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Interactome Analysis Identifies Novel Targets of Phosphoinositide 3-kinase (PI3K) that Mediate
Astrocyte Neuroprotection
by
Samih Ahmad Alqawlaq
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Laboratory Medicine and Pathobiology University of Toronto
Biochemical studies demonstrate that ZC3H14 utilizes five evolutionarily conserved
tandem cysteine3histidine (CCCH) zinc fingers, which binds to polyadenosine (polyA)
RNA with high affinity and specificity[147]. There are at least four known alternatively
spliced variants of ZC3H14, which give rise to four distinct protein isoforms [148].
Isoforms 1-3 have a N-terminal proline tryptophan isoleucine (PWI), which mediates
entry through the nuclear pore; thus, these isoforms are expected to localized within the
cytoplasm as well as the nucleus [149]. Isoform 4 lacks key domains needed to interact
with the nuclear pore and is therefore localized mainly within the cytoplasm [148].
Through binding with the polyA tails, ZC3H14 carries out a range of post-transcriptional
modifications on premature transcripts [147, 150]. These include control of PolyA tails
length [151], nuclear export [149], and mRNA splicing [152]. Mutation of the ZC3H14
impairs neural function in Drosophila and has been associated with cognitive deficits in
humans [153]. ZC3H14 knockout mouse models show anatomical changes in brain
ventricles as well as working memory deficits [154]. Thus far, no studies have reported
a ZC3H14 interaction with PI3K, or other kinases.
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The THO complex 1 (Thoc1) encodes a nuclear matrix protein that also binds to PolyA
tails of mRNA [155]. As part of the THO ribonucleoprotein complex, Thoc1 protein is
recruited to premature RNA transcripts, which allows access to various RNA processing
and export machinery [156, 157]. Impaired Thoc1 function negatively impacts
transcription elongation and nuclear export, which also affects mRNA stability in the
cytoplasm [157]. A 2018 study reported that ZC3H14 interacts with THO complex to
coordinately control RNA processing, poly(A) tail length, and consequently mRNA
stability [155]. The same study showed that mRNA targets of ZC3H14 and THOC1
include the postsynaptic density protein 95 (Psd95), ATP synthase lipid-binding protein
(Atp5g1) and microtubule-associated protein tau (MAPT). Conversely, knockdown of
ZC3H14 or THO components lead to decreased levels of mature transcripts and
accumulation of Atp5g1 and Psd95 pre-mRNA in the cytoplasm. The gene encodes a
subunit of mitochondrial ATP synthase, which catalyzes ATP synthesis [158]. Thus,
ZC3H14 and THOC1’s functions may have a direct impact on cell energy synthesis and
survival.
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141. Li, J., et al., Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase. Nature, 2002. 418: p. 793.
142. Thapar, R. and A.P. Denmon, Signaling pathways that control mRNA turnover. Cell Signal, 2013. 25(8): p. 1699-710.
143. Ming, X.F., et al., Parallel and independent regulation of interleukin-3 mRNA turnover by phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase. Mol Cell Biol, 2001. 21(17): p. 5778-89.
144. Benjamin, D., et al., BRF1 protein turnover and mRNA decay activity are regulated by protein kinase B at the same phosphorylation sites. Mol Cell Biol, 2006. 26(24): p. 9497-507.
145. Graham, J.R., et al., mRNA degradation plays a significant role in the program of gene expression regulated by phosphatidylinositol 3-kinase signaling. Mol Cell Biol, 2010. 30(22): p. 5295-305.
146. Wigington, C.P., et al., The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA. J Biol Chem, 2016. 291(43): p. 22442-22459.
147. Kelly, S.M., et al., Recognition of polyadenosine RNA by zinc finger proteins. Proc Natl Acad Sci U S A, 2007. 104(30): p. 12306-11.
148. Leung, S.W., et al., Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein. Gene, 2009. 439(1-2): p. 71-8.
149. Marfatia, K.A., et al., Domain analysis of the Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, Nab2p. Dissecting the requirements for Nab2p-facilitated poly(A) RNA export. J Biol Chem, 2003. 278(9): p. 6731-40.
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151. Kelly, S.M., et al., A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length. RNA, 2014. 20(5): p. 681-8.
