disease management as well as promising targets for disease modifying therapies. Both strategies to target tau, C9orf72 or TDP-43-mediated toxicity as well as approaches to enhance GRN expression will be discussed (Wauters et al., In: Disease-Modifying Targets in Neurodegenerative Disorders - Paving the Way for Disease-Modifying Therapies Edited by Baekelandt, V., Lobbestael, E., editors (1st Edition - Elsevier): 199-250 (2017).

II.4 Laboratory of Peripheral nerve systems (PNS) diseases

Neurogenetics (P. De Jonghe, J. Baets, S. Weckhuysen and co-workers)

A. Epilepsy (with key contribution of S. Weckhuysen)

We reviewed STXBP1 and the epileptic encephalopathies related to mutations in this gene. STXBP1 is an essential protein for presynaptic vesicle release. Mutations in STXBP1 have been


associated with a series of (epileptic) neurodevelopmental disorders collectively referred to as STXBP1-encephalopathy (STXBP1-E). In this review we hypothesize about the potential of STXBP1 as a therapeutic target in the field of epileptic encephalopathies. Areas covered: A state of the art overview on current understanding of the pathophysiologic mechanism underlying STXBP1-E is presented. Possibilities of different treatment modalities are discussed including unbiased compound screening, specific protein-protein interaction inhibition and gene therapy, consisting either of gene suppletion or upregulation of gene expression. Expert opinion: Current treatment for STXBP1-E is largely limited to seizure control and future therapies will need to target the developmental aspects of the disease as well. Both in vitro- and animal models used to study the pathophysiology of STXBP1-E could be further optimized as a model for compound screening. They should reflect both the hyper excitable state and the psychomotor delay of STXBP1-E. Specific protein-protein interaction and gene therapy are promising future treatment options that need to be investigated further. We suggest a parallel research strategy on basic pathophysiology and compound development with both fields working in close collaboration with the patient/clinical community. (Stamberger,H. et al Expert Opinion on Therapeutic Targets 2017).

Mutations in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been associated with a spectrum of epilepsies and neurodevelopmental disorders. In a collaborative study reported the phenotypes of 71 patients and reviewed 130 previously reported patients. We found that (i) encephalopathies with infantile/childhood onset epilepsies (≥3 months of age) occur almost as often as those with an early infantile onset (<3 months), and are thus more frequent than previously reported; (ii) distinct phenotypes can be seen within the late onset group, including myoclonic-atonic epilepsy (two patients), Lennox-Gastaut not emerging from West syndrome (two patients), and focal epilepsies with an electrical status epilepticus during slow sleep-like EEG pattern (six patients); and (iii) West syndrome constitutes a common phenotype with a major recurring mutation (p.Arg853Gln: two new and four previously reported children). Other known phenotypes include Ohtahara syndrome, epilepsy of infancy with migrating focal seizures, and intellectual disability or autism without epilepsy. To assess the response to antiepileptic therapy, we retrospectively reviewed the treatment regimen and the course of the epilepsy in 66 patients for which well-documented medical information was available. We find that the use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening. Regarding the genetic findings, truncating mutations were exclusively seen in patients with late onset epilepsies and lack of response to sodium channel blockers. Functional characterization of four selected missense mutations using whole cell patch-clamping in tsA201 cells-together with data from the literature-suggest that mutations associated with early infantile epilepsy result in increased sodium channel activity with gain-of-function, characterized by slowing of fast inactivation, acceleration of its recovery or increased persistent sodium current. Further, a good response to sodium channel blockers clinically was found to be associated with a relatively small gain-of-function. In contrast, mutations in patients with late-onset forms and an insufficient response to sodium channel blockers were associated with loss-of-function effects, including a depolarizing shift of voltage-dependent activation or a hyperpolarizing shift of channel availability (steady-state inactivation). Our clinical and experimental data suggest a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy.(Wolff, M. et al Brain 2017). De novo in-frame deletions and duplications in the SPTAN1 gene, encoding the non-erythrocyte αII spectrin, have been associated with severe West syndrome with hypomyelination and pontocerebellar atrophy. In a collaborative effort we aimed at comprehensively delineating the phenotypic spectrum associated with SPTAN1 mutations. Using different molecular genetic


techniques, we identified 20 patients with a pathogenic or likely pathogenic SPTAN1 variant and reviewed their clinical, genetic and imaging data. SPTAN1 de novo alterations included seven unique missense variants and nine in-frame deletions/duplications of which 12 were novel. The recurrent three-amino acid duplication p.(Asp2303_Leu2305dup) occurred in five patients. Our patient cohort exhibited a broad spectrum of neurodevelopmental phenotypes, comprising six patients with mild to moderate intellectual disability, with or without epilepsy and behavioural disorders, and 14 patients with infantile epileptic encephalopathy, of which 13 had severe neurodevelopmental impairment and four died in early childhood. Imaging studies suggested that the severity of neurological impairment and epilepsy correlates with that of structural abnormalities as well as the mutation type and location. Out of seven patients harbouring mutations outside the α/β spectrin heterodimerization domain, four had normal brain imaging and three exhibited moderately progressive brain and/or cerebellar atrophy. Twelve of 13 patients with mutations located within the spectrin heterodimer contact site exhibited severe and progressive brain, brainstem and cerebellar atrophy, with hypomyelination in most. We used fibroblasts from five patients to study spectrin aggregate formation by Triton-X extraction and immunocytochemistry followed by fluorescence microscopy. αII/βII aggregates and αII spectrin in the insoluble protein fraction were observed in fibroblasts derived from patients with the mutations p.(Glu2207del), p.(Asp2303_Leu2305dup) and p.(Arg2308_Met2309dup), all falling in the nucleation site of the α/β spectrin heterodimer region. Molecular modelling of the seven SPTAN1 amino acid changes provided preliminary evidence for structural alterations of the A-, B- and/or C-helices within each of the mutated spectrin repeats. We conclude that SPTAN1-related disorders comprise a wide spectrum of neurodevelopmental phenotypes ranging from mild to severe and progressive. Spectrin aggregate formation in fibroblasts with mutations in the α/β heterodimerization domain seems to be associated with a severe neurodegenerative course and suggests that the amino acid stretch from Asp2303 to Met2309 in the α20 repeat is important for α/β spectrin heterodimer formation and/or αII spectrin function. (Syrbe, S. et al Brain 2017). Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. In a collaborative study we reported biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease. (Santiago-Sim, T. et al American Journal of Human Genetics 2017) In a collaborative study we evaluated the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling.We reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function. We identified 19 patients with de novo mutations in DNM1 and a


sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function. We can conclude that the phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention. (von Spiczak, S. et al Neurology 2017)

In a collaborative study we described the clinical and whole genome sequencing (WGS) study of a non-consanguineous Italian family in which two siblings, a boy and a girl, manifesting a severe epileptic encephalopathy (EE) with skeletal abnormalities, carried novel SLC35A3 compound heterozygous mutations. Both siblings exhibited infantile spasms, associated with focal, and tonic vibratory seizures from early infancy. EEG recordings showed a suppression-burst (SB) pattern and multifocal paroxysmal activity in both. In addition both had quadriplegia, acquired microcephaly, and severe intellectual disability. General examination showed distal arthrogryposis predominant in the hands in both siblings and severe left dorso-lumbar convex scoliosis in one. WGS of the siblings-parents quartet identified novel compound heterozygous mutations in SLC35A3 in both children. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine transporter. With this study, we add SLC35A3 to the gene list of epilepsies. Neurological symptoms and skeletal abnormalities might result from impaired glycosylation of proteins involved in normal development and function of the central nervous system and skeletal apparatus. (Marini, C. et al American Journal of Medical Genetics - Part A 2017)

