Down regulation of trk but not p75NTR gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer’s disease

Down regulation of trk but not p75NTR gene expression in singlecholinergic basal forebrain neurons mark the progression ofAlzheimer’s disease

Stephen D. Ginsberg,*,�,� Shaoli Che,*,� Joanne Wuu,§ Scott E. Counts§ and Elliott J. Mufson§

*Center for Dementia Research, Nathan Kline Institute, �Department of Psychiatry �Department of Physiology & Neuroscience, New

York University School of Medicine, Orangeburg, New York, USA

§Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA

Abstract

Dysfunction of cholinergic basal forebrain (CBF) neurons of

the nucleus basalis (NB) is a cardinal feature of Alzheimer’s

disease (AD) and correlates with cognitive decline. Survival

of CBF neurons depends upon binding of nerve growth

factor (NGF) with high-affinity (trkA) and low-affinity

(p75NTR) neurotrophin receptors produced within CBF neu-

rons. Since trkA and p75NTR protein levels are reduced

within CBF neurons of people with mild cognitive impair-

ment (MCI) and mild AD, trkA and/or p75NTR gene

expression deficits may drive NB degeneration. Using

single cell expression profiling methods coupled with cus-

tom-designed cDNA arrays and validation with real-time

quantitative PCR (qPCR) and in situ hybridization, individual

cholinergic NB neurons displayed a significant down regu-

lation of trkA, trkB, and trkC expression during the pro-

gression of AD. An intermediate reduction was observed in

MCI, with the greatest decrement in mild to moderate AD as

compared to controls. Importantly, trk down regulation is

associated with cognitive decline measured by the Global

Cognitive Score (GCS) and the Mini-Mental State Exam-

ination (MMSE). In contrast, there is a lack of regulation of

p75NTR expression. Thus, trk defects may be a molecular

marker for the transition from no cognitive impairment (NCI)

to MCI, and from MCI to frank AD.

Keywords: microarray, mild cognitive impairment, neurotro-

phin, nucleus basalis, RNA amplification, trkA.

J. Neurochem. (2006) 97, 475–487.

Cholinergic basal forebrain (CBF) neurons of the nucleusbasalis (NB) provide the major cholinergic innervation to thecortex and hippocampus, and play a key role in memory andattentional behaviors (Bartus et al. 1982; Mesulam et al.1983; Baxter and Chiba 1999). CBF neurons undergoselective degeneration including the loss of presynapticcholinergic enzymes [e.g. choline acetyltransferase (ChAT)]during the later stages of Alzheimer’s disease (AD) that isassociated with disease duration and degree of cognitiveimpairment (Wilcock et al. 1982; Bierer et al. 1995).Degeneration of the CBF system suggests that deficits incortical cholinergic transmission mediated via NB neuronsmay contribute to the severe cognitive abnormalities seen inadvanced AD (Whitehouse et al. 1982; Mufson et al. 2003).Despite intense interest in the cholinobasal cortical projectionsystem, the molecular and cellular mechanisms underlyingNB neurodegeneration during the progression of AD remainunclear. Notably, CBF survival appears to require appropriate

binding, internalization, and retrograde transport of theprototypic neurotrophin, nerve growth factor (NGF), whichis synthesized and secreted by cells in the cortex (Sofroniew

Received November 22, 2005; revised manuscript received January 3,2006; accepted January 9, 2006.Address correspondence and reprint requests to Stephen D. Ginsberg,

Center for Dementia Research, Nathan Kline Institute, New York Uni-versity School of Medicine, 140 Old Orangeburg Road, Orangeburg,NY, USA. E-mail: [email protected] used: AD, Alzheimer’s disease; BDNF, brain derived

neurotrophic factor; BSA, bovine serum albumin; CBF, cholinergic basalforebrain; ChAT, choline acetyltransferase; ECD, extracellular domain;EST, expressed sequence-tagged cDNA; GCS, Global Cognitive Score;MCI, mild cognitive impairment; MMSE, Mini-Mental State Examina-tion; NB, nucleus basalis; NCI, no cognitive impairment; NGF, nervegrowth factor; NFT, neurofibrillary tangle; NHS, normal horse serum;PBS, phosphate-buffered saline; PHF, paired helical filament; qPCR,real-time quantitative PCR; ROS, Religious Orders Study; SDS, sodiumdodecyl sulfate; TC, terminal continuation; TK, tyrosine kinase domain.

Journal of Neurochemistry, 2006, 97, 475–487 doi:10.1111/j.1471-4159.2006.03764.x

� 2006 The AuthorsJournal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 475–487 475

et al. 2001; Mufson et al. 2003). NGF delivery attenuatesCBF neurodegeneration and improves learning and memoryin animal models of neurodegeneration, excitotoxicity, aging,and amyloid toxicity (Tuszynski 2002). Moreover, agedtransgenic mice engineered to produce anti-NGF antibodiesexhibit CBF neurodegeneration and inclusions that resemblethe pathologic hallmarks of AD (Capsoni et al. 2000; Rubertiet al. 2000), indicating the importance of appropriate growthfactor synthesis for CBF viability in vivo and the develop-ment of AD-like pathology associated with neurotrophindysregulation.

NGF exerts functional consequences for cholinergic NBneuronal survival by interacting with at least two neurotro-phin receptors, the low-affinity pan-neurotrophin receptorp75NTR and the high-affinity NGF-specific receptor tyrosinekinase, trkA (Kaplan and Miller 2000; Teng and Hempstead2004). TrkB and trkC are also localized to CBF neurons,albeit at lower levels than trkA (Salehi et al. 1996; Mufsonet al. 2002). Trk receptors, along with p75NTR, are producedwithin CBF neurons and transported anterogradely to thecortex where they bind NGF and other members of thisfamily of neurotrophins (Kaplan and Miller 2000; Howe andMobley 2004).