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37
Chapter 2: Rationale, purpose, Hypothesis and Aims
38
Rationale, Hypothesis and Aims
2.1 Rationale
Until the early 1980’s, glial cells had been thought of as simply structural, binding
together neuronal tissue, including the retina, and hence their name (glia is derived from
the Greek word for glue). This concept became challenged by growing evidence
demonstrating that glial cells, and especially astrocytes, sustain neuronal functions and
homeostasis in diverse and complex ways [1]. One important facet among these
functions is the astrocyte capacity to alleviate the impact of metabolic stress in neurons
through secreted pro-survival factors. This observation is of particular importance in the
context of neurodegenerative diseases, since excessive stimulation by neurochemicals,
such as glutamate, leads to death of neurons in a number of diseases including
Alzheimer’s disease, and glaucoma. However, we currently lack an understanding of
the basic neuronal pathways involved in transducing these astrocyte signals, which
prevents us from leveraging them for future treatment or diagnostic strategies.
Studying ACM provides a window into the inner workings of astrocyte-neuron
communication and associated protective outcomes in neurons. ACM contains a
complex mixture of proteins, lipids, neurochemicals and nucleic-acid containing
exosomes [2-5]. To explore ACM components, we isolated and cultured primary retinal
astrocytes as previously described [6, 7], and subsequently collected ACM for analysis.
39
Astrocytes cultured with this protocol exhibit characteristic astrocyte morphology and a
range of specific markers, including GFAP and GS (Figure 2-1).
Figure 2-1. Characterization of primary retinal astrocytes
Astrocyte markers (I) GFAP (green), (II) GS (green) are shown in separate immunofluorescence trials in
cultured astrocytes; DAPI is used as a co-stain (blue). (III) Immuno blots of GS and GFAP in cultured
astrocytes.
We used cultured primary retinal astrocytes to collect ACM and assess its
neuroprotective effects against metabolic stress in an in vitro model, enabling high
throughput screening of involving signaling mechanisms (methods described in Chapter
40
3). ACM was shown to be highly protective against glutamate-induced metabolic stress
in transformed hippocampal neurons (Ht22), and the effect was demonstrated to have a
concentration-dependent effect (Figure 2-2).
Figure 2-2. Assessing ACM neuroprotection against metabolic stress in Ht22 cells
(I) ACM protection in Ht22 cells against 5 mM glutamate injury is demonstrated by increased metabolic
activity of neurons pre-conditioned with ACM, compared to control media. (II) ACM activity is maintained
when diluted to 25% of its volume, beyond which the neuroprotective activity is virtually lost (n=3, **
p<0.05).
However, the combined neuroprotective effect of ACM is complex, due to the wealth of
other biologics in ACM, including growth factors. Identifying the key ligands that mediate
ACM activity can be tackled through numerous approaches, including proteomic,
genomic, and pharmacological technologies. However, due to the complexity of the
ACM mixture, ‘omic’ type analyses carry with them the potential to generate large
41
biological datasets that are challenging to analyze. For example, preliminary data
generated by our group showed that boiling or trypsinizing ACM eliminates its
neuroprotective activity, strongly suggesting a protein component to the activity. This
thinking led us to initially carry out global mass spectroscopy and RNAseq analysis on
ACM and astrocytes, respectively. The analysis was unsuccessful due to two reasons:
1) the necessary presence of serum in ACM to maintain the protective activity, which
masked astrocyte-secreted proteins with its abundance, and 2) lack of a strong control
strategy for ranking sufficiently specific candidates. Eliminating serum from ACM,
through a series of optimization studies, failed to generate a suitable defined media
replacement.
An alternative approach is the use of chemical genetics screens using tool compounds
to identify specific targets induced by ACM. This approach has several advantages: 1)
in contrast with global screens, tool compounds, have defined targets that can elucidate
specific signaling pathways and ligands of interest. 2) Combining tool compound
libraries with robotic capabilities allow high throughput analyses of specific targets
simultaneously. 3) Tool compounds screens are modular in the sense that they can be
scaled up or down, based on experimental goals, to profile a wide range of tool
compound library sizes. 4) Finally, these screens can provide functional information, in
the sense that the primary endpoint is ACM induced protection. Thus, the use of tool
compound screens can identify necessary signaling pathways through which the
neuroprotection is transduced. Therefore, this strategy was ultimately chosen to identify
key neuroprotective signaling pathways activated by ACM.
42
Among the screened targets are kinases, which transduce extracellular signals
efficiently through phosphorylation of downstream targets. Kinases mediate
phosphorylation reactions by transferring the gamma phosphate of Adenosine
Triphosphate (ATP) onto hydroxyl groups of various lipids, sugars or amino acids[8].
Kinases are essential players in signaling pathways and are responsible for maintaining
cellular homeostasis, survival, and growth [9]. The current project focuses on a key pro-
survival kinase, PI3K, a key mediator of astrocyte-neuron communication. Further,
identifying the binding partners of kinases such as PI3K can provide insight into the
neuroprotective signaling pathway and its activators. Analyzing these interactions in
neurons following ACM exposure can provide valuable information into how the
neuroprotection is transduced along a specific signaling pathway, such as PI3K. Thus,
proteomic analysis was employed in this project to identify how PI3K binding patterns
change in neurons following ACM exposure.