In a collaborative study we aimed to analyze whether KCNQ2 R201C and R201H variants, which show atypical gain-of-function electrophysiologic properties in vitro, have a distinct clinical presentation and outcome. Ten children with heterozygous, de novo KCNQ2 R201C or R201H variants were identified worldwide, using an institutional review board (IRB)-approved KCNQ2 patient registry and database. We reviewed medical records and, where possible, interviewed parents and treating physicians using a structured, detailed phenotype inventory focusing on the neonatal presentation and subsequent course. Nine patients had encephalopathy from birth and presented with prominent startle-like myoclonus, which could be triggered by sound or touch. In seven patients, electroencephalography (EEG) was performed in the neonatal period and showed a burst-suppression pattern. However, myoclonus did not have an EEG correlate. In many patients the paroxysmal movements were misdiagnosed as seizures. Seven patients developed epileptic spasms in infancy. In all patients, EEG showed a slow background and multifocal epileptiform discharges later in life. Other prominent features included respiratory dysfunction (perinatal respiratory failure and/or chronic hypoventilation), hypomyelination, reduced brain volume, and profound developmental delay. One patient had a later onset, and sequencing indicated that a low abundance (~20%) R201C variant had arisen by postzygotic mosaicism. Heterozygous KCNQ2 R201C and R201H gain-of-function variants thus present with profound neonatal encephalopathy in the absence of neonatal seizures. Neonates present with nonepileptic myoclonus that is often misdiagnosed and treated as seizures. Prognosis is poor. This clinical presentation is distinct from the phenotype associated with loss-of-function variants, supporting the value of in vitro functional screening. These findings suggest that gain-of-function and loss-of-function variants need different targeted therapeutic approaches. (Mulkey, S.B. Epilepsia 2017)


Recurrent and prolonged seizures are harmful for the developing brain, emphasizing the importance of early seizure recognition and effective therapy. Amplitude-integrated electroencephalography (aEEG) has become a valuable tool to diagnose epileptic seizures, and, in parallel, genetic etiologies are increasingly being recognized, changing the paradigm of the workup and management of neonatal seizures. In a multicenter descriptive study, we reported, clinical data and aEEG findings of 9 newborns with KCNQ2 mutations. Refractory seizures occurred in the early neonatal period with similar seizure type, including tonic features, apnea, and desaturation. A distinct aEEG seizure pattern, consisting of a sudden rise of the lower and upper margin of the aEEG, followed by a marked depression of the aEEG amplitude, was found in 8 of the 9 patients. Prompt recognition of this pattern led to early treatment with carbamazepine in the 2 most recent cases. Early recognition of the electroclinical phenotype by using aEEG may thus direct genetic testing and a precision medicine approach with sodium channel blockers in neonates with KCNQ2 mutations. (Vilan, A. et al Neonatology 2017)

To ascertained possible determinants of carbamazepine (CBZ)- and oxcarbazepine (OXC)-induced hyponatremia in a large cohort of people with epilepsy. We collected data on serum sodium levels in people with epilepsy who were attending a tertiary epilepsy center while on treatment with CBZ or OXC. We defined hyponatremia as Na+ ≤134 mEq/L and severe hyponatremia as Na+ ≤128 mEq/L. We identified 1,782 people who had used CBZ (n = 1,424) or OXC (n = 358), of whom 50 were treated with both drugs. Data on sodium level measurements were available in 1,132 on CBZ and in 289 on OXC. Hyponatremia occurred in 26% of those taking CBZ and 46% of those taking OXC. This was severe in 7% in the CBZ group and 22% in the OXC group. Hyponatremia was symptomatic in 48% and led to admissions in 3%. Age over 40 years, high serum levels of CBZ and OXC, and concomitant use of other antiepileptic drugs were the main risk factors for hyponatremia in both treatment groups. Female patients on OXC were at a higher risk than male patients of hyponatremia. The risk of hyponatremia on CBZ was significantly associated with the risk of hyponatremia on OXC within a subgroup that used both drugs consecutively. We can conclude that hyponatremia is a common problem in people taking CBZ or OXC. Regular ascertainment of sodium levels in those taking either drug is recommended and results should be acted on. (Berghuis,B. et al Epilepsia 2017)

The classic epileptic encephalopathies, including infantile spasms (IS) and Lennox-Gastaut syndrome (LGS), are severe seizure disorders that usually arise sporadically. De novo variants in genes mainly encoding ion channel and synaptic proteins have been found to account for over 15% of patients with IS or LGS. The contribution of autosomal recessive genetic variation, however, is less well understood. In a collaborative study of two large consortia we implemented a rare variant transmission disequilibrium test (TDT) to search for autosomal recessive epileptic encephalopathy genes in a cohort of 320 outbred patient-parent trios that were generally prescreened for rare metabolic disorders. In the current sample, our rare variant transmission disequilibrium test did not identify individual genes with significantly distorted transmission over expectation after correcting for the multiple tests. While the rare variant transmission disequilibrium test did not find evidence of a role for individual autosomal recessive genes, our current sample is insufficiently powered to assess the overall role of autosomal recessive genotypes in an outbred epileptic encephalopathy population.(Epi4K Consortium et al European Journal of Human Genetics 2017)

B. Spastic paraplegias and Spinocerebellar Ataxias (with key contribution of J. Baets) Spastic paraplegia type 5 (SPG5) is a rare subtype of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative disorders defined by progressive neurodegeneration of the corticospinal tract motor neurons. SPG5 is caused by recessive mutations in the gene CYP7B1 encoding oxysterol-7α-hydroxylase. This enzyme is involved in the degradation of cholesterol into primary bile acids. CYP7B1 deficiency has been shown to lead to accumulation of neurotoxic oxysterols. We participated in a multicentre study, contributing to the detailed clinical and


biochemical analysis in 34 genetically confirmed SPG5 cases from 28 families, studying dose-dependent neurotoxicity of oxysterols in human cortical neurons. Our patients participated in a randomized placebo-controlled double blind interventional trial targeting oxysterol accumulation in serum of SPG5 patients. Clinically, SPG5 manifested in childhood or adolescence (median 13 years). Gait ataxia was a common feature. SPG5 patients lost the ability to walk independently after a median disease duration of 23 years and became wheelchair dependent after a median 33 years. The overall cross-sectional progression rate of 0.56 points on the Spastic Paraplegia Rating Scale per year was slightly lower than the longitudinal progression rate of 0.80 points per year. Biochemically, marked accumulation of CYP7B1 substrates including 27-hydroxycholesterol was confirmed in serum (n = 19) and cerebrospinal fluid (n = 17) of SPG5 patients. Moreover, 27-hydroxycholesterol levels in serum correlated with disease severity and disease duration. Oxysterols were found to impair metabolic activity and viability of human cortical neurons at concentrations found in SPG5 patients, indicating that elevated levels of oxysterols might be key pathogenic factors in SPG5. We thus performed a randomized placebo-controlled trial (EudraCT 2015-000978-35) with atorvastatin 40 mg/day for 9 weeks in 14 SPG5 patients with 27-hydroxycholesterol levels in serum as the primary outcome measure. Atorvastatin, but not placebo, reduced serum 27-hydroxycholesterol from 853 ng/ml [interquartile range (IQR) 683-1113] to 641 (IQR 507-694) (-31.5%, P = 0.001, Mann-Whitney U-test). Similarly, 25-hydroxycholesterol levels in serum were reduced. In cerebrospinal fluid 27-hydroxycholesterol was reduced by 8.4% but this did not significantly differ from placebo. As expected, no effects were seen on clinical outcome parameters in this short-term trial. In this study, we define the mutational and phenotypic spectrum of SPG5, examine the correlation of disease severity and progression with oxysterol concentrations, and demonstrate in a randomized controlled trial that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in serum of SPG5 patients. We thus demonstrate the first causal treatment strategy in hereditary spastic paraplegia. (Schöls, L. et al Brain 2017)