Defining the molecular and cellular mechanisms underly-ing the pathophysiological role of NGF receptors in theselective vulnerability of cholinergic neurons of the NB andthe progression of dementia remains elusive. Characterizingthese mechanisms may lead to the development of rationaltherapies for the amelioration of CBF cellular degeneration,intervention for clinical symptoms, and early diagnosis ofmild cognitive impairment (MCI) and/or AD. To derivecognitively based molecular mechanisms of NGF receptordysfunction from individual cholinergic NB neurons, tissuesamples were harvested post-mortem from cases clinicallycharacterized with no cognitive impairment (NCI), MCI, andAD from the Religious Orders Study (ROS), an ongoinglongitudinal clinicopathological study of aging and AD inolder Catholic nuns, priests, and brothers (Mufson et al.2000, 2002; Bennett et al. 2002). Antemortem cognitivemeasures including the global cognitive score (GCS),comprised of a battery of 19 different neuropsychologicaltests (Bennett et al. 2002), and the Mini-Mental StateExamination (MMSE) were correlated with gene levelexpression of p75NTR, trkA, trkB, and trkC derived fromindividual cholinergic NB neurons.

Materials and methods

Clinical and pathological evaluation of ROS subjects

In order to enter the ROS cohort, subjects are judged by an

examining neurologist to not have any coexisting clinical or

neurologic condition(s) contributing to cognitive impairment.

Neuropsychological tests were chosen to measure a range of

cognitive abilities with emphasis on those affected by aging and AD.

Cognitive testing was performed under the auspices of a trained

neuropsychologist, and scores were available within the last year of

death. The 19 tests that constitute the GCS are listed in

Supplemental Table 1, and they comprised a composite GCS score

for each subject in addition to the individual scores on the respective

cognitive tests. A board-certified neurologist with expertise in the

evaluation of the elderly made a clinical diagnosis for each ROS

participant based upon review of all clinical data and physical

examination. Subjects were categorized as NCI (n ¼ 12; mean age

81.0 ± 9.1 years), MCI insufficient to meet criteria for dementia

(n ¼ 10; 81.9 ± 4.3), or AD (n ¼ 12; 84.5 ± 6.9) (Table 1). Details

of the clinical and neuropsychological evaluation for the ROS

cohort have been published previously (Mufson et al. 2000, 2002;Bennett et al. 2002). This study was performed in accordance with

IRB guidelines administrated by the Rush University Medical

Center and the New York University School of Medicine/Nathan

Kline Institute.

At autopsy, one hemisphere was immersion-fixed in a 4%

paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.2 for

24 h at 4�C, cryoprotected, and cut frozen at a section thickness of

40 lm (Mufson et al. 2000, 2002; Chu et al. 2001; Counts et al.2006). From the opposite hemisphere, tissue from cortex, hippo-

campus, and brainstem structures were harvested and prepared for

paraffin embedding. Tissue sections were stained for the visualiza-

tion of senile plaques and neurofibrillary tangles using thioflavine-S,

antibodies directed against paired helical filament (PHF) tau (gift

from Peter Davies) and a modified Bielschowsky silver stain

(Mufson et al. 2000, 2002; Bennett et al. 2002). Additional sectionswere stained for Lewy bodies using commercially available

antibodies directed against ubiquitin and alpha-synuclein (Mufson

et al. 2000, 2002; Bennett et al. 2002). The remaining tissue slabs

were frozen at )80�C. A pathological diagnosis was made while

the neuropathologist was blinded to the clinical diagnosis. Neuro-

pathological designations were based on the NIA Reagan and

CERAD criteria (Mirra et al. 1991; Hyman and Trojanowski 1997).

In addition, a Braak score (Braak and Braak 1991) was tabulated for

each case. Exclusion criteria included stroke and Parkinson’s

disease. ApoE genotyping was performed by PCR analysis (Mufson

et al. 2000). The majority of AD cases from the ROS cohort used in

this study are mild to moderate AD based upon neuropathological

and cognitive criteria. End-stage AD subjects were not overly

represented in this study. Currently, consensus criteria for the

clinical classification of MCI are being developed (Winblad et al.2004). The present MCI population was defined as subjects with

impaired neuropsychological test scores who were not found to have

dementia by the examining neurologist (Mufson et al. 2000;

Bennett et al. 2002; DeKosky et al. 2002), similar to the criteria

used by independent experts in the field to describe subjects who are

not cognitively normal but do not meet established criteria for

dementia (Petersen 2004; Winblad et al. 2004).

Accession of CBF NB neurons and immunocytochemistry

Acridine orange histofluorescence (Ginsberg et al. 1997, 1998;

Mufson et al. 2002) and bioanalysis (Agilent 2100, Palo Alto, CA,

USA) (Ginsberg and Che 2002, 2004) were performed on each brain

utilized in this study to ensure that high quality RNAwas present in

tissue sections prior to performing downstream genetic analyses.

476 S. D. Ginsberg et al.

Journal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 475–487� 2006 The Authors

Table

1C

linic

al,

dem

ogra

phic

,and

neuro

path

olo

gic

alchara

cte

ristics

Clin

icalD

iagnosis

Gro

up

com

parison

Pairw

ise

com

parisons*

NCI

(N¼

12)

MCI

(N¼

10)

AD

(N¼

12)

Tota

l(N

¼34)

Ageatdeath

(year)

:m

ean

±S

D(r

ange)

81.0

±9.1

(66–92)

81.9

±4.3

(75–92)

84.5

±6.9

(69–94)

82.4

±7.1

(66–94)

p¼

0.3

4a

–

Number(%

)

ofmales:

6(5

0%

)3

(30%

)5

(45%

)14

(42%

)p¼

0.6

8b

–

Education(y

ear)

:m

ean

±S

D(r

ange)

17.5

±4.8

(8–24)

18.8

±2.3

(16–22)

16.3

±4.1

(6–20)

17.6

±3.9

(6–24)

p¼

0.4

1a

–

GCS

:m

ean

±S

D(r

ange)

0.5

±0.3

(0.0

–1.1

)0.2

±0.2

()0.2

,0.4

))

0.9

±0.5

()1.6

,–

0.4

)0.0

±0.7

()1.6

,1.1

)p<

0.0

001

a(N

CI,

MC

I)>

AD

MMSE

:m

ean

±S

D(r

ange)

27.6

±1.5

(25–30)

26.6

±2.8

(20–30)

14.0

±9.7

(0–25)

22.4

±8.8

(0–30)

p<

0.0

001

a(N

CI,

MC

I)>

AD

PMI

(h):

mean

±S

D(r

ange)

12.4

±10.7

(3.2

–33.5

)7.8

±4.7

(3.6

–16)

6.9

±3.2

(3–12)

7.4

±3.2

(3–33.5

)p¼

0.7

0a

–

Number(%

)with

ApoE

e4allele

:

2(1

7%

)4

(40%

)6

(60%

)12

(37%

)p¼

0.1

3b

–

Braakscore

:0

10

01

I/II

50

16

p¼

0.0

22

aN

CI

<(M

CI,

AD

)

III/

IV6

84

18

V/V

I0

27

9

NIA-R

eagan

diagnosis

(lik

elih

ood

of

AD

):

No

AD

00

00

Low

83

112

p¼

0.0

04

aN

CI

<A

D

Inte

rmedia

te3

74

14

Hig

h0

04

4

aK

ruskal–

Walli

ste

st,

bF

isher’s

exact

test.