Mass spectrometry (MS) enables high throughput identification and functional
annotation of proteins in a cell, tissue, or organisms. It is an analytical technique that
measures the mass-to-charge ratio (m/z) of an ion, generated following fragmentation of
a protein sample with high energy. It is used to identify known as well as novel
macromolecules, including proteins, and provide detailed information about their
structures. Quantitative mass spectrometry (MS) provides an effective approach to
concurrent identification and determination of concentration ratios of proteins from
different samples, eliminating inter-run variability[10]. One of these labeling methods is
featured in the current work: the isobaric tag for relative and absolute
43
quantitation (iTRAQ) technology. The technique employs isotope labeled tags that can
be covalently bonded to the N-termini and side chain amines of proteins. [11]. Due to
the isobaric design employed in iTRAQ labeling, differentially labeled peptides appear
as single peaks in MS-MS scans, which reduces the likelihood of peak
overlapping. This allows the relative quantification of proteins pooled from up to 8
different samples.
2.2 Global Hypothesis
I hypothesize that ACM mediated neuroprotection against metabolic stress is driven by
astrocyte-secreted factors. These factors activate pro-survival signaling pathways, such
as PI3K, which can be dissected and targeted to enhance neuronal survival under
stress conditions.
2.3 Purpose and aims
The purpose of the current research is to identify key signals induced by astrocyte-
secreted factors involved in protecting neurons from metabolic stress. To achieve this
goal, I will address three main aims:
1. Identify ACM-induced neuronal mechanisms, such as kinases, which can be
utilized to expand key protective signaling pathways.
a. Carry out a tool compound screen to identify key kinases and receptors
along ACM-mediated neuroprotection pathway in the glutamate sensitive
cell line Ht22.
44
b. Validate screen hits independently in Ht22 cells in primary neurons.
c. Confirm involvement of key kinases using immunoblotting.
2. Identify ACM-regulated target interactors in neurons, including downstream
effectors and upstream receptors.
a. Generate interactome data on a candidate signaling hub protein in Ht22
cells treated with ACM using mass spectroscopy.
b. Guide bioinformatic analysis to identify ACM-regulated interactors.
c. Confirm interactions using co-immunoprecipitation and immunoblotting.
3. Validate the neuroprotective activity of candidate kinases and interactors in in
vitro and in vivo metabolic stress models.
a. Assess the effect of stimulating identified protein-protein interactions on
ACM neuroprotection.
b. Assess the impact of knocking down identified interactors on ACM
neuroprotection.
Together these aims will provide new insights into how astrocytes protect neurons from
metabolic stress through secreted factors and induced neuronal pathways.
45
Chapter 2 References
1. Alqawlaq, S., J.G. Flanagan, and J.M. Sivak, All roads lead to glaucoma: Induced retinal injury cascades contribute to a common neurodegenerative outcome. Exp Eye Res, 2018.
2. Baldwin, K.T. and C. Eroglu, Molecular mechanisms of astrocyte-induced synaptogenesis. Current opinion in neurobiology, 2017. 45: p. 113-120.
3. Livne-Bar, I., et al., Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury. J Clin Invest, 2017.
4. Hughes, E.G., S.B. Elmariah, and R.J. Balice-Gordon, Astrocyte secreted proteins selectively increase hippocampal GABAergic axon length, branching, and synaptogenesis. Molecular and cellular neurosciences, 2010. 43(1): p. 136-145.
5. Wang, G., et al., Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4): potential mechanism of apoptosis induction in Alzheimer disease (AD). J Biol Chem, 2012. 287(25): p. 21384-95.
6. Livne-Bar, I., et al., Pharmacologic inhibition of reactive gliosis blocks TNF-α-mediated neuronal apoptosis. Cell Death Dis, 2016. 7(9): p. e2386.
7. Nahirnyj, A., et al., ROS Detoxification and Proinflammatory Cytokines Are Linked by p38 MAPK Signaling in a Model of Mature Astrocyte Activation. Plos One, 2013. 8(12).
8. Fabbro, D., S.W. Cowan-Jacob, and H. Moebitz, Ten things you should know about protein kinases: IUPHAR Review 14. Br J Pharmacol, 2015. 172(11): p. 2675-700.
9. Duncan, J.S., et al., Regulation of cell proliferation and survival: convergence of protein kinases and caspases. Biochim Biophys Acta, 2010. 1804(3): p. 505-10.