CHIP, the protein encoded by STUB1, is a central component of cellular protein homeostasis and interacts with several key proteins involved in the pathogenesis of manifold neurodegenerative diseases. This gives rise to the hypothesis that mutations in STUB1 might cause a far more multisystemic neurodegenerative phenotype than the previously reported cerebellar ataxia syndrome. We contributed our data to whole exome sequencing data-sets from n = 87 index subjects of two ataxia cohorts that were screened for individuals with STUB1 mutations. In-depth phenotyping by clinical evaluation and neuroimaging was performed in mutation carriers. Four novel STUB1 mutations in three affected subjects from two index families (frequency 2/87 = 2.3%). All three subjects presented with a severe multisystemic phenotype including severe dementia, spastic tetraparesis, epilepsy, and autonomic dysfunction in addition to cerebellar ataxia, plus hypogonadism in one index patient. Diffusion tensor imaging revealed degeneration of manifold supra- and infratentorial tracts. These findings provide clinical and imaging support for the notion that CHIP is a crucial converging point of manifold neurodegenerative processes, corresponding with its universal biological function in neurodegeneration. Further, our data reveal the second STUB1 family with ataxia plus hypogonadism reported so far, demonstrating that Gordon Holmes syndrome is indeed a recurrent manifestation of STUB1. However, it does not present in isolation, but as part of a broad multisystemic neurodegenerative process. This supports the notion that STUB1 disease should be conceptualized not by historical or clinical syndromic names, but as a variable multisystemic disease defined by disturbed function of the underlying STUB1 gene, which translates into a multidimensional gradual spectrum of variably associated clinical signs and symptoms. (Hayer, S.N. et al Orphanet Journal of Rare Diseases 2017)

Despite extensive efforts, half of patients with rare movement disorders such as hereditary spastic paraplegias and cerebellar ataxias remain genetically unexplained, implicating novel genes and unrecognized mutations in known genes. Non-coding DNA variants are suspected to account for a substantial part of undiscovered causes of rare diseases. In a multicenter study we contributed to the


identification of mutations located deep in introns of POLR3A to be a frequent cause of hereditary spastic paraplegia and cerebellar ataxia. First, whole-exome sequencing findings in a recessive spastic ataxia family turned attention to intronic variants in POLR3A, a gene previously associated with hypomyelinating leukodystrophy type 7. Next, screening of a cohort of hereditary spastic paraplegia and cerebellar ataxia cases (n = 618) for mutations in POLR3A identified compound heterozygous POLR3A mutations in ∼3.1% of index cases. Interestingly, >80% of POLR3A mutation carriers presented the same deep-intronic mutation (c.1909+22G>A), which activates a cryptic splice site in a tissue and stage of development-specific manner and leads to a novel distinct and uniform phenotype. The phenotype is characterized by adolescent-onset progressive spastic ataxia with frequent occurrence of tremor, involvement of the central sensory tracts and dental problems (hypodontia, early onset of severe and aggressive periodontal disease). Instead of the typical hypomyelination magnetic resonance imaging pattern associated with classical POLR3A mutations, cases carrying c.1909+22G>A demonstrated hyperintensities along the superior cerebellar peduncles. These hyperintensities may represent the structural correlate to the cerebellar symptoms observed in these patients. The associated c.1909+22G>A variant was significantly enriched in 1139 cases with spastic ataxia-related phenotypes as compared to unrelated neurological and non-neurological phenotypes and healthy controls (P = 1.3 × 10-4). In this study we demonstrate that (i) autosomal-recessive mutations in POLR3A are a frequent cause of hereditary spastic ataxias, accounting for about 3% of hitherto genetically unclassified autosomal recessive and sporadic cases; and (ii) hypomyelination is frequently absent in POLR3A-related syndromes, especially when intronic mutations are present, and thus can no longer be considered as the unifying feature of POLR3A disease. Furthermore, our results demonstrate that substantial progress in revealing the causes of Mendelian diseases can be made by exploring the non-coding sequences of the human genome. (Minnerop, M. et al Brain 2017)

The genetic causes of many rare inherited motoneuron diseases and ataxias (MND and ATX) remain largely unresolved, especially for sporadic patients, despite tremendous advances in gene discovery. Whole exome data is often available for patients, but it is rarely evaluated for unusual inheritance patterns, such as uniparental disomy (UPD). UPD is the inheritance of two copies of a chromosomal region from one parent, which may generate homozygosity for a deleterious recessive variant from only one carrier-parent. Detection of UPD-caused homozygous disease-causing variants is detrimental to accurate genetic counseling. Whole-exome sequencing can allow for the detection of such events. In a collaborative study we systematically studied the exomes of a phenotypically heterogeneous cohort of unresolved cases (n = 96 families) to reveal UPD events hindering a diagnosis and to evaluate the prevalence of UPD in recessive MND and ATX. One hereditary spastic paraplegia case harbored homozygous regions spanning 80% of chromosome 16. A homozygous disease-causing mutation in the SPG35 disease gene was then identified within this region. This study demonstrates the ability to detect UPD in exome data of index patients. Our results suggest that UPD is a rare mechanism for recessive MND and ATX. (Bis, D.M. et al Molecular Genetics & Genomic Medicine 2017)

C. Inherited Peripheral Neuropathies (Taskforce Spierziekten/Perifere Neuropathieën) Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. We are a partner in a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal


model-derived potential biomarkers in skin biopsy mRNA extracts. In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-I is changing over time and correlates with disease progression. In summary, we provided evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A. (Fledrich, R. et al Journal of Neurology, Neurosurgery and Psychiatry 2017) Distal hereditary motor neuropathy is a heterogeneous group of inherited neuropathies characterized by distal limb muscle weakness and atrophy. Although at least 15 genes have been implicated in distal hereditary motor neuropathy, the genetic causes remain elusive in many families. To identify an additional causal gene for distal hereditary motor neuropathy we joined forces with a research team in Taiwan. The Taiwanese team performed exome sequencing for two affected individuals and two unaffected members in a Taiwanese family with an autosomal dominant distal hereditary motor neuropathy in which mutations in common distal hereditary motor neuropathy-implicated genes had been excluded. The exome sequencing revealed a heterozygous mutation, c.770A > G (p.His257Arg), in the cytoplasmic tryptophanyl-tRNA synthetase (TrpRS) gene (WARS) that co-segregates with the neuropathy in the family. Further analyses of WARS in an additional 79 Taiwanese pedigrees with inherited neuropathies and 163 index cases from Australian, European, and Korean distal hereditary motor neuropathy families identified the same mutation in another Taiwanese distal hereditary motor neuropathy pedigree with different ancestries and one additional Belgian distal hereditary motor neuropathy family of Caucasian origin. Cell transfection studies demonstrated a dominant-negative effect of the p.His257Arg mutation on aminoacylation activity of TrpRS, which subsequently compromised protein synthesis and reduced cell viability. His257Arg TrpRS also inhibited neurite outgrowth and led to neurite degeneration in the neuronal cell lines and rat motor neurons. Further in vitro analyses showed that the WARS mutation could potentiate the angiostatic activities of TrpRS by enhancing its interaction with vascular endothelial-cadherin. Taken together, these findings establish WARS as a gene whose mutations may cause distal hereditary motor neuropathy and alter canonical and non-canonical functions of TrpRS. (Tsai, P-C. et al Brain 2017) We contributed to the study of a Dutch research group that ascertained a consanguineous family from Pakistan to have a novel deafness-dystonia syndrome with motor regression, ichthyosis-like features and signs of sensory neuropathy. By applying a combined strategy of linkage analysis and whole-exome sequencing in the presented family, a homozygous nonsense mutation, c.4G>T (p.Glu2*), in FITM2 was identified. FITM2 and its paralog FITM1 constitute an evolutionary conserved protein family involved in partitioning of triglycerides into cellular lipid droplets. Despite the role of FITM2 in neutral lipid storage and metabolism, no indications for lipodystrophy were observed in the affected individuals. In order to obtain independent evidence for the involvement of FITM2 in the human pathology, downregulation of the single Fitm ortholog, CG10671, in Drosophila melanogaster was pursued using RNA interference. Characteristics of the syndrome, including progressive locomotor impairment, hearing loss and disturbed sensory functions, were recapitulated in Drosophila, which supports the causative nature of the FITM2 mutation. Mutation-based genetic counseling can now be provided to the family and insight is obtained into the potential impact of genetic variation in FITM2. (Zazo-Seco,C. et al Disease Models & Mechanisms 2017) We participated in a workshop that brought together nineteen clinicians with a special interest in Charcot-Marie-Tooth disease (CMT) (5 adult and 2 paediatric neurologists, 9 orthopaedic surgeons, 2 therapists, 1 specialist in rehabilitation medicine) and three patient representatives from 8