*With

Bonfe

rroni-ty

pe

corr

ection.

Down regulation of trk 477

� 2006 The AuthorsJournal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 475–487

RNase-free precautions were used throughout the experimental

procedures, and solutions were made with 18.2 mega Ohm

RNase-free water (Nanopure Diamond, Barnstead, Dubuque, IA,

USA).

Tissue sections were processed for p75NTR immunocytochemistry

using a monoclonal antibody raised against human p75NTR

(Schatteman et al. 1988; Mufson et al. 1989b, 2002; Counts et al.2004). p75NTR immunoreactivity colocalizes with approximately

95% of all CBF neurons within the human NB (Mufson et al.1989a,b), and is an excellent phenotypic marker for these cells. Our

laboratory group has also identified CBF neurons for microaspira-

tion using neurofilament immunoreactivity, ChAT immunoreactivity,

and cresyl violet staining as part of preliminary studies and separate

single cell analyses of the basal forebrain (Ginsberg and Che 2002,

2004; Mufson et al. 2002, 2004). CBF neurons selected for

microaspiration were localized to the anterior subfield of the NB

located ventral to the anterior commissure (Mufson et al. 2002). Theanterior NB subfield (Mufson et al. 1989a) can be identified readily

under the dissecting microscope by an investigator blinded to case

demographics, ensuring the aspiration of CBF neurons.

Immunocytochemistry was performed as described previously

(Mufson et al. 1989b, 2002; Counts et al. 2004). Following several

rinses in phosphate-buffered saline (PBS, pH 7.2) tissue sections

were incubated for 20 min in a Tris-buffered saline (pH 7.4)

solution containing 0.1 M sodium periodate (Sigma, St Louis, MO,

USA) to inhibit endogenous peroxidase staining. Tissue sec-

tions were incubated for 1 h in a PBS solution containing 0.3%

Triton X-100, 3% normal horse serum (NHS) and 2% bovine serum

albumin (BSA). Primary antibody (monoclonal p75NTR, 1 : 60 000)

was applied for 4 h at 22�C with constant agitation. The diluent for

the primary antibody contained 0.4% Triton X-100, 1% NHS and

1% BSA. Sections were processed with the ABC kit (Vector

Laboratories, Burlingame, CA, USA) and developed in a 0.2 M

sodium acetate imidazole buffer (pH 7.4) with 2.5% nickel II sulfate

(Sigma), 0.05% 3¢ 3¢ diaminobenzidine (DAB, Sigma) and 0.005%

hydrogen peroxide (pH 7.2) (Chu et al. 2001; Mufson et al. 2002).Immunostained tissue sections were stored in RNase-free PBS at

4�C until neurons were microaspirated for cDNA array analysis

within 72 h.

Single cell microaspiration and Terminal Continuation (TC)

RNA amplification

Microaspiration and TC RNA amplification procedures have been

described in detail elsewhere (Ginsberg and Che 2002, 2004; Che

and Ginsberg 2004) and are diagrammed in Fig. 1. Linearity and

fidelity of the TC RNA amplification procedure has been published,

including the use of CBF neurons as input sources of RNA (Che and

Ginsberg 2004; Ginsberg 2005). Moreover, variability between

single cell expression profiles and reproducibility of expression

levels has been evaluated extensively by our laboratory group and

published previously (Che and Ginsberg 2004; Ginsberg and Che

2004; Ginsberg 2005). Briefly, individual p75NTR-immunoreactive

NB neurons were microaspirated from paraformaldehyde-fixed

40 lm thick frozen cut sections of the basal forebrain using a

micromanipulator and microcontrolled vacuum source (Eppendorf,

Westbury, NY, USA) attached to an inverted microscope (E800,

Nikon, Japan). The amplification of RNA from individual NB

neurons was performed using a new terminal continuation (TC)

RNA amplification methodology (Che and Ginsberg 2004; Ginsberg

and Che 2004; Ginsberg 2005). The TC RNA amplification protocol

is available at http://cdr.rfmh.org/pages/ginsberglabpage.html. Indi-

vidual, not pooled, CBF neurons were extracted in 250 lL of Trizol

reagent (Invitrogen, Carlsbad, CA, USA). RNAs were reverse

transcribed in the presence of the poly d(T) primer (10 ng/lL) and

(a)

(b)

(c) (d)

Fig. 1 (a–b) Photomicrographs of a microaspirated NB neuron. (a)

Representative p75NTR–immunoreactive NB neuron prior to microas-

piration. The arrow points to the tip of a micropipette. (b) Same tissue

section shown in A illustrating the site of the microaspirated neuron

(asterisk). (c) Schematic overview of the molecular experimental

design. (d) Description of the TC RNA amplification method. A TC

primer and a poly d(T) primer are added to the mRNA population to be

amplified (green rippled line). First strand synthesis (blue line) occurs

as an mRNA-cDNA hybrid is formed following reverse transcription.

After an RNase H digestion step to remove the original mRNA tem-

plate strand, second strand synthesis (red line) is performed using Taq

polymerase. The resultant double stranded (ds) product is utilized as

template for in vitro transcription, yielding linear RNA amplification of

antisense orientation (yellow rippled lines).



