© 2015. Published by The Company of Biologists Ltd.

Title Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor

intracellular domain fragment.

Authors

Dusan Matusica [1, 4], Fabienne Alfonsi [1], Bradley J Turner [2], Tim J Butler [1], Stephanie R

Shepheard [3], Mary-Louise Rogers [3], Sune Skeldal [1], Clare K Underwood [1], Marie

Mangelsdorf [1], Elizabeth J Coulson* [1].

Affiliations

[1] Queensland Brain Institute, The University of Queensland, Brisbane, Qld 4072, Australia

[2] The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville

3052, Vic 3051, Australia

[3] Department of Human Physiology, Centre for Neuroscience, Flinders University, GPO Box

2100, Adelaide, SA 5001, Australia

[4] Department of Anatomy & Histology, Centre for Neuroscience, Flinders University, GPO

Box 2100, Adelaide, SA 5001, Australia

Address/telephone/fax/e-mail of corresponding author

* Elizabeth J Coulson, Queensland Brain Institute, The University of Queensland, Brisbane, 4072

Qld, Australia. phone: +61 7 33466392, fax: +61 7 33466301, email: e.coulson@uq.edu.au

Conflict of interest Patents covering the use of c29 as a therapeutic in a range of neurological

conditions are held by the University of Queensland.

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JCS Advance Online Article. Posted on 26 October 2015

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia

(Career Development Fellowship 569601 to EJC and project grant 10012610), the Australian

Research Council and NuNerve Pty Ltd (Linkage project grant LP10012610) and the Goodenough

and Wantoks bequest. CKU and MM were recipients of an Australian Postgraduate Award and the

Ross Maclean Fellowship, respectively. DM was supported by Centre for Neuroscience, Flinders

University and the Flinders Medical Centre Research Foundation grants. SRS was the recipient of

an Australian Rotary Health (Neville & Jeanne York) MND PhD Scholarship and M-LR was

supported by a Rosalind Nicholson MNDRIA Grant. Robert Rush (Flinders University, Adelaide,

Australia) kindly provided the TrkB antibody used for western blotting and immunoprecipitation

experiments. Moses Chao (Skirball Institute, New York, USA) kindly provided the 9992 p75NTR

antibody for western blotting. We thank Stephan Weise and Michael Sendtner (Würzburg

University, Germany) for assistance in establishing the NGF-killing motor neuron assay. NSC-34

cells were kindly provided by Dr. Neil Cashman (University of British Columbia). We thank other

members of the Coulson laboratory past and present for helpful discussions and Rowan Tweedale

for critical reading of the manuscript and editorial assistance.

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Abstract

The neurotrophin receptor (p75NTR) can mediate neuronal apoptosis in disease or following trauma,

and facilitate survival through interactions with Trk receptors. Here we tested the ability of a

p75NTR-derived trophic cell-permeable peptide, c29, to inhibit p75NTR-mediated motor neuron death.

Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29

treatment of SOD1G93A mice, a common model of amyotrophic lateral sclerosis, resulted in

increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident

with this, c29 treatment of these mice reduced the production of p75NTR cleavage products.

Although c29 treatment inhibited mature- and pro-nerve growth factor-induced death of cultured

motor neurons, and these ligands induced the cleavage of p75NTR in motor-neuron-like NSC-34

cells, there was no direct effect of c29 on p75NTR cleavage. Rather, c29 promoted motor neuron

survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that

low levels of BDNF were present, an effect that was replicated in vivo in SOD1G93A mice. We

conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in

vivo.

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Introduction

The selective death of motor neurons is the central pathology of neurodegenerative diseases such as

amyotrophic lateral sclerosis and spinal muscular atrophy (Murray et al., 2010). Motor neurons are

also vulnerable following axonal injury, with motor neuron dysfunction resulting in paralysis

(Branco et al., 2007). During development, motor neurons are dependent on growth factors such as

the neurotrophins for their survival and function. In developing motor neurons, brain-derived

neurotrophic factor (BDNF) and neurotrophins (NT) 3 and 4 mediate cell survival through their

cognate Trk receptors, TrkB or C (Kaplan and Miller, 2000). Similarly, after nerve lesion and in

animal models of motor neuron disease, BDNF and NT3 can protect and repair motor neurons

(Yuen and Mobley, 1995; Wiese et al., 2004; Henriques et al., 2010). During development and

following injury, the p75 neurotrophin receptor (p75NTR) has been shown to mediate motor neuron

death (Seeburger et al., 1993; Lowry et al., 2001; Dechant and Barde, 2002; Ibanez and Simi, 2012)

in response to nerve growth factor (NGF) (Sedel et al., 1999; Ernfors, 2001) or its immature

precursor (proNGF), released by activated astrocytes (Pehar et al., 2004; Domeniconi et al., 2006).