countries (UK, Italy, Netherlands, Belgium, Germany, Sweden, US and Australia) for the 221st ENMC International workshop on foot surgery in Charcot-Marie-Tooth (CMT) disease on 10–12 June 2016 in Naarden, The Netherlands. Foot surgery is often performed in patients with CMT as foot deformities are frequent complications of this disease. Several procedures have been described to correct severe foot deformities in CMT and surgical approaches appear to vary between centres in the same country and between different countries. The aims of the workshop were to review the current surgical approaches, to agree on some basic principles and recommendations for foot surgery in CMT patients (adults and children) and to identify areas for further research. (Reilly, M. et al Neuromuscular Disorders 2017) D. Muscle Disorders (Taskforce Spierziekten/Perifere Neuropathieën) Mutations in the gene coding for protein O-mannosyl-transferase 2 (POMT2) are known to cause severe congenital muscular dystrophy, and recently, mutations in POMT2 have also been linked to a milder limb-girdle muscular dystrophy (LGMD) phenotype, named LGMD type 2N (LGMD2N). Only four cases have been reported so far.ClinicalTrials.gov ID: NCT02759302 As partners in a multicenter study we report 12 new cases of LGMD2N, aged 18-63 years. Muscle involvement was assessed by MRI, muscle strength testing and muscle biopsy analysis. Other clinical features were also recorded. Presenting symptoms were difficulties in walking, pain during exercise, delayed motor milestones and learning disabilities at school. All had some degree of cognitive impairment. Brain MRIs were abnormal in 3 of 10 patients, showing ventricular enlargement in one, periventricular hyperintensities in another and frontal atrophy of the left hemisphere in a third patient. Most affected muscle groups were hip and knee flexors and extensors on strength testing. On MRI, most affected muscles were hamstrings followed by paraspinal and gluteal muscles. The 12 patients in our cohort carried 11 alleles with known mutations, whereas 11 novel mutations accounted for the remaining 13 alleles. We describe the first cohort of patients with LGMD2N and show that unlike other LGMD types, all patients had cognitive impairment. Primary muscle involvement was found in hamstring, paraspinal and gluteal muscles on MRI, which correlated well with reduced muscle strength in hip and knee flexors and extensors. The study expands the mutational spectrum for LGMD2N, with the description of 11 novel POMT2 mutations in the association with LGMD2N. (Østergaard, ST. et al J Neurol Neurosurg Psychiatry (2017)

Sporadic late-onset nemaline myopathy (SLONM) is a rare, late-onset muscle disorder, characterized by the presence of nemaline rods in muscle fibers. Phenotypic characterization in a large cohort and a comprehensive overview of SLONM are lacking. In a large collaborative study we described the clinico-pathological features, treatment and outcome in a large cohort of 76 patients with SLONM, comprising 10 new patients and 66 cases derived from a literature meta-analysis (PubMed, 1966-2016), and compared these with 15 reported HIV-associated nemaline myopathy (HIV-NM) cases. In 6 SLONM patients, we performed a targeted next-generation sequencing (NGS) panel comprising 283 myopathy genes. SLONM patients had a mean age at onset of 52 years. The predominant phenotype consisted of weakness and atrophy of proximal upper limbs in 84%, of proximal lower limbs in 80% and both in 67%. Other common symptoms included axial weakness in 68%, as well as dyspnea in 55% and dysphagia in 47% of the patients. In 53% a monoclonal gammopathy of unknown significance (MGUS) was detected in serum. The mean percentage of muscle fibers containing rods was 28% (range 1-63%). In 2 cases ultrastructural analysis was necessary to detect the rods. The most successful treatment in SLONM patients (all with MGUS) was autologous peripheral blood stem cell therapy. A targeted NGS gene panel in 6 SLONM patients (without MGUS) did not reveal causative pathogenic variants. In a comparison of SLONM patients with and without MGUS, the former comprised significantly more males, had more rapid disease progression, and more vacuolar changes in muscle fibers. Interestingly, the muscle biopsy of 2 SLONM patients with MGUS revealed intranuclear rods, whereas this feature was not seen in any of the biopsies from patients without paraproteinemia. Compared to the overall


SLONM cohort, significantly more HIV-NM patients were male, with a lower age at onset (mean 34 years). In addition, immunosuppression was more frequently applied with more favorable outcome, and muscle biopsies revealed a significantly higher degree of inflammation and necrosis in this cohort. Similar to SLONM, MGUS was present in half of the HIV-NM patients. SLONM presents a challenging, but important differential diagnosis to other neuromuscular diseases of adult onset. Investigations for MGUS and HIV should be performed, as they require distinct but often effective therapeutic approaches. Even though SLONM and HIV-NM show some differences, there exists a large clinico-pathological overlap between the 2 entities. (Schnitzler, LJ. Et al Orphanet J Rare Dis. 2017)

Peripheral Neuropathy (V. Timmerman, and co-workers) We wrote an update for BRAIN summarizing all published mutations in three small heat shock proteins (HSPBs); so far 32 mutations occur in HSPB1 and were reported in 169 patients, nine mutations have been described in HSPB8 in 68 patients, and one mutation in HSPB3 in two patients. The phenotypic spectrum of HSPB mutations may not be restricted to axonal CMT disease (CMT2) or distal hereditary motor neuropathy (dHMN), but mutations were also reported in rare patients with ALS. Only recently, a few patients with HSPB mutations were reported to develop distal myopathy at a later stage of the motor neuropathy. This indicates that mutations in multifunctional HSPB’s can give rise to multiple neurodegenerative and neuromuscular phenotypes (Adriaenssens et al. 2017, Brain). Recent studies have shown a direct and/or indirect correlation between gene mutations and autophagy. Autophagy, a cellular homeostatic process, is required for removal of cell aggregates, long-lived proteins and dead organelles from the cell in double-membraned vesicles destined for the lysosomes. Autophagy is evolutionarily conserved and essential for the survival and proper functioning of the cell. It has been shown to be affected in various common neurodegenerative diseases of both the central and peripheral nervous system. We reviewed genes involved in hereditary neuropathies which are linked to autophagy and proposed disruption of the autophagic flux as emerging common disease mechanism. We highlighted different steps of autophagy linked to these genes and discussed possibilities and challenges of targeting this pathway (Haidar & Timmerman, 2017, Frontiers in Molecular Neuroscience).