10. Wiese, S., et al., Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics, 2007. 7(3): p. 340-50.
11. Ross, P.L., et al., Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics, 2004. 3(12): p. 1154-69.
46
Chapter 3: Interactome Analysis Identifies RNA
Binding Protein ZC3H14 as a Novel Interactor of
Phosphoinositide 3-kinase (PI3K) and a Mediator of
Astrocyte Neuroprotection
(Please note that this chapter incorporates material from a manuscript in preparation for
publication by the authors below)
47
Interactome Analysis Identifies RNA Binding Protein ZC3H14 as a Novel Interactor of Phosphoinositide 3-kinase (PI3K) and a Mediator of Astrocyte Neuroprotection
Samih Alqawlaq, Izhar Livne-Bar, Declan Williams, Sara W Leung, Darren Chan, Anita
H Corbett, Gerold Schmitt-Ulms, Jeremy M Sivak
Candidate’s role: contributed to creating, designing and performing experiments, data
collection and analysis, as well as manuscript assembly and editing
3.1 Abstract
In a homeostatic state, astrocytes can support neuronal survival and function through a
range of secreted signals that protect against neurotoxicity, oxidative stress, and
apoptotic cascades. Thus, the analysis of astrocyte conditioned media (ACM) may
provide valuable insight into the nature of these protective mechanisms, and how they
might be promoted. Previously, we characterized a potent neuroprotective activity
mediated by ACM in neurons and the retina in metabolic stress models. However, the
molecular entity and mechanism underlying this activity remained unclear. Here, a
chemical genetics screen revealed phosphoinositide 3-kinase (PI3K) as a central player
transducing ACM-mediated neuroprotection. To identify additional proteins contributing
to the protective activity, endogenous neuronal PI3K was immunoprecipitated from
astrocytes exposed to ACM or control media, and MS/MS analyses were undertaken.
MS data analyses pointed toward only five additional proteins that co-
immunoprecipitated with PI3K and were regulated by the ACM signal. These hits
included expected PI3K interactors, such as the platelet-derived growth factor receptor
48
A (PDGFRA), and novel interactors, such as the zinc finger CCCH-type containing 14
(ZC3H14). ZC3H14 has recently emerged as an important RNA binding protein that
modifies poly-adenosine tail lengths on nascent mRNA transcripts. In downstream
validation studies we show that Platelet Derived Growth Factor-BB (PDGF-BB) strongly
IPI00466258.2 Isoform 1 of SH3 domain-containing kinase-binding protein 1
25 0.786 0.989 1.009 0.934 0.556 0.126
IPI00986371.1 60S ribosomal protein L27a-like
20 1.679 0.8 1.379 0.83 0.846 0.258
64
Additional bioinformatic analyses further narrowed down the list of interactors to achieve
three main criteria: 1) capturing quantifiable hits with at least 2 PSMs, 2) filtering out
non-specific binders using the negative control as a reference, and 3) including
reproducible hits across the three triplicates. A total of 122 proteins were quantified from
three or more MS3 spectra having reporter ion signals representing all six PIK3R1
immunoprecipitates (all three CFM treated replicates and all three ACM treated
replicates). Of these, 90 proteins had negative control/ACM3 iTRAQ ratios of less than
0.35, indicating that they were co-enriched with PI3KR1 specifically. Further, only 26
candidate PI3KR1 interactors fit the reproducibility criteria of having median reporter ion
signals varying less than 20% among all three ACM replicates (Figure 3-3A).
65
Figure 3-3. A summary of ACM-regulated PI3K interactions.
(A) Overview of PI3K interactors and subsequent bioinformatic analysis. (B) A total of five PI3K
protein-protein interactions were upregulated following ACM treatment. The receptor tyrosine
kinase Pdgfra, and adaptor proteins Ywhaz and Ywhae have been previously established as
PI3K interactors through a variety of sources. In addition two novel ACM induced interactors
were detected; the poly-A RBPs ZC3H14 and THOC1.
Beyond identifying specific PI3K interactors, we also investigated how PI3K binding
preferences shifted following ACM treatment. Thus, we investigated interactors that, in
addition to having high reproducibility and specificity ratios (previously defined), have a
CFM/ACM ratio between 0.65 and 1.35, indicating ACM regulated binding with PI3K.
Remarkably, a total of only 5 interactors met these criteria, all showing increased
binding with PI3K following ACM treatment (Table 2). Three ACM-induced interactors
66
are known based on established literature, including the platelet-derived growth factor
receptor alpha (PDGFRA), and two 14-3-3 adaptor proteins [23, 24]. However, two of
the identified ACM-induced binders are novel, previously unknown PI3k interactors
(Figure 3-3B). These novel interactors include the Isoform 1 of Zinc finger CCCH
domain-containing protein 14 (Zc3h14) and Isoform 1 of THO complex subunit 4
(Thoc1). Intriguingly, both of these novel interactors are involved in mRNA
polyadenylation and processing.