However, p75NTR is a multifunctional receptor for both mature and pro-neurotrophins, capable of

regulating different biological effects in response to its ligands (Roux and Barker, 2002; Blochl and

Blochl, 2007; Underwood and Coulson, 2007). In addition to its death-signaling capability, p75NTR

also acts as a co-receptor that can assist Trk receptors to bind their ligands with higher affinity and

specificity, thereby enhancing their trophic functions (Hempstead et al., 1991; Barker, 2007). An

important mechanism in this process appears to be the generation of biologically active receptor

fragments following enzymatic cleavage of p75NTR (Ceni et al., 2010; Kommaddi et al., 2011;

Matusica et al., 2013).

In a two-step process, p75NTR is enzymatically initially cleaved by an α-secretase (ADAM17) that

removes the extracellular domain of the receptor, leaving a membrane-bound C-terminal fragment.

This fragment is subsequently cleaved by γ-secretase, releasing the intracellular domain into the

cytoplasm (Jung et al., 2003; Kanning et al., 2003). The intracellular domain contains two regions

that mediate intracellular signaling via protein-protein interactions: the juxtamembrane domain, and

a tumor necrosis factor receptor-like death domain (Roux and Barker, 2002; Skeldal et al., 2011).

Increased metalloprotease cleavage of p75NTR has been associated with the death of sciatic neurons

following injury (DiStefano et al., 1993; Murray et al., 2003), and evidence of increased levels of

p75NTR proteolytic cleavage has been reported using urine analysis in both SOD1 transgenic mice

and people living with amyotrophic lateral sclerosis (Shepheard et al., 2014). Elevated levels of the

C-terminal and intracellular domain fragments have also been reported in the degenerating motor

axons of SOD1G93A transgenic mice (Perlson et al., 2009), and both p75NTR cleavage fragments

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have been linked to death signaling (Skeldal et al., 2011). Although the nature of p75NTR fragment

signaling appears to be cell-type specific (Vicario et al., 2015), these studies indicate that the

proteolytic cleavage of p75NTR may be a key mechanism by which p75NTR mediates death signaling

after motor neuron injury and disease.

We have previously shown that the juxtamembrane region of p75NTR has the ability to induce cell

death when bound to the cell membrane (Coulson et al., 2000; Underwood et al., 2008). We also

demonstrated that a soluble peptide mimic (c29) of the juxtamembrane intracellular domain

fragment of p75NTR can act as a dominant negative inhibitor of p75NTR death signaling (Coulson et

al., 2000). Recently, we have shown that the intracellular domain fragment of p75NTR and the c29

peptide are able to increase NGF survival signaling through interaction with TrkA receptors

(Matusica et al., 2013). This in turn leads to increased neurite outgrowth at low NGF

concentrations, and enhanced survival of sympathetic neurons undergoing growth factor withdrawal

(Matusica et al., 2013). Here we investigated whether c29 plays a similar role in TrkB-dependent

cells, determining the role of p75NTR cleavage in neurotrophin-induced motor neuron death and

exploring whether c29 can inhibit this process in vitro and in vivo.

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Materials and methods

c29 and control peptide synthesis

A 29 amino acid peptide of the juxtamembrane ‘Chopper’ domain (Coulson et al., 2000)

(KRWNSCKQNKQGANSRPVNQTPPPEGEKL) and a randomly scrambled version (SC:

SKGQVCRNQPGQNKPEPANKSWKETPLRN) were synthesized as N-terminal fusions to a non-

naturally occurring TAT-like protein transduction domain (PTD) peptide (YARAAARNARA; (Ho

et al., 2001) using t-boc chemistry then purified using reverse phase HPLC by Dr. James I. Elliott

(Yale University). For axotomy experiments, PTD was conjugated to c29 via a disulfide-link

(Coulson et al., 2000) by Auspep Pty. Ltd. No difference in functionality was seen between the two

conjugation methods in cell culture assays, and no effects were seen in cells treated with PTD

peptide alone, peptides without carrier or disulfide-linked peptides that had been reduced with DTT.

For immunoprecipitation experiments, the unconjugated c29 peptide was labeled on the amino

terminus with biotin via a six-carbon spacer. Except for these immunoprecipitation experiments,

‘c29’ refers to the PTD-conjugated form of the c29 peptide. The biotinylated control peptide

mimicking the p75NTR extracellular juxtamembrane domain LC1 (H-RGTTDNLIGGSC-NH2) was

manufactured by Auspep Pty Ltd.

Motor neuron axotomy

The Institutional Animal Ethics Committee approved all experiments involving the use of animals,

in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific

Purposes. Ulnar nerve axotomy was performed on newborn Wistar rat pups as previously described

(Lowry et al., 2001). Peptides (PTD-c29 and PTD only; 100nM) were applied via a 1 mm3 cube of

pluronic gel foam that was sutured to the proximal axonal stump (Cheema et al., 1996). After 5

days the animals were perfused with a 4% solution of paraformaldehyde in phosphate buffer.

Cervical spinal cords were removed, after which serial sections were cut at 40 µm, mounted onto

gelatin-coated slides and stained in 0.1% cresyl violet. Details of the counting procedure have been

described previously (Lowry et al., 2001). Briefly, motor neurons in the spinal cord displaying a

prominent nucleolus were counted in every fifth section. The percentage neuronal survival after

axotomy was calculated based on the nucleolar number on the axotomized side as a percentage of

the nucleolar number in the contralateral side. Data were analyzed by two-tailed t-test.