TC primer (10 ng/lL) in 1X first strand buffer (Invitrogen), 1 mM

dNTPs, 5 mM DTT, 20 U of RNase inhibitor and 5 U reverse

transcriptase (Superscript III, Invitrogen). The synthesized single

stranded cDNAs were converted into double stranded cDNAs by

adding into the reverse transcription reaction the following: 10 mM

Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.5 U RNase H

(Invitrogen) in a total volume of 99 lL. Samples were placed in a

thermal cycler and second strand synthesis proceeded as follows:

RNase H digestion step 37�C, 10 min; denaturation step 95�C,3 min; annealing step 50�C, 3 min; elongation step 75�C, 30 min 5

U (1 lL) Taq polymerase (PE Biosystems, Foster City, CA, USA)

was added to the reaction at the initiation of the denaturation step

(i.e. hot start) (Che and Ginsberg 2004). The reaction was

terminated with 5 M ammonium acetate. The samples were extracted

in phenol:chloroform:isoamyl alcohol (25 : 24 : 1) and ethanol

precipitated with 5 lg of linear acrylamide (Ambion, Austin, TX,

USA) as a carrier. The solution was centrifuged at 14 000 r.p.m and

the pellet washed once with 95% ethanol and air-dried. The cDNAs

were resuspended in 20 lL of RNase free H2O and drop dialyzed on

0.025 lm filter membranes (Millipore, Billerica, MA, USA) against

50 mL of 18.2 MegaOhm RNase-free H2O for 2 h. The sample was

collected off the dialysis membrane and hybridization probes were

synthesized by in vitro transcription using 33P incorporation in

40 mM Tris (pH 7.5), 7 mM MgCl2, 10 mM NaCl, 2 mM spermidine,

5 mM of DTT, 0.5 mM of ATP, GTP, and CTP, 10 lM of cold UTP,

20 U of RNase inhibitor, T7 RNA polymerase (1000 U, Epicentre,

Madison, WI, USA), and 40 lCi of 33P-UTP (GE Healthcare,

Piscataway, NJ, USA). The reaction was performed at 37�C for 4 h.

Radiolabeled TC RNA probes were hybridized to custom-designed

cDNA arrays without further purification.

Custom-designed cDNA array platforms and array

hybridization

Array platforms consisted of 1 lg of linearized cDNA purified from

plasmid preparations adhered to high-density nitrocellulose

(Hybond XL, GE Healthcare). Each cDNA and/or expressed

sequence-tagged cDNA (EST) was verified by sequence analysis

and restriction digestion. cDNA clones and ESTs from mouse, rat,

and human were employed. All of the neurotrophin and neurotro-

phin receptor clones were derived from human sequences. Approxi-

mately 220 cDNAs/ESTs were utilized on the current array platform.

The majority of genes are represented by one transcript on the array

platform. However, several genes have representation at 3¢ and 5¢regions, including the high-affinity neurotrophin receptors (trkA,

trkB, and trkC) to assess relative expression levels from separate

regions of the gene and evaluate potential RNA degradation

(Ginsberg et al. 2000). For example, ESTs that encode the tyrosine

kinase domain (TK) and extracellular domain (ECD) were employed

for trkA, trkB, and trkC.

Arrays were prehybridized (2 h) and hybridized (12 h) in a

solution consisting of 6X SSPE, 5X Denhardt’s solution, 50%

formamide, 0.1% sodium dodecyl sulfate (SDS), and denatured

salmon sperm DNA (200 lg/mL) at 42�C in a rotisserie oven

(Ginsberg and Che 2002, 2004; Ginsberg 2005). Following

hybridization, arrays were washed sequentially in 2X SSC/0.1%

SDS, 1X SSC/0.1% SDS and 0.5X SSC/0.1% SDS for 20 min each

at 42�C. Arrays were placed in a phosphor screen for 24 h and

developed on a phosphor imager (GE Healthcare).

Data collection and statistical analysis

Hybridization signal intensity was quantified by subtracting back-

ground using empty vector (pBs). Expression of TC amplified RNA

bound to each linearized cDNA (approximately 220 cDNAs/ESTs)

was expressed as a proportion of the total hybridization signal

intensity of the array (a global normalization approach). Global

normalization effectively minimizes variation due to differences in

the specific activity of the synthesized TC probe as well as the

absolute quantity of probe present (Eberwine et al. 2001; Ginsbergand Che 2004; Ginsberg 2005). Data analyzed in this manner does

not allow the absolute quantitation of mRNA levels. However, an

expression profile of relative changes in mRNA levels was

generated.

Demographic and clinical characteristics (age, sex, years of

education, GCS, MMSE, ApoE allele, and PMI) and neuropatho-

logic classifications (Braak score and NIA-Reagan diagnosis) were

compared among clinical diagnostic groups by the Kruskal–Wallis

test and Fisher’s exact test, with Bonferroni-type correction for

pairwise comparisons. Expression levels were clustered and dis-

played using a bioinformatics and graphics software package

(GeneLinker Gold, Predictive Patterns, Kingston, ON). As multiple

cells were measured in each subject, between-subject versus within-

subject (between-cell) variation in gene expression levels was

analyzed by variance component analysis and intraclass correlation

coefficients, which ranged between 0.4 and 0.6 for p75 NTR, trkA,

trkB and trkC. The association between gene expression levels and

subject characteristics (diagnostic groups as well as other demogra-

phic, clinical, and neuropathological variables) was evaluated via

mixed models repeated measures analyses, which accounts for both

between-subject and within-subject variation (SAS Institute 1999).

In the analyses, we used random intercept, fixed effect covariate,

Kenward-Roger denominator degrees of freedom, unstructured

covariance structure, and log-transformed gene expression levels

(SAS Institute 1999). The level of statistical significance was set at

0.01 (two-sided).

qPCR

qPCR was performed on unfixed, microdissected frozen tissue at the

level of the anterior NB ventral to the decussation of the anterior

commissure (n ¼ 7 NCI; n ¼ 6 MCI, and n ¼ 8 AD). Micro-

punched tissue was also obtained from the caudate nucleus from the

same subjects as a control brain region since striatal trkA-

immunoreactive neurons are not selectively vulnerable in AD

(Boissiere et al. 1997; Mufson et al. 1997, 2000; Chu et al. 2001).Microdissected tissues contain an admixture of cholinergic neurons,

non-cholinergic neurons, glia, and vasculature. PCR primers were

designed for five genes, p75NTR, trkA, trkB, trkC, and the

housekeeping gene glyceraldehyde-3-phosphate dehydrogenase

(GAPDH). Due to the high homology of the trk receptors (Shelton

et al. 1995), TaqMan hydrolysis probes were designed (trkA, trkB,

and trkC) for gene quantification. The p75NTR and GAPDH primer

sequences have been used by our group and employ SYBR green

dye chemistry as a reporter (Ginsberg and Che 2004, 2005). Primer

sequences are reported in Supplemental Table 2. Samples were run

on a real-time PCR cycler (7900HT, ABI) as per the manufacturers

instructions. Standard curves and cycle threshold (Ct) were measured

using standards obtained from total human brain RNA. Relative

changes in PCR product synthesis was analyzed by one-way ANOVA



with post hoc analysis (Neumann-Keuls test; level of statistical

significance was set at 0.05) for individual comparisons. Amplicon

specificity was evaluated by subcloning the amplicon products (Zero

Blunt, Invitrogen) and performing sequence analysis.