67
Table 2 List of ACM-induced PI3K interactors and corresponding CFM:ACM fold
changes
Accession Description PSM
Sum of Peptides
CFM1/ ACM3
CFM2/ ACM3
CFM3/ ACM3
Average fold
change Negative/ACM
IPI00461416.4 Isoform 1 of Zinc finger CCCH
domain-containing protein 14 (Zc3h14)
29 99 0.48 0.56 0.54 0.53 0.186
IPI00844650.1 Isoform 1 of Alpha-
type platelet-derived growth factor
receptor (Pdgfra)
24 70 0.62 0.52 0.45 0.53 0.392
IPI00114407.2 Isoform 1 of THO
complex subunit 4 (Thoc1)
15 60 0.31 0.44 0.38 0.38 0.03
IPI00116498.1 14-3-3 protein
zeta/delta (Ywhaz)
7 52 0.42 0.51 0.42 0.45 0.129
IPI00118384.1 14-3-3 protein epsilon (Ywhae)
6 41 0.42 0.51 0.43 0.45 0.143
68
3.4.4 ZC3H14 complexes with PI3k
Among the ACM-regulated interactors is the RBP ZC3H14, which we report here as a
PI3K interactor for the first time. ZC3H14 is an RBP that has been shown to stabilize
messenger RNA(mRNA) transcripts through modifying their poly-Adenosine (polyA) tail
length [25-27]. Thus, observing PI3K-ZC3H14 interaction fits with the role of PI3K/AKT
pathway in promoting cell survival through boosting protein synthesis [2, 28]. To confirm
the interaction between PI3k and ZC3H14 biochemically, Co-Ip were carried out by
immunoprecipitating PI3K and probing for ZC3H14 (Figure 3-4A). ZC3H14 has three
known isoforms; isoform 1, which was identified in the interactome, as well as isoform 2
and 3 all co-immunoprecipitated with PI3k. In order to confirm the identity of the eluted
band, ZC3H14 was knocked down (Figure 3-4B). A separate PI3k IP was carried out in
ZC3H14 knockdown Ht22 cell lysates compared to scrambled control, and followed by
probing for ZC3H14. Depletion of ZC3H14 led to loss of the eluted band (Figure 3-4C).
Finally, a reverse Co-Ip of the two proteins was carried out by immunoprecipitating
ZC3H14, followed by probing for PI3K p85, which further supported the interaction
(Figure 3-4D).
69
Figure 3-4. ZC3H14 is a novel PI3K interactor.
(A) Co-immunoprecipitation (Co-Ip) of PI3K was carried out in Ht22 cells and probed with an
antibody to ZC3H14 to verify interaction between the two proteins (I). Successful IP of PI3K was
confirmed by blotting for PI3K (II). (B) The efficiency of knockdown was verified by
immunoblotting for ZC3H14 in corresponding lysates, compared to control. (C) ZC3H14 was
knocked down in Ht22 cells. Eluates from the IP in knockdown lysates show a missing ZC3H14
band, compared to control. (D) As a further validation for the interaction, a reverse Co-IP was
carried out by immunoprecipitating ZC3H14 and probing for PI3K. Successful ZC3H14 IP is
demonstrated by immunoblotting for ZC3H14.
70
3.4.5 PDGF induces neuroprotective PI3K recruitment of ZC3H14
The MS/MS interactome results suggested that PDGFRA binding to PI3K was
increased following ACM addition. To validate this finding, we first investigated whether
exogenous PDGF activates the PI3K pathway in HT22 cells. Addition of recombinant
PDGF-BB rapidly induced robust AKT phosphorylation at 5 and 50ng/mL, indicating
activation of the PI3k pathway (Figure 3-5A). This is consistent with the ACM-induced
activation previously shown in Figure 1E, and is consistent with PDGF-BB as a
neuroprotective component of ACM. Next, we investigated whether recombinant PDGF-
BB can account for the neuroprotection of ACM in Ht22 cells. Thus, we returned to the
glutamate injury model previously described for the chemical genetics screen. The
experiment demonstrated a robust protection effect mediated by recombinant PDGF-BB
at 50 ng/mL (Figure 3-5B).