Identification of novel mutations in small heat shock proteins

In collaboration with neurologists in France, we studied the phenotypic spectrum of distal HMN caused by mutations in HSPB1 and HSPB8 and investigated the functional consequences of newly discovered variants. Among 510 unrelated patients living in France we identified mutations in HSPB1 (28 index patients/510; 5.5%) and HSPB8 (4 index patients/510; 0.8%) genes. Patients have slowly progressive distal and proximal weakness in lower limbs, mild lower limbs sensory involvement, foot deformities, progressive distal upper limb weakness, mildly raised serum creatine kinase levels and CNS involvement. We identified 12 HSPB1 and 4 HSPB8 mutations, including respectively 5 and 3 not previously reported. Transmission was either dominant, recessive or de

novo. Three missense mutations in HSPB1 (Pro7Ser, Gly53Asp, Gln128Arg) cause hyperphosphorylation of neurofilaments, while the C-terminal mutant Ser187Leu triggers protein aggregation. Two frameshift mutations (Leu58fs, Ala61fs) create a premature stop codon leading to proteasomal degradation. Two mutations in HSPB8 (Lys141Met/Asn) show increased binding to BAG3. We demonstrate that HSPB1 and HSPB8 mutations are a major cause of inherited motor axonal neuropathy. Mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins (Echaniz-Laguna et al. 2017, Human Mutation).

Mutant HSPB1 causes loss of translational repression by binding to an RNA binding protein.


Most mutations in HSPB1 target the highly conserved α-crystallin domain, while other mutations affect the C- or N-terminal regions or its promotor. Mutations inside the α-crystallin domain have been shown to enhance the chaperone activity of HSPB1 and increase the binding to client proteins. However, the P182L missense mutation, located outside and downstream of the α-crystallin domain, behaves differently. This specific mutation results in a severe phenotype affecting exclusively the motor neurons of the peripheral nervous system. We identified that the P182L mutant protein has a specifically increased interaction with the RNA binding protein poly(C)binding protein 1 (PCBP1), and results in a reduction of its translational repressive activity. RNA immunoprecipitation followed by RNA sequencing on mouse brain lead to the identification of PCBP1 mRNA targets. These targets contain larger 3’- and 5’-UTRs than average and are enriched in an RNA motif consisting of the CTCCTCCTCCTCC consensus sequence. Interestingly, next to the clear presence of neuronal transcripts among the identified PCBP1 targets we identified known genes associated with peripheral neuropathies and spastic paraplegias. We therefore concluded that HSPB1 can mediate translational repression through interaction with an RNA binding protein further supporting its role in neurodegenerative disease (Geuens et al. 2017, Acta Neuropathologica Communications).

HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate ER stress-

induced apoptosis.

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. In collaboration with researchers from Ireland we demonstrated that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing HSPB1, and that this is due to an enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. We showed that cells overexpressing CMT-related HSPB1 mutations exhibited an increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants (Kennedy et al. 2017, Cell Death and Disease).

Development of a KI-KO mouse model for HSPB8

To delineate the molecular deficits and functional consequences of HSPB8 mutations we generated and published our results of a knock-in model for the K141N mutation mimicking the dHMN phenotype. This work took 10 years of continuous research performed by several team members, starting from the design of the KI-KO gene targeting construct to phenotyping. We now observed that homozygous knock-in mice (HspB8K141N/K141N) develop a progressive axonopathy resulting in locomotor deficits. At the ultrastructural level, mice accumulate the mutant HspB8 protein and display degenerative patterns similar to dHMN patients. Interestingly, these animals also develop a progressive myofibrillar myopathy as observed in some rare patients with HSPB8 mutations. Strikingly, the homozygous HspB8 knock-out animals (HspB8-/-) do not show any sign of axonopathy and display a much milder myopathy than the HspB8 knock-in animals (Bouhy et al. 2018, Acta Neuropathologica).

III. METABOLIC CONDITIONS WITH NEUROLOGICAL COMPLICATIONS

With the retirement of Prof Marescau, the Laboratory of Neurochemistry and Behaviour decided to discontinue the study of biochemical and pathophysiological parameters linked to hereditary disorders of the urea cycle and related pathways in December 2017.

IV. PHENOTYPICAL AND BIOCHEMICAL CHARACTERIZATION OF (TRANSGENIC) ANIMAL

MODELS FOR NEURODEGENERATIVE AND OTHER NEUROLOGICAL CONDITIONS


In-depth behavioural phenotyping of (transgenic) mouse models contributes to the unraveling of the influence of gene alterations and gene-environment interactions on the pgenotype of different forms of mental retardation and neurodegeneration, as well as our knowledge of underlying pathophysiological mechanisms and the identification of therapeutic targets for the development of novel treatment strategies. Aging is the key risk factor for Alzheimer's disease (AD). In addition, the amyloid-beta (Aβ) peptide is considered a critical neurotoxic agent in AD pathology. However, the connection between these factors is unclear. We aimed to provide an extensive characterization of the gene expression profiles of the amyloidosis APP23 model for AD and control mice and to evaluate the effect of aging on these profiles. We also correlated our findings to changes in soluble Aβ-levels and other pathological and symptomatic features of the model. We observed a clear biphasic expression profile. The first phase displayed a maturation profile, which resembled features found in young carriers of familial AD mutations. The second phase reflected aging processes and showed similarities to the progression of human AD pathology. During this phase, the model displayed a clear upregulation of microglial activation and lysosomal pathways and downregulation of neuron differentiation and axon guidance pathways. Interestingly, the changes in expression were all correlated to aging in general, but appeared more extensive/accelerated in APP23 mice (Janssen et al., 2017 Biochim Biophys Acta). Tauopathies include a variety of neurodegenerative diseases associated with the pathological aggregation of hyperphosphorylated tau, resulting in progressive cognitive decline and motor impairment. The underlying mechanism for motor deficits related to tauopathy is not yet fully understood. Here, we use a novel transgenic tau mouse line, Tau 58/4, with enhanced neuron-specific expression of P301S mutant tau to investigate the motor abnormalities in association with the peripheral nervous system. Using stationary beam, gait, and rotarod tests, motor deficits were found in Tau 58/4 mice already 3 months after birth, which deteriorated during aging. Hyperphosphorylated tau was detected in the cell bodies and axons of motor neurons. At the age of 9 and 12 months, significant denervation of the neuromuscular junction in the extensor digitorum longus muscle was observed in Tau 58/4 mice, compared to wild-type mice. Muscle hypotrophy was observed in Tau 58/4 mice at 9 and 12 months. Using electron microscopy, we observed ultrastructural changes in the sciatic nerve of 12-month-old Tau 58/4 mice indicative of the loss of large axonal fibers and hypomyelination (assessed by g-ratio). We conclude that the accumulated hyperphosphorylated tau in the axon terminals may induce dying-back axonal degeneration, myelin abnormalities, neuromuscular junction denervation, and muscular atrophy, which may be the mechanisms responsible for the deterioration of the motor function in Tau 58/4 mice. Tau 58/4 mice represent an interesting neuromuscular degeneration model, and the pathological mechanisms might be responsible for motor signs observed in some human tauopathies (Yin Z, Valkenburg F et al. 2017). Various P301S tau transgenic mouse models are used to model frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, a correlation between cognitive deficits and cellular tau pathology at different ages in different related mouse models has not been described in literature yet. We aimed to investigate the correlation of tau neuropathology with age-, genotype- and mouse line-related deficits in motor function, behavior and cognition. We assessed Tau58/2 and 58/4 models with the 0N4R P301S mutation concerning motor function, cognition and other behavioral aspects with the SHIRPA primary screen, neuromotor assessments, behavioral and cognitive paradigms. The spatiotemporal development of tau pathology was investigated immunohistochemically. We observed neuromotor impairments in both models starting from age 3 months. On electron microscopy, neurofibrillary pathology was visible in the spinal cords in mice