In situ hybridization

Tissue slabs containing the entire NB were immersion fixed in 4%

paraformaldehyde and frozen sectioned at a thickness of 40 lm for

in situ hybridization histochemistry (Mufson et al. 1996; Chu et al.2001). Probes were generated against human p75NTR and trkA.

Briefly, a p75NTR probe was generated from 1.5 kilobase (kb) cDNA

complementary to human p75NTR mRNA (Johnson et al. 1986) andsubcloned into pBs (Higgins and Mufson 1989). Transcription was

performed in a solution containing 4 mM Tris-HC1 (pH 7.5), 6 mM

MgCl2, 2 mM DTT, 5 U RNasin, 400 mM of ATP, GTP, and UTP,

25 mM of 35S-CTP (800 Ci/mmol; GE Healthcare), 2 mg of

linearized template and 10 U T7 RNA polymerase. Prehybridization

was performed at 56�C for 1 h in a solution consisting of 50%

formamide, 0.5 M NaCl, 25 mM Pipes buffer (pH 6.8), 10 mM

EDTA, 250 mM DTT, 5X Denhardt’s solution, 0.2% SDS, 10%

dextran sulfate, and tRNA (500 mg/mL). Subsequent hybridization

was performed for 16 h at 56�C followed by stringent washing

conditions (Higgins and Mufson 1989; Mufson et al. 1996). Slideswere air-dried and exposed to X-ray film (DuPont Cronex 4, MES

Services, Fulton, IL, USA) for 24–48 h, dipped into Kodak NTB-2

emulsion (Eastman Kodak, Rochester, NY, USA; diluted 1 : 1 with

distilled water), exposed for 4–6 days at 4�C, and developed for

autoradiography. Selected slides were counterstained with cresyl

violet for cellular visualization. The material presented for p75NTR

in situ hybridization was generated from for an earlier study

(Mufson et al. 1996).A trkA cRNA probe was generated from a 545 basepair (bp)

fragment of cDNA coding for the ECD of human trkA (Shelton

et al. 1995). TrkA cDNA was obtained from post-mortem human

brain using PCR and subcloned into pGEM )7Zf(–) vector in the

XbaI/BamHI sites (Shelton et al. 1995). This plasmid was linearized

with either SacI to serve as a template for T7 RNA polymerase

(antisense) or SphI to serve as template for Sp6 RNA polymerase

(sense), respectively. In vitro transcription was performed in the

presence of ATP, GTP, UTP and biotin-14-CTP, RNasin, transcrip-

tion buffer, and T7 or Sp6 RNA polymerase for 2 h at 37�C (Chu

et al. 2001). The reaction was stopped by the addition of 1 lL of 0.5

EDTA. Biotin-labeled trkA cRNA was purified by ethanol precipi-

tation and resuspended in 30 lL of diethyl pyrocarbonate treated

water. Prehybridization was conducted for 2 h at 37�C using

hybridization buffer (50% deionized formamide, 10% dextran

sulfate, 10% Denhardt’s solution, 30% 2X SSC) containing heat

denatured torula yeast RNA (0.1 mg/mL) and 0.2% non-fat milk

(Chu et al. 2001). Following rinses in 2X SSC, tissue sections of the

NB are incubated in hybridization buffer with heat denatured

biotinylated trkA probe (1 lg/mL) and torula yeast RNA (0.1 mg/

mL) at 41�C for 18 h (Chu et al. 2001). TrkA mRNAwas visualized

using either autoradiographic labeling or using a biotinylated

reaction product visualized by the ABC method (Vector Laborat-

ories) with 0.025% DAB, 1% nickel II sulfate and 0.0025%

hydrogen peroxide (Mufson et al. 2000; Chu et al. 2001). The

tissue sections examined for trkA in situ hybridization were derived

from material used as part of an earlier report (Chu et al. 2001).

Control experiments included using sense probes, pretreatment of

tissue sections with RNase, and processing the sections without

biotinylated secondary antibodies.

Results

A total of 174 single cholinergic NB neurons were analyzedin 34 post-mortem human brains, with an average of 5–6cells per subject (range 2–11). Subjects in this study werecompatible among the three diagnostic groups in age, gender,post-mortem interval (PMI), years of education, and ApoE4status (Table 1). Post-mortem neuropathologic examinationrevealed that 50% (6/12) of NCI cases were classified asBraak stages III-IV and none as Braak stages V-VI; 80% (8/10) of MCI cases were classified as Braak stages III-IV and20% (2/10) as stages V-VI; 34% (4/12) of AD caseswere classified as Braak stages III-IV and 58% (7/12) asstages V-VI (Table 1). In addition, 6 NCI and 1 AD caseswere identified as Braak stages 0–II (Table 1). Using NIA-Reagan criteria for pathological diagnosis of AD, 73% ofNCI and 30% of MCI cases were classified as having a lowlikelihood of AD, with 27% of NCI and 70% of MCI cases ashaving an intermediate likelihood of AD. In contrast, the ADcases were classified as having an intermediate (44%) or highlikelihood (44%) of AD (Table 1).