Since PDGFRA and ZC3H14 were both components of the PI3K complex in the ACM
interactome, we investigated whether PDGF treatment affects PI3k-ZC3H14 binding. A
PI3K Co-IP was carried out, as previously described, following 20 ng/mL PDGF-BB and
vehicle treatment. For each IP, equal concentrations of cell lysate, antibody, and beads
were used. The resulting blots demonstrate a dramatic increase in ZC3H14 elution. This
observation is supported by complimentary ZC3H14 depletion in the unbound fraction of
PDGF treated cells compared to the vehicle control (Figure 3-5C). In comparison, the
PI3K depletion and elution were consistent for each condition (Figure 3-5C). As a
functional validation, knocking down ZC3H14 rendered Ht22 cells insensitive to PDGF-
71
mediated neuroprotection (Figure 3-5D). Together, these data suggest PDGF treatment
increases PI3K recruitment of ZC3H14 to mediate the neuroprotective signal.
Figure 3-5. PDGF is enriched in ACM and induces PI3K recruitment of ZC3H14.
(A) Recombinant PDGF treatment produces a strong p-akt signal in Ht22 cells at 5 ng/mL and
50 ng/mL, indicating PI3K pathway activation. (B) A robust protection effect is mediated by
recombinant PDGF-BB at 50 ng/mL against glutamate-induced metabolic stress (n=3). (C)
PDGF treatment leads to increased association between PI3K and ZC3H14 in coIP eluates.
Following PI3K IP the ZC3H14 band is increased in PDGF treated cells compared to control.
Immunoblotting of PI3K was also carried out to confirm equal amounts of captured PI3K in both
conditions. (D) ZC3H14 knockdown eliminates PDGF-mediated neuroprotection against
glutamate injury in Ht22 cells (n=3)(**p < 0.01; bars are S.E.M).
72
3.5 Discussion
Analysis of ACM is an established approach to investigating astrocyte-neuron
interactions and identifying novel astrocyte-derived neuroprotective factors against
metabolic injuries [1, 29-31]. This study used a combination of chemical genetics
screening and mass spectroscopy to identify key signaling pathways and ligands
involved in ACM-mediated neuroprotection against metabolic stress. The chemical
genetics screen identified PI3K as the main signaling hub for ACM activity; we further
validated this finding in primary cortical neurons. Following a highly specific PI3K
immunoprecipitation in crosslinked Ht22 lysates, we used mass spectroscopy to
generate a PI3K interactome with the goal of identifying upstream effectors and
downstream targets. The interactome lead to the identification of a novel PI3K-ZC3H14
interaction, which was upregulated by ACM addition in Ht22 cells. Further, the
interactome showed that PI3K-PDGFr interaction was also upregulated by ACM,
strongly implicating PDGF as a key ligand in ACM-mediated neuroprotection. Follow up
validation studies showed that PDGF protected neurons from glutamate metabolic
stress in vitro, and increased PI3K recruitment of ZC3H14. Finally, we showed that
ZC3H14 knockdown eliminated PDGF-mediated neuroprotection, which highlights the
importance of ZC3H14 in coordinating neuroprotective signals against metabolic stress.
PDGF is secreted primarily by macroglia, including astrocytes and retinal Muller cells,
as well as by neurons, mediating short-range paracraine communication between
astrocytes and neurons [32]. It has also been shown to protect neurons against
oxidative stress and NMDA-induced metabolic stress in primary neurons and retinal
73
ganglion cells [33-37]. Activation of the PI3K-AKT pathway with NFs such as PDGF is
known to promote cell survival through inactivation of apoptotic factors [6, 38], activation
of metabolic regulators [39, 40], and activation of mammalian target of rapamycin
(mTOR), which leads to increased translation of growth proteins and further anti-
apoptotic signaling [38, 41]. Less known is PI3K’s protective role through interacting
with a range of RBPs, with the adapter protein 14-3-3 functioning as a mediator,
ultimately leading to stabilizing of mRNA transcripts [42, 43].
RBPs play an important role in post-transcriptional gene regulation through physical
interactions with adenine-(or adenosine) and uridine-rich elements of RNA [44, 45]. An
example of these RBPs is ZC3H14, which binds polyadenosine (polyA) tails and carries
out a range of post-transcriptional modifications on premature transcripts [46, 47].
These include control of PolyA tail length [27], nuclear export [48], and mRNA splicing
[49]. A 2018 study reported that ZC3H14 interacts with THO complex 1 (THOC1) to
coordinately control RNA processing, poly(A) tail length, and consequently mRNA
stability [25]. THOC1 was among the ACM-regulated novel PI3K interactors in our
screen. Morris and Corbett also showed that mRNA targets of ZC3H14 and THOC1
include the postsynaptic density protein 95 (Psd95), ATP synthase lipid-binding protein
(Atp5g1) and microtubule-associated protein tau (MAPT), which are integral to neuronal
function and metabolism. Further, altering ZC3H14 expression impairs neural function in
Drosophila and has been associated with cognitive deficits in humans [50], anatomical
changes in mouse brain ventricles as well as working memory deficits [26]. Thus, our
74
findings provide a novel insight into the role of PI3K and ZC3H14 in regulating neuronal
function and survival (Figure3- 6).