aged 3 months; however, AT8 immunoreactivity was not yet observed at this age. Behavioral abnormalities and memory deficits occurred at a far later stage (>9 months) when tau pathology was fully disseminated throughout the brain. Spatiotemporally, tau pathology spreads from the spinal cord via the midbrain to the frontal cortex, while the hippocampus was relatively spared, thus explaining the late-onset of cognitive deficits. Our findings indicate the face- and construct validity of both Tau58 models to investigate tauopathies. These novel, promising models might provide insights into the pathologic effects of tau species in vivo, and may consequently facilitate the development of new therapeutic targets to delay or halt the processes occurring in neurodegenerative tauopathies (Valkenburg et al. to be submitted to Neurodegenerative diseases). High-frequency repetitive transcranial magnetic stimulation (HF-rTMS) is currently accepted as an evidence-based treatment option for treatment-resistant depression (TRD). Additionally, HF-rTMS showed beneficial effects on psychomotor retardation in patients. The classical HF-rTMS paradigms however are unlikely to replace electroconvulsive therapy, a more potent alternative for TRD albeit with important side-effects. Therefore, recent studies have investigated 'accelerated' HF-rTMS protocols demonstrating promising clinical responses in patients with TRD. Since the neuronal effects of accelerated HF-rTMS are underinvestigated, we evaluate here the possible metabolic and neurochemical effects of this treatment alternative. More specifically, we measured the effect on brain glucose metabolism and monoamines/metabolites, as well as on the spontaneous motor activity in rats. We found that brain glucose metabolism and monoamines remained generally unaffected after accelerated HF-rTMS, with the exception of reduced total striatal 5-hydroxyindolacetic acid (a metabolite of serotonin) levels. Interestingly, when compared to sham stimulation, the velocity, the total distance traveled as well as the percentage of movement, as measured by the open-field test, were significantly enhanced after accelerated HF-rTMS showing an increased motor activity. Our current results indicate that the accelerated HF-rTMS-induced increase in motor activity in rats, may be related to the striatal neurochemical effect (El Arfani et al. 2017). Review: Pathophysiological mechanisms underlying Alzheimer's disease (AD) remain insufficiently documented for the identification of accurate diagnostic markers and purposeful target discovery and development. Nonhuman primates (NHPs) have important translational value given their close phylogenetic relationship to humans and similar developmental paths in (neuro)anatomy, physiology, genetics, and neural functions, as well as cognition, emotion, and social behavior. Areas covered: This review deals with the past and future role of NHP-based research in AD pathophysiology, diagnosis and drug discovery, and touches upon ethical and legal aspects. Expert opinion: Aging NHPs are not complete phenocopies of human AD. Conceivably, no other species or experimental model will ever develop the full spectrum of AD-typical alterations. Nevertheless, partial - and even negative - models can increase knowledge of disease mechanisms. Modeling complex brain disorders should not be based on a single model or species. Understanding brain diseases relies on knowledge of healthy brain functioning, and given their close phylogenetic relationship to humans, NHPs serve excellent tools in this respect. NHP-based studies remain essential in the development and validation of radiopharmaceuticals for early diagnostic imaging biomarkers, as well as in the efficacy and safety evaluation of new therapeutic approaches, with active immunization or vaccination approaches as front runners (Van Dam & De Deyn, 2017) Though neuroglobin confers neuroprotection against Alzheimer’s disease (AD) pathology, its expression gets downregulated in late-stage AD. Here, we provide evidence that this decrease is associated with the AD-linked angiopathy. While wild-type mice of different ages show an upregulated cerebral neuroglobin expression upon whole-body hypoxia, APP23 mice exhibit a decreased transcription. Interestingly, transcription of cytoglobin, of which the involvement in the amyloid pathology remained to be elucidated, follows a similar pattern. To further unravel the


underlying mechanism, we studied REST/NRSF expression levels after identifying a recognition site in the regulatory region of both globins. Neuroglobin-cytoglobin-REST/NRSF expression correlations were detected mainly in the cortex. This raises the possibility of REST/NRSF being an upstream regulator (Van Acker et al. 2017, FEBS Letter) ANK3 encodes AnkyrinG, a member of the Ankyrin family that is expressed in several different isoforms in many tissues. A unique serine-rich domain and tail domain present in the two largest isoforms of AnkG (270 and 480 kDa), restrict AnkG to the axon initial segment and nodes of Ranvier of myelinated neurons. At these sites, AnkG is a master regulator, coordinating the strict clustering of components necessary for proper action potential initiation and propagation along the axon. These components include voltage-gated sodium channels, potassium channels and members of the L1 cell adhesion molecule family. Genetic variation in the ANK3 gene has been linked to a range of neuropsychiatric and neurodevelopmental disorders, including schizophrenia, bipolar disorder, intellectual disability and autism spectrum disorders. Here, we study the effect of reduced expression of the brain-specific isoforms of Ank3 on cognition and behavior using a mouse model. In three independent behavioral tests (Open field test, Elevated plus maze and Social interaction test) we found evidence for increased anxiety in our Ank3 mouse model. Besides, we observed specific neuroanatomical defects in heterozygous knockout mice, including a smaller cingulate cortex, granular retrosplenial cortex, primary motor cortex and fimbria of the hippocampus. Our results suggest that reduced expression of the brain-specific isoforms of AnkG has an effect on behavior in mice. (van der Werft et al. 2017, Behav Brain Res). Cognitive functioning in the Morris Water Maze (MWM) is assumed to be reflected by path length. In this study, the interference of motor deficits, as a confounding factor on cognitive functioning, was assessed by means of a lateralization study with hemicerebellectomized (HCX) mice. This model is characterized by motor deficits restricted to the lesion side, allowing comparison within the model itself (left vs. right), rather than the effect of the manipulation on this measure (experimental vs. control). Spatial learning was assessed after left or right hemicerebellectomy in adult mice by means of two MWM designs in which the location of the starting positions was altered for one condition in the adapted (Adap) MWM experiment, hypothesizing that motor impairments ipsilateral to the lesion side result in a difference in path length. When the starting positions were equal for both conditions in the traditional (Trad) MWM experiment, path length during the acquisition phase and spatial memory were more affected for the left HCX, while these effects disappeared after mirroring the starting positions in the Adap MWM, implying that motor phenotype and corresponding increase in task difficulty are responsible for the contradictory results in the Trad MWM experiment. the differences found in the latter experiment were circumvented in the adapted MWM protocol, and therefore, excluding the motor deficit as a confounding factor on cognitive MWM parameters (De Coninck et al., 2017, Somatosens Mot Res.). Over the past year, the Rodent Behavioural Research Unit has, amongst others, been involved in the (behavioural) phenotyping of the following mouse models and/or the following research lines:

- Setup-up and behavioural and (neuro)pathological evaluation of an APP23 x ApoE knockout mouse model for mixed dementia.

- Development and evaluation of an hemicerebellectomy mouse model - Evaluation of sleep and circadian locomotor rhythm in the APP23 mouse model and control

littermates of various ages using the in-house developed EEG/EMG-activity - Evaluation of pharmacologically induced epilepsy in the APP23 mouse model and control

littermates of various ages using the in-house developed EEG/EMG-activity - Evaluation of pharmacologically induced epilepsy in the TAU58/4 mouse model and control

littermates of various ages using the in-house developed EEG/EMG-activity - Evaluation of Fragile X associated mouse models


- Evaluation of FTD-related mouse models - Evaluation of Down syndrome mouse models - Evaluation of temporal lobe epilepsy rat models - Evaluation of hippocampal-related memory deficits in mouse models of vestibulary

araflexia.