Single cholinergic NB cell expression profile analysisusing custom-designed cDNA array platforms revealeddifferential regulation of neurotrophin receptor mRNAs.Other classes of transcripts were evaluated, includingsynaptic-related markers, glutamatergic neurotransmission,protein phosphatases/kinases, among other gene classes thatwill comprise a separate report due to the extensive amountof data. Significant down regulation of trkA, trkB, and trkCwas observed in individual neurons microaspirated from ADand MCI brains as compared to NCI (Fig. 2) (see Supple-mental Results). Moreover, down regulation was found fortwo separate ESTs for each trk gene (e.g. ESTs targeted to theECD and TK domain) (Fig. 2). Individual cholinergic NBneurons from MCI brains displayed reduced levels of trkA,trkB, and trkC as compared to NCI (Fig. 2). Withincholinergic NB neurons, several trk ESTs displayed signifi-cantly higher expression levels in MCI than AD (e.g.trkAECD, trkBECD, and trkCTK), indicating that MCI cho-linergic NB neurons exhibit intermediate levels of trkA, trkB,and trkC compared to NCI and AD. By contrast, nosignificant differences in relative expression levels forp75NTR were observed across clinical groups (Fig. 2). Nochanges in p75NTR expression levels were observed in CBFneurons identified by neurofilament immunoreactivity andcresyl violet staining (Ginsberg and Che 2002, 2004),consistent with the present observations. In addition, regu-lation of ChAT mRNA was not observed across clinicalconditions (Fig. 2a). Taken together, these findings indicate a



relative selectivity in the alteration of high-affinity neurot-rophin receptors within single NB neurons during theprodromal stage of AD. Furthermore, no regulation ofGAPDH was observed across clinical groups (Fig. 2a).

Similar to the expression profile analysis shown in Fig. 2,where an intermediate expression level or ‘step down effect’from NCI to MCI to AD occurred, post-mortem trk geneexpression levels from individual cholinergic NB neuronswere also related to two antemortem cognitive assessmentmeasures. A significant association was found betweendecreased trkA (p ¼ 0.0008), trkB (p ¼ 0.0001), and trkC(p ¼ 0.003) levels and lower composite GCS scores(Fig. 3a). By contrast, no relationship was observed betweenGCS scores and p75NTR expression. Similar results werefound for trk array profiles and MMSE scores (Fig. 3b) (seeSupplemental Results). These observations were not facili-tated by low GCS or MMSE scores observed in more severe

AD cases examined in this study. Rather, these data indicatethat virtually all of the MCI and AD cases displayintermediate and lower expression levels, respectively, thatcontribute to the overall decrement in expression levels(Fig. 3).

Validation of the results obtained by array analysis wasperformed by qPCR. Results were reported as mean Ct ± sd.A high Ct level equates to low expression levels. Evaluationof Ct data did not reveal differential regulation of p75NTR

expression across NCI, MCI, and AD (Fig. 4a). Similar tothe observations reported on the custom-designed arrays,down regulation of trkA (Fig. 4b), trk B (Fig. 4c), and trkC(Fig. 4d) was observed in MCI and AD compared to NCI(see Supplemental Results for Ct values). Moreover, asignificant intermediate or ‘step down effect’ between trkAexpression levels in MCI and AD was found by qPCR(Fig. 4b), consistent with the custom-designed array results.Due to the low expression levels of both trkB and trkC in thebasal forebrain tissue samples assayed by qPCR, an obser-vation consistent with previous reports (Salehi et al. 1996;Mufson et al. 2002), discrimination of potential expressionlevel differences between MCI and AD (as evidenced bytrkA qPCR) was not possible. No differences in GAPDHexpression levels were found in NCI, MCI, and AD subjects(data not shown). In contrast to the marked down regulationof trkA in NB tissue dissections, no differential regulation oftrkA was observed in the striatum obtained from the samecase materials, indicating a regional selectivity to the downregulation of trkA (Fig. 4e). Although there is considerablehomology between the trkA, trkB, and trkC genes (Sheltonet al. 1995), the TaqMan primers used in these qPCR studiesdemonstrated virtually no cross reactivity between neurotro-phin receptors (Fig. 4f).

Array results were further validated in tissue sections ofthe basal forebrain using in situ hybridization histochemistry.Probes for both p75NTR and trkA predominantly labeled largemultipolar NB neurons, with little or no labeling of glial cellsor surrounding neuropil. Consistent with results obtainedfrom single cell data acquired on custom-designed cDNAarray platforms and regional tissue microdissections forqPCR, down regulation of trkA expression levels wasapparent in NB tissue sections from MCI and AD brains incomparison with NCI brains (Fig. 5a–c). Additionally, nosignificant differences in p75NTR labeling were found acrossclinical groups (Figs 5d, e).

Discussion

Creating a molecular fingerprint of single neurons that areselectively vulnerable requires their precise localizationwithin a defined brain region. Therefore, resolution at thelevel of homogeneous neuronal populations is necessary tocreate an expression profile for affected cells such ascholinergic NB neurons. Simultaneous quantitative assess-

(b)

(a)

Fig. 2. Expression profile analysis of p75NTR, trkA, trkB, trkC, ChAT

and GAPDH derived from individual NB neurons from NCI, MCI and

AD subjects. (a) Dendrogram with a color coded scale illustrating

relative expression levels. No significant differences are found for

ChAT, p75NTR and GAPDH gene expression. In contrast, statistically

significant down regulation (asterisk) of trkA, trkB, and trkC is

observed in MCI and AD. ESTs identifying ECD and TK domains

display down regulation. The decrement of trk gene expression in MCI

is intermediate relative to AD, indicating a step down effect in

expression levels from NCI to MCI to AD. (b) Representative custom-

designed arrays illustrating expression level differences between NCI,

MCI, and AD. Three individual cases are depicted for each condition.



ment of multiple transcripts by microaspiration, RNAamplification, and custom-designed cDNA microarray ana-lysis provides a paradigm whereby the genetic signature ofanatomically defined cells within a specific brain region canbe differentiated from neighboring structures (Che andGinsberg 2004; Ginsberg and Che 2004, 2005). Thisexperimental design allows for rigorous quantitative analysesof vulnerable cell types during the progression of clinicalimpairment (Galvin and Ginsberg 2004, 2005).