Figure 3-6. Summary of the mechanisms of ACM neuroprotection against
metabolic stress.
(1) PDGF, a component of ACM, binds to PDGFr in neurons, (2) phosphorylating PI3K,
which in turn binds to the RBP ZC3H14. (3) Activated ZC3H14 then binds with target
mRNAs’ polyA tails, (4) leading to increased stability of pro-survival and metabolic
mRNAs.
Future studies will investigate whether ACM-mediated neuroprotection can be
linked to the stability of mRNA targets of ZC3H14 and THOC1. Exploring this novel
interaction further will provide a better understanding of how astrocytes communicate
with neurons to maintain their survival. Further, it may ultimately lead to the
development of novel therapeutic approaches for the treatment of neurodegenerative
diseases.
75
3.6 ACKNOWLEDGEMENTS
CIHR, NSERC, TWGH Foundation Glaucoma Research Chair (JS), VSRP Program
(SA).
76
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Supplementary Tables
Supplementary Table 3. A list of PI3K antibodies used in immunoprecipitation
screen, along with molecular weight of target peptide, and corresponding IgG
isotype
Ab # Antibody description and catalogue number Size (kD)
IgG isotype
Ab 1 Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody 4228
60 and 85
Rabbit IgG
Ab 2 PI3 Kinase p85 (19H8) Rabbit mAb 4257 85 Rabbit IgG
Ab 3 PI3 Kinase p110α (C73F8) Rabbit mAb 4249 110 Rabbit IgG
Ab 4 PI3 Kinase p110β (C33D4) Rabbit mAb 3011 110 Rabbit IgG
Ab 5 PI3 Kinase Class III (D4E2) Rabbit mAb 3358 100 Rabbit IgG
Ab 6 PI3 Kinase p110γ (D55D5) Rabbit mAb 5405 110 Rabbit IgG
show anatomical changes in brain ventricles as well as working memory deficits [22].
This evidence further emphasizes the role of ZC3H14 in maintaining normal neuronal
functioning, as seen in its impact on anatomy of neuronal tissue, not just their functions.
90
Thirdly, ZC3H14 has been shown to control mRNA transcripts critical to metabolism,
such as the ATP5g1, which is essential to mitochondrial output [23, 24]. Altered stability
of ATP5g1 may therefore hinder mitochondrial functions, which has negative
implications on neuronal function and survival. Mitochondria are abundantly present in
neuronal tissue; in the eye, mitochondria are particularly dense within unmyelinated
RGC axons in the retina, in order to sustain energetic needs of neurons [25]. To provide
context, up to 90% of the ATP produced by mitochondria is used to maintain action
potentials and neuronal survival [26]. In addition to being the cell’s powerhouse,
mitochodnria also serve as an integration centre for inflammatory and apoptotic signals
in neurons. Thus, one future direction would be to investigate a possible link between
altered ZC3H14 expression and mitochondrial health. An example of this direction
would be to correlate the rate of ZC3H14 mutations with mitochondrial dysfunction in
neurodgenerative diseases, such as glaucoma and Alzheimer’s disease. Another
direction would be to explore the effect of upregulating ZC3H14 expression on neuronal
rescue in the context of metabolic stress.
Another PI3K binder that emerged from the interactome is the RBP THOC1. THOC1
encodes a nuclear matrix protein and is recruited to premature RNA transcripts to
perform various post-transcriptional processing functions [27, 28]. THOC1 also forms a
complex with ZC3H14 coordinately control RNA processing, poly(A) tail length, and
consequently mRNA stability, including ATP5g1 [23]. The scope of the current project
did not include the characterization of THOC1-PI3K interaction; however, a proposed
future direction would be to explore this interaction. Because it is known that THOC1
91
forms a complex with ZC3H14, it would be insightful to demonstrate whether PI3K
interacts directly with THOC1.