V. STUDY OF SPONGIFORM ENCEFALOPATHIES (P. CRAS AND CO-WORKERS)

The research of task force 2 focusses on the role of neuroprotective mechanisms in transmissible spongiform encefalopathies, i.e., Creutzfeldt-Jakob Disease (CJD); bovine spongiform encephalopathy (BSE) and scrapie. Since 1998 a brain bank containing both fixated and frozen brain tissue of neuropathologically confirmed CJD patients, as well as CSF of neuropathologically confirmed, probable and possible CJD patients is established.

In addition, the group has contributed to cervical dystonia, as well as stroke rehabilitation research over the past year.

Cervical dystonia

Cervical dystonia (CD) is a movement disorder characterized by involuntary muscle contractions leading to an abnormal head posture or movements of the neck. Dysfunctions in somatosensory integration are present and previous data showed enlarged postural sway in stance. Postural control during quiet sitting and the correlation with cervical sensorimotor control were investigated. Postural control during quiet sitting was measured via body sway parameters in 23 patients with CD, regularly receiving botulinum toxin treatment and compared with 36 healthy controls. Amplitude and velocity of displacements of the center of pressure (CoP) were measured by two embedded force plates at 1000 Hz. Three samples of 30 s were recorded with the eyes open and closed. Disease-specific characteristics were obtained in all patients by the Tsui scale, Cervical Dystonia Impact Profile (CDIP-58) and Toronto Western Spasmodic Rating Scale (TWSTRS). Cervical sensorimotor control was assessed with an infrared Vicon system during a head repositioning task. Body sway amplitude and velocity were increased in patients with CD compared to healthy controls. CoP displacements were doubled in patients without head tremor and tripled in patients with a dystonic head tremor. Impairments in cervical sensorimotor control were correlated with larger CoP displacements (rs ranged from 0.608 to 0.748). Postural control is impaired and correlates with dysfunction in cervical sensorimotor control in patients with CD. Treatment is currently focused on the cervical area. Further research towards the potential value of postural control exercises is recommended (De Pauw et al., 2017 Exp Brain Res).

Idiopathic cervical dystonia (CD) is a focal dystonia characterized by an abnormal tilted or twisted head position. This abnormal head position could lead to a distorted perception of the visual vertical and spatial orientation. The aim of this cross-sectional study was to investigate whether the perception of the visual vertical is impaired in patients with CD. The subjective visual vertical test (SVV) was measured in 24 patients with CD and 30 controls. The SVV test is conducted in a completely darkened room. A laser bar is projected on an opposing white wall, which is deviated from the earth's gravitational vertical. Participants were seated with their head unrestrained and were instructed to position this bar vertically. The deviations in degrees (°) are corrected for the side of laterocollis in order to measure the E-effect. We found that patients were able to position the laser bar as equally close to the earth's gravitational vertical as controls (+ 0.67° SD ± 2.12 vs + 0.29° SD ± 1.08, p = 0.43). No E-effect was measured. Notwithstanding the abnormal position of the head, the perception of the visual vertical in patients with idiopathic CD is intact, possibly because of central neural compensatory mechanisms (De Pauw et al., 2017, Acta Neurol Belg).

Patients with idiopathic adult-onset cervical dystonia (CD) experience an abnormal head posture and involuntary muscle contractions. Although the exact areas affected in the central nervous system remain uncertain, impaired functions in systems stabilizing the head and neck are apparent


such as the somatosensory and sensorimotor integration systems. The aim of the study is to investigate cervical sensorimotor control dysfunction in patients with CD. Cervical sensorimotor control was assessed by a head repositioning task in 24 patients with CD and 70 asymptomatic controls. Blindfolded participants were asked to reposition their head to a previously memorized neutral head position (NHP) following an active movement (flexion, extension, left, and right rotation). The repositioning error (joint position error, JPE) was registered via 3D motion analysis with an eight-camera infrared system (VICON ® T10). Disease-specific characteristics of all patients were obtained via the Tsui scale, Cervical Dystonia Impact Profile (CDIP-58), and Toronto Western Spasmodic Rating Scale.Patients with CD showed larger JPE than controls (mean difference of 1.5°, p < .006), and systematically 'overshoot', i.e. surpassed the NHP, whereas control subjects 'undershoot', i.e. fall behind the NHP. The JPE did not correlate with disease-specific characteristics. Cervical sensorimotor control is impaired in patients with CD. As cervical sensorimotor control can be trained, this might be a potential treatment option for therapy, adjuvant to botulinum toxin injections (De Pauw et al., 2017, Brain Behav).

Stroke rehabilitation

To investigate the effects of a three month active cycling program followed by coaching on physical activity in subacute stroke patients. Patients (n = 59; mean age =65.4 ± 10.3) aged ≤80 years with first stroke and able to cycle at 50 revolutions/minute enrolled 3-10 weeks post stroke. Patients were randomly allocated to three month active cycling group (n = 33) or to a control group (n = 26), 3 x 30 minutes training/week. Afterwards, the active cycling group was randomized into a coaching (n = 15) versus non-coaching group (n = 16) for nine months. Physical activity was measured by objective and self-reported measures, which were taken before/after the active cycling program and during six and 12 months, except the Baecke-questionnaire, which was used at baseline and 12 months. A significant difference was found in Baecke/sport (95% confidence interval: 0.06, 2.24; p = 0.039) between the active cycling group and the control group, in patients with severe motor function deficits at baseline. Patients in the control group performed significant less sports at 12 months (mean Baecke/sportbaseline =3.07 ± 1.21, mean Baecke/sport12months = 1.43 ± 0.98; p = 0.01). Furthermore, all groups showed significant changes over time in all measures at three months (except: Physical Activity Scale for Individuals with Physical Disabilities, diary/Mets*minutes-moderate) and 12 month and additionally in a subgroup with severe motor function deficits (except diary Mets*minutes-sedentary). When active cycling combined with education is used in subacute patients with severe motor function deficits, more sport participation might be observed after one year. No other significant group differences were found over time. In all groups, however, patients showed significant improvement over time in physical activity measures. Future work is needed to explore the most effective coaching approach after an aerobic training program. Implications for Rehabilitation The active cycling program combined with education is applicable in subacute stroke patients as it required little stand-by assistance due to chip cards, the intensity was gradually built and the involvement of caregivers in the educational sessions. This training approach also revealed applicable in severely impaired stroke patients and might facilitate sport participation on the long-term. This randomized controlled study aims to quantify physical activity after stroke by using a combination of objective and self-report measures, which revealed detailed information about different aspects of physical activity levels. There is a need for coaching approaches that facilitate aerobic exercise after ending a supervised program. A coaching approach needs to guide patients in adopting aerobic exercise as a part of a lifestyle change and needs to be less time consuming (Vanroy et al., 2017, Disabil Rehabil.). To examine the effects of 3 months of aerobic training (AT) followed by coaching on aerobic capacity, strength, and gait speed after subacute stroke. DESIGN: Randomized controlled trial. SETTING: Inpatient rehabilitation center. PARTICIPANTS: Patients (N=59; mean age ± SD, 65.4±10.3y; 21 women (36%); Barthel Index ≤50 in 64% of patients) with first stroke and able to