The present study combines custom-designed cDNA arrayanalysis with qPCR and in situ hybridization derived fromindividual neurons and microdissected regions of the anteriorportion of the cholinergic NB and demonstrates that geneexpression for the high-affinity neurotrophin receptors trkA,trkB, and trkC is significantly down regulated during theclinical progression of AD. Although a discrepancy in termsof fold difference is observed between the array and qPCRassays, this is a common occurrence due to several potentialfactors including RNA quantity and sensitivity of the specificplatform (Ginsberg 2005; Ginsberg et al. 2006). An import-ant factor is whether or not both methods show a similardirection, i.e. no change, up regulation, or consistent downregulation, as observed in the present study. Specifically, trkreceptor expression was reduced in the NB of subjects withMCI compared to NCI, with even further reductionsobserved in AD. These results suggest that the onset ofneurotrophic dysfunction in CBF neurons occurs during theearliest stages of cognitive decline, and that deficits in trkexpression are associated with the clinical presentation of the

disease. In support of this hypothesis, down regulation of thetrk genes correlate with comprehensive (GCS) and individual(MMSE) measures of cognitive decline across the clinicaldiagnostic groups. In contrast, there is a lack of regulation ofp75NTR expression. This is intriguing, as phenotypic silen-cing of both trkA and p75NTR protein expression has beenreported (Mufson et al. 1989b, 2000) in contrast to the stableexpression of ChAT gene expression (present study) andChAT protein (Gilmor et al. 1999) within NB neurons duringthe prodromal stage of AD. These differential alterations ingene/protein regulation are also reflected in the corticalprojection sites of the cholinergic NB neurons. For example,cortical trkA protein expression is decreased, whereasp75NTR protein levels remain stable during the progressionof AD (Counts et al. 2004). Since both receptors areproduced within NB neurons and anterogradely transportedto the cortex, the possibility exists that the transport of trkAand/or the translation to protein is altered as opposed top75NTR by the disease process. TrkA binding to NGF is acrucial factor for signal transduction associated with cho-linergic neuronal survival (Kaplan and Miller 2000), thusreduction of trkA (as well as trkB and trkC) may haveimportant consequences related to cholinergic basocorticaldysfunction as well as cognitive decline during the transitionfrom MCI to AD.

Retrograde transport of NGF bound to activated trkAreceptors via signaling endosomes appears to be an importantmechanism for delivery of NGF signals to target basalforebrain neurons (Howe and Mobley 2004). These binding

(a)

(b)

Fig. 3 Scattergrams demonstrating the relationship of trk gene

expression levels with GCS and MMSE scores. These data are log-

transformed and analyzed using a mixed models repeated measures

method. (a) Highly significant associations are found whereby

decreased trkA (p ¼ 0.0008), trkB (p ¼ 0.0001), and trkC (p ¼ 0.003)

levels are observed relative to lower GCS scores in AD and MCI as

compared to NCI. Intermediate expression levels are found in MCI

relative to NCI and AD. Thus, as disease progresses and GCS scores

drop, lower trkA, trkB, and trkC levels are found within individual CBF

neurons. Each color coded data point (green square, NCI; blue tri-

angle, MCI; red circle, AD) represents relative expression level values

for an individual case. (b) Correlation of gene expression levels with

MMSE scores. Highly significant associations are demonstrated

whereby decreased trkA, trkB, and trkC levels are observed relative to

lower MMSE scores in MCI and AD as compared to NCI, further val-

idating the results garnered from the GCS scores.



events result in the retrograde transport of the boundneurotrophin ligand to CBF consumer neurons and theinitiation of downstream cellular signal transduction relatedto cell survival (Counts and Mufson 2005). TrkA proteinlevels in cholinergic NB neurons are significantly reduced,along with decreased cortical levels early in the progressionof AD (Boissiere et al. 1997; Mufson et al. 1997, 2000; Chuet al. 2001). TrkA gene expression levels have been shownto be down regulated in end-stage AD patients, althoughcorrelation with antemortem cognitive measures and evalu-ation of trkB/trkC was not investigated (Mufson et al. 2002).In contrast, the cholinergic phenotype of these neurons aswell as cortical ChAT activity are preserved in people withMCI and mild AD compared to the dramatic reduction ofthese cholinergic markers in late stage AD (Mufson et al.2003; Counts et al. 2004; Counts and Mufson 2005).

Moreover, recent profiling studies indicate that the receptorsfor the putative cholinergic survival neuropeptide galanin(GALR1, GALR2 and GALR3) are unchanged in NBneurons in prodromal AD (Counts et al. 2006). Thesefindings suggest a phenotypic down regulation of NGFreceptors, but not a frank loss, of cholinergic neurons duringthe prodromal stage of AD. Therefore, a defect in trkAmRNA expression in NB neurons early in the course of ADcould impact protein translation, ligand receptor binding, andretrograde transport of NGF leading to cholinergic cellulardegeneration and cognitive decline during the course of thedisease (Chu et al. 2001; Counts and Mufson 2005).

The persistence of p75NTR protein expression in the cortexin MCI and AD (Counts et al. 2004) may reflect severalpossible mechanisms. For example, a compensatory responsein remaining p75NTR-containing NB neurons may occur to

Fig. 4 (a) qPCR validation of p75NTR, trkA, trkB and trkC expression

using basal forebrain and striatal dissections. No differences are

observed in p75NTR expression levels across NCI (black), MCI (blue),

and AD (red) basal forebrain, validating array observations. (b) A

significant decrease in trkA expression within MCI (asterisk) and AD

(double asterisk) vs. NCI basal forebrain tissue was observed. (c)

Down regulation of trkB in MCI (asterisk denotes p ¼ 0.012) and AD

(asterisk denotes p ¼ 0.008) as compared to NCI was observed. Due

to the low expression levels of trkB in the basal forebrain tissue

samples assayed by qPCR, evaluation of expression level differences

between MCI and AD was not possible. (d) Similar to c, down regu-

lation of trkC expression in MCI (asterisk denotes p ¼ 0.020) and AD

(asterisk denotes p ¼ 0.007) was observed. The low expression levels

of trkC in the basal forebrain precluded an assessment of expression

level differences between MCI and AD. (e) TrkA expression does not

differ between NCI, MCI, and AD from striatal dissections indicating

the regional and cellular selectivity of neurotrophin receptor down

regulation observed in the basal forebrain and individual NB neurons.