4.3 PDGFr as a key receptor in ACM-mediated neuroprotection
The current study also shows that PI3K-ZC3H14 association was upregulated through
activation of PDGFr with PDGF-BB in Ht22 cells. Exogenous PDGF-BB was also
sufficient to reproduce both ACM-induced AKT phosphorylation as well as ACM-
mediated neuroprotection in Ht22 cells. A number of other studies showed that
astrocyte-mediated neuroprotection relieson growth factors, including vascular
endothelial growth factors (VEGF) and fibroblast growth factor (FGF), and the insulin-
like growth factor-1 (IGF-1) neurons [29-32]. Neurons of the central nervous system
(CNS) and sensory tissues like the retina, rely on NF support from neighboring glia and
target sites to modulate their function from developmental stages through maturity [33,
34]. NFs maintain neuronal survival through inhibition of apoptotic pathways, promoting
anti-oxidant activities, and boosting cellular bioenergetics [35]. Conversely, NF
deprivation induces the intrinsic apoptotic pathway, causing reduced ATP production,
reactive oxygen species generation, cytochrome C release, and subsequent caspase
activation [36]. However, to our knowledge this is the first study describing a
neuroprotective role of astrocyte-induced PDGFr activation. PDGF is a dimer of A- and
B-chains (AA, BB, or AB) which signals through tyrosine kinase receptors (RTKs)
(PDGFRα and PDGFRβ [37, 38].
92
In a neuronal context PDGF is secreted primarily by macroglia, including astrocytes and
retinal Muller cells, as well as by neurons, mediating short-range paracrine
communication between astrocytes and neurons [39]. It has also been shown to protect
neurons against oxidative stress and NMDA-induced metabolic stress in primary
neurons and retinal ganglion cells [40-44]. In human, PDGF is highly expressed in white
matter in humans following stroke, suggesting that PDGF may be involved in either
regeneration or protection of damaged neurons [45]. Thus, PDGF involvement in
astrocyte-mediated neuroprotection in the current study adds further evidence to the
role PDGF in neuroprotection against metabolic stress. However, there are some
considerations for the use of PDGF as a neuroprotective biologic in aerogeneration.
One of PDGF’s roles is to promote new blood vessel maturation; thus, the impact of
PDGF on retinal vasculature needs to be considered carefully, given the risk of aberrant
vascularization[46] . Furthermore, unlike the homogenous cell line used to asses PDGF
neuroprotective potential, neuronal tissue is highly complex and heterogenous. As a
result, administering PDGF will affect surrounding glia and connective tissue, initiating
signaling cascades in a number of cell populations. The collective outcome of this
signaling wave needs to be addressed carefully in future studies to assess PDGF’s
safety in neuronal tissue.
4.4 Considerations for future direction
Taken together, this study provides a case for the use of interactomes in
exploring signalling pathways involved in biological activities of interest, such as
neuroprotection or regeneration. This approach can be especially powerful if a specific
93
protein is known to be critical to an activity; in this case, the protein can serve as a bait
with which the rest of the pathway is captured. However, a few points are important to
point out with respect to the interactome data. While the interaction between ZC3H14
and PI3K has been implied using evidence mass spectroscopy and IP studies, it is
unclear whether the interaction occurs directly or as part of a larger complex. PI3K
interacts with a number of targets along the AKT-mTOR pathway (see Chapter 1).
Therefore, it is important to investigate the involvement of other targets downstream of
PI3K in transducing ACM signalling in neurons through ZC3H14 binding. A likely
downstream target is mTOR, which has been shown to be involved in a range of
neuroprotection and regeneration models. Secondly, while using ACM derived from
primary astrocytes, for consistency the PI3K interactome performed in this work was
carried out using the transformed hippocampal cell line Ht22, which is a well-established
model in neurodegenerative research [47-49]. While useful and convenient,
immortalized cell lines are genetically altered, and are prone to cumulative mutations
with passaging, which may introduce functional and phenotypic differences compared to
their primary counterparts. Therefore, one future direction of this project is to confirm the
PI3K-ZC3H14 interaction in vivo using brain or retinal models. We have generated
immunofluorescence images of retina sections showing localization of ZC3H14 in the
GCL layer; we also demonstrate co-localization with PI3K following Kainic acid injury
(Figure 4-1). Further, it is essential to confirm whether PDGF administration increases
the PI3K association with ZC3H14, as observed in Ht22 cells. Similarly, it would be
useful to assess the neuroprotective effect of PDGF administration in ZC3H14 knockout
models under metabolic stress conditions.
94
Figure 4- 1 Immunostaining of ZC3H14, and AKT in retinal sections treated with
vehicle and Kianic acid (1hr).
In conclusion, neurodegeneration and neuroprotection are extremely complex
processes that involve hundreds of biological factors and signalling pathways in delicate
orchestration. Thus, it is more than likely that ZC3H14’s role in protecting neurons is
complemented with a range of other factors and pathways. Future work can expand on
the current work using proteomic analyses that can identify other binding partners of
ZC3H14, to generate a snapshot of this RBP in its natural complex. Using this
approach, one can then compare global functional patterns of this ZC3H14 and its
partners in the normal and disease settings. This ‘bird eye’ view takes into account the
complexity of neurodegenerative disease, and may ultimately bring us closer to
incorporating ZC3H14 targeting in a neuroprotective therapy.
95
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