cycle at 50 revolutions/min were enrolled in the study 3 to 10 weeks after stroke onset. INTERVENTIONS: Patients were randomly allocated to a 3-month active cycling group (ACG, n=33) and education, or to a control group (CG, n=26). Afterward, patients in the ACG were randomly assigned either to a coaching (n=15) or to a noncoaching group (n=16) for 9 months. MAIN OUTCOME MEASURES: Aerobic capacity, isometric knee extension strength, and gait ability and speed were measured before and after intervention and during follow-up at 6 and 12 months. RESULTS: A nonsignificant difference was found in workload (Wattpeak) (P=.078) between ACG and CG after 3 months. Furthermore, after 3 months of cycling and after 9 months of coaching, all groups showed significant changes over time (P≤.027) in peak oxygen consumption, Wattpeak, leg strength, and gait speed. Also, significant changes over time (P<.001) were found in the ACG and the CG in patients with walking inability at baseline. CONCLUSIONS: No significant differences between training groups were found over time. Although our study did not have objective exercise data from the training device during follow-up, the 3-month active cycling (AC) program combined with education sessions seemed an applicable method in subacute stroke rehabilitation. New long-term AT interventions should focus on coaching approaches to facilitate training after a supervised AC program (Vanroy et al., 2017, Arch Phys Med Rehabil).

VI. CELLULAR AND NETWORK NEUROBIOLOGY (M. GIUGLIANO AND CO-WORKERS)

During 2017, it was proposed that the Lab for Theoretical Neurobiology and Neuroengineering incorporated another unit at the Department of Biomedical Sciences of the University of Antwerp. This merge aims at strengthening and widening its research activities and substantially increase the full-time professorial human resources. In 2018, a new unit will be thus launched, under the new name of Molecular, Cellular, and Network Excitability Lab (MCNE), with Prof. M. Giugliano as its spokeperson. This lab is today fully equipped with world-class infrastructures and unique expertise in cellular electrophysiology, cellular neurotechnologies, computer simulations, and high-throughput biosignals analysis. It is routinely engaged in top notch fundamental and translational investigations, independently and in collaboration with a number of centers in Belgium and worldwide. MCNE’s research interest include: ● Cellular and Molecular Excitability

○ Cellular biophysics of action potential initiation in (neo)cortical neurons

○ Downstream transfer of spike-train correlation in (neo)cortical neurons

○ The role of the extracellular matrix in cortical neuronal connectivity

○ Channelopathies and ion channel variants in heterologous expression systems

○ Molecular mechanisms of channel gating

● Network Excitability

○ Emergence of coordinated electrical activity in (neo)cortical neuronal networks

○ Timescales of excitability and phase transition in (neo)cortical neuronal activity

○ Understanding delayed QT effects of drugs and toxins

● Neurotechnology, biological technology, and drug screening

○ The interaction between carbon nanomaterials and neuronal cells membrane

○ Human iPSC as a model for drug safety

○ Optocapacitance

○ Zebrafish models of long QT

● Computational Neuroscience

○ In silico electrophysiology of somatosensory cortical microcircuits


MCNE is fully licensed to perform experiments with tissue collected from small rodents and it has an official authorization for operating an L2 laboratory. This is required for handling adeno-associated viral vectors for Optogenetics and Cellular Imaging applications in Neuroscience. MCNE represents the first Belgian laboratory to join the Human Brain Project FET-Flagship consortium with renewal of its funding for the phase s (2018-2020), funded by the European Commission. All areas of expertise have led to publications in highly ranked international journals, including, in the recent past the journal Nature and, in the current reporting year the prestigious European Journal of Neuroscience. The unit successfully organized in July 2017, the world congress of the Organization for Computational Neuroscience, which attracted more than 500 participants from all around the world. This placed once more our University and IBB on the map of international excellence. TNBN’s unique expertise include:

- Patch-clamp electrophysiology in acute rat brain slices and primary neuronal cell cultures; - Silicon probe multiunit recordings in anaesthetised and awake behaving mice; - In vitro & In vivo intracellular electrophysiology; - In vitro & In vivo substrate-integrated microelectrode arrays for electrophysiology

(McDonald et al. 2017, MRS Communications) - Differential Interference Contrast Microscopy for in vitro intracellular electrophysiology; - (AAV-mediated) Optogenetics modulation of cellular & network excitability and plasticity; - Genetically-encoded Calcium sensors and their confocal imaging in live neuronal networks; - Wide-field LED photoactivation and Galvo-based laser patterned photoactivation; - Data analysis, mathematical modelling, and numerical simulations.

A renewed vision for the future has been already drafted, for MCNE. It aims at accompanying standard cellular neuroscience research on rodent animal models with the ambitious activity of tackling fundamental priorities of human cortical electrophysiology. Thanks to links established with UZA and UZ Leuven, intact cytoarchitecture of ex vivo adult tissue samples, obtained from resective neurosurgery in human patients will become accessible, with more advantages than in

vitro human stem cells reprogramming. Its fundamental significance of this approach became apparent after Giugliano’s discovery in the recent past of unanticipated quantitative differences of human vs. rodent L2/3 pyramidal cells. The unit further aims at formulating and validating single cell mathematical models and network-level descriptions on these experiments. In silico cellular electrophysiology will be used comparatively to guide the priority for cortical cell type experimental investigation. The unit ultimately aims at linking brain (patho)physiological network rhythms and synchronization to cellular biophysical properties, upon a systematic and comparative characterization of the dynamical transfer function of human cortical cells. Such an explicitly link is possible by the quantitative framework of “sparsely synchronized oscillations”. The hypothesis is that, when probed by more accurate protocols than conventional methods, the electrical response properties of human cortical neurons and microcircuits reveal considerable differences from those of rodents, differing from basic textbook knowledge. This vision is relevant and timely for the design of novel therapeutic strategies, given the rapidly ageing population and the associated neurological medical challenges. The unit further aims at studying the molecular mechanism of patient-specific channelopathies and translate this knowledge to the clinic and ultimately to the patient to improve therapy. This is a multidisciplinary approach using non-animal models such as human induced pluripotent stem cell derived cardiac myocytes and neurons. Besides studying channelopathies, which are clinically relevant, we aim at improving in vitro drug-safety assays relevant for the industry. These assays assess electrophysiological cardio- and neuro-toxic effects of drugs and lead-compounds. We will


also continue our investigations using the zebrafish model of cardiac physiology and optocapacitance. Because of the increasing focus on the modulation of channel kinetics by lipids, we will use the developed assays to study the effect of changes in lipid metabolism on cellular and network excitability. These assays will allow to evaluate both epigenetic modifiers such as drugs as genetic variations in endogenous lipid metabolism (e.g. mutations in CYP enzymes). The most important 2017 discovery of TNBN in fundamental neuroscience research, came from the introduction and validation of the so-called dynamical response properties of cortical neurons (Linaro et al. 2017, Eur J Neurosci). These quantify the upper biophysical limits of the rapidity by which a single cortical pyramidal neurons relay time-varying information. Such a novel experimental characterization was pioneered by the lab and sparkled great interest and it extended to a more realistic conductance cellular stimulation paradigm. By such an approach, it became possible to quantify the precise extent at which cells can track fast temporal foreground modulations over a noisy conductance background. This is an important generalization of the conventional frequency – current curve, used as a classic text-book neuronal phenotype characterization over the past decades. Moreover, such an enhanced input-output characterization of cell excitability has been shown to be key in explaining not only network-wide phenomena, such as self-sustained rhythms and transient responses, but more importantly to interpret the correlation transfer as well.

I SCIENTIFIC RESULTS IBB 2017 - bornbunge.be · may be related to the aetiology of suicidal ideation in older adults (Rymo et al., 2017, Acta ... We provide consensus guidelines for

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