(f) Control demonstrating a robust signal using trkA TaqMan primers

and a trkA plasmid (black) as an input source. In contrast, a virtually

undetectable signal is generated using trkA TaqMan primers and trkB

plasmid (gray) as an input source, indicating that trkA primers do not

cross react with trkB.



stabilize cortical receptor levels. This is unlikely since therewas no change in p75NTR gene expression. Alternatively, abuild up of p75NTR protein could occur in the cortex due to aretrograde transport defect or due to the de novo expressionof p75NTR within cortical neurons in AD (Mufson andKordower 1992). Interestingly, deficits in trkA and retro-grade transport of NGF to CBF consumer neurons occurs inend stage AD (Mufson et al. 1997), in a segmental trisomymouse model of Down’s syndrome (Cooper et al. 2001), andin aged rats with cognitive impairment (Cooper et al. 1994;De Lacalle et al. 1996). Moreover, aged rats with mildcognitive impairment display a silencing of trkA expressionwithin CBF neurons prior to cholinergic atrophy or loss oftrkA containing neurons (Saragovi 2005). These deficits areenhanced in trkA-positive cholinergic neurons in aged ratswith severe cognitive impairment (Saragovi 2005), suggest-ing that trkA down-regulation is associated with cognitiveimpairment seen in aging as well as AD. In addition,transgenic mice engineered to produce anti-NGF antibodiesdisplay AD-like neuropathology within CBF neurons (Cap-soni et al. 2000; Ruberti et al. 2000). Taken together, thesefindings suggest that early defects in trkA receptor expres-sion may be a precursor (and a potential biomarker) to theextensive cell loss observed within the CBF in end stage AD(Whitehouse et al. 1982; Mufson et al. 1989b).

There is evidence to suggest that an imbalance in the ratioof trkA and p75NTR, in part, may lead to cell death bypromoting unscheduled cell cycle re-entry and apoptosis(Yoon et al. 1998; Naumann et al. 2002). p75NTR-mediatedapoptosis involves the activation of cell cycle regulatorymolecules, and a link between aberrant cell cycle re-entry ofpost-mitotic neurons and apoptosis has been established inCBF neurons in MCI and early AD (Yang et al. 2003).Several studies suggest that proper neurotrophin receptorsignaling depends upon interactions with the NGF precursorprotein, proNGF. For example, the ratio of proNGF to matureNGF is increased in cortex obtained from MCI and ADsubjects and correlates with cognitive decline (Peng et al.2004). It has been hypothesized that proNGF bound to trkAinitiates cell survival activity (Fahnestock et al. 2004),whereas proNGF bound to p75NTR induces apoptosis(Pedraza et al. 2005). Additional findings indicate that thepro-apoptotic effect of p75NTR-mediated proNGF signaling isdependent upon interactions between p75NTR and theneurotensin receptor sortilin (Nykjaer et al. 2004). Thesedata suggest that the ratio of trkA to p75NTR receptors maydetermine whether neurons survive or degenerate whenexposed to NGF or proNGF. Thus, a �50% reduction incortical trkA that occurs at the onset of AD may signify arelative increase in pro-apoptotic p75NTR signaling incholinergic NB neurons. Interestingly, brain derived neuro-trophic factor (BDNF) and its precursor protein proBDNF,which bind to the trkB receptor are significantly decreased inMCI and AD cortex compared to NCI (Peng et al. 2005).These results suggest that multiple defects in neurotrophinreceptor expression and neurotrophin signaling play a keyrole in NB degeneration that may exacerbate functionaldeficits and lead to advanced pathology including synapticdysfunction and cognitive decline early in the pathogenesisof AD.

By utilizing state-of-the-art molecular approaches formultiple mRNA assessments in concert with clinicopatho-logical correlations in a range from normal senescence tofrank dementia, this study provides unique insights intospecific alterations in neurotrophin receptor gene expressionwithin cholinergic NB neurons during the clinical progres-sion of AD. The loss of trk expression in MCI suggests thattrk reduction plays a role in the early stages of cholinergicNB cellular dysfunction contributing to cognitive deficits andto the ultimate demise of these neurons in the later stages ofAD. Thus, early defects in trk expression may providemarkers for the identification of individuals with MCI and/orin the prodromal stage(s) of AD. Interestingly, a phase Iclinical trial whereby genetically modified autologous fibro-blasts that secrete human NGF were grafted directly into theNB region improved cognition in mild AD patients (Tus-zynski et al. 2005). The efficacy of this treatment mayinvolve increased trk expression, which is positively regu-lated by NGF (Holtzman et al. 1992; Li et al. 1995).

(a)

(b)

(c)

(e)

(d)

Fig. 5 Validation of cDNA array results using in situ hybridization

histochemistry directed against p75NTR and trkA within the anterior

NB. (a) Biotinylated probes against trkA demonstrated a pronounced

down regulation of trkA gene expression in MCI (b) and AD (c) as

compared to NCI (a), consistent with array observations. Intermediate

MCI expression levels were difficult to discern based solely upon

in situ hybridization results. (d) Radioisotopic probes generated

against p75NTR demonstrated no significant differences in expression

levels between aged controls (d) and AD (e) subjects. Panels (d) and

(e) were adapted from reference (Mufson et al. 1996).



Ultimately, genetic fingerprinting of CBF neurons willprovide a foundation for the development of novel pharma-cotherapeutic intervention(s) to aid in ameliorating orpreventing age and disease-related cognitive decline. Takentogether, the current single cell gene array observations(validated independently with qPCR and in situ hybridizationmeasures) in post-mortem human tissues may reflect aspecific molecular signature underlying cholinergic NBneuronal dysregulation during the early stages of dementiaand progressing towards frank AD.

Acknowledgements

This work was supported by grants from the NIH (AG10161,

AG10688, AG14449, AG21661, AG26032, and NS43939),

Alzheimer’s Association and Illinois Department of Public Health.

We are indebted to the altruism and support of the participants in the

ROS. A list of participating groups can be found at the website:

http://www.rush.edu/rumc/page-R12394.html. We thank Drs David

A. Bennett, director of the ROS clinical core, Julie Schneider,

director of the ROS neuropathology core, Sue Leurgans for

statistical consultation, and Ralph A. Nixon for critical review of

the manuscript. We also thank Ms. Irina Elarova, Ms. Shaona Fang,

Mr Marc D. Ruben and Dr Nadeem Mohammad for expert technical

assistance.

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Down regulation of trk but not p75NTR gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer’s disease

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