SOD1 animal experiments

Treatments

Male and female transgenic mice with the G93A human SOD1 mutation (SOD1G93A; line B6SJL-

TgN [SOD1-G93A] 1Gur; Jackson Laboratories), and in some cases age-matched C57Bl6 mice,

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were used. Some cohorts of mice were administered peptides by a single intraperitoneal injection (5

mg/kg) and sacrificed 60-90 min later by perfusion (for histology) or cervical dislocation

(biochemical analyses). Alternatively, mice were treated systemically with peptides starting from 9

weeks of age (the time at which p75NTR expression is upregulated in spinal motor neurons; Lowry et

al., 2001). The animals were randomly assigned a treatment group and implanted with a 28 day

subcutaneous osmotic minipump (Alzet model 1004) delivering ~5 mg/kg/day PTD-c29 (flow rate:

0.11 µl/h) or vehicle and the pumps were replaced monthly until death/euthanasia.

Behavioral analyses

Mice were weighed weekly until close to end stage when they were monitored daily. Urine

discharged during weighing was collected and frozen. Testing of motor function using a rotarod

device began at 10 weeks of age. Each weekly session consisted of three trials on the elevated

accelerating rotarod (Ugo Basile) beginning at 5 rpm/min and accelerating to 20 rpm/min over 20 s

for a total duration of 3 min. To determine mortality in a reliable and humane fashion, we used an

artificial end point, defined as 80% minimum original weight threshold. The moribund mice were

scored as “dead” and were euthanized. Survival analysis of the SOD1G93A transgenic mice was

performed using a log-rank (Mantel-Cox) test and analysis of motor neuron cell numbers in spinal

cord sections was performed using a student’s t-test. Between-group analyses of weight were

performed by repeated measure ANOVA followed by a Sidak’s test of analysis of the differences

between groups. A two-tailed Mann Whitney test was used to compare the time it took mice to lose

10% of their maximal weight. Rotarod data were analyzed by ANOVA with a Sidak’s multiple

comparison test.

Biochemical analysis of p75NTR extracellular domain fragment.

Urine was collected from SOD1G93A and wildtype mice during weighing, using metabolic cages or

from the bladder after euthanasia. However, insufficient urine for analysis was obtained from some

mice at each time point. For peptide-treated mice, urine was collected from 6 c29-treated and 6

saline-treated SOD1 animals on an at least weekly basis. For analysis, we pooled data obtained

from consecutive samples such that many data points contain data derived from two samples from

the same animals but collected a week or less apart. A sandwich ELISA was used to measure the

extracellular domain fragment of p75NTR (p75NTR ECD) in urine as previously described (Shepheard

et al., 2014). Briefly, ELISA plates (96-well plates, Costar Corning) were coated for 18h with anti-

p75NTR ECD MLR1 (4µg/ml in 25mM sodium carbonate 25mM, sodium hydrogen carbonate,

0.01% thimerosol, pH 9.6) at 4°C. Wells were blocked with sample buffer comprising phosphate

buffered saline (PBS), 2% bovine serum albumin (BSA; Sigma) and 0.01% thimerosol, pH 7.4, for

1 h at 37°C. Recombinant human mouse p75NTR ECD (control antigen, aa:20-243, R&D Systems)

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and urine samples were diluted in sample buffer and incubated for 20h at room temperature in the

antibody coated wells. For detection, wells were incubated in goat anti-p75NTR ECD (R&D

Systems) for 1 h followed by a bovine anti goat IgG-HRP antibody (Jackson ImmunoResearch) for

1 h at room temperature. Visualization was via a peroxidase reaction developed using the color

reagent 3,3’,5,5’-tetramethylbenzidine (TMB, Bio-Rad) and stopped with 2 M sulfuric acid.

Between steps, plates were washed 4 times with wash buffer (PBS, 0.05% Tween20, 0.01%

thimerosol, pH 7.4). Plates were read at 450 nm with a PerkinElmer Victor-x4 Plate Reader. Mouse

creatinine was used as a standard for urinary protein measurements and was assessed using a

creatinine detection kit (Enzo Life Sciences) as previously described (Shepheard et al., 2014).

p75NTR ECD specific ELISA results were analyzed by one-way ANOVA, with a Bonferroni’s

multiple comparison test.

Peptide distribution and motor neuron survival

To assess the biodistribution of c29 and motor neuron survival in treated post natal day 115

SOD1G93A mice, animals were first anesthetized and then intracardially perfused with PBS,

followed by 4% paraformaldehyde in PBS. Spinal cords were dissected and post-fixed in 4%

paraformaldehyde at room temperature for 2h.

Fixed spinal cord samples were cryoprotected overnight by immersion in 20% sucrose/PBS,

embedded in optimal cutting temperature compound (OCT; Tissue Tek; Raymond Lamb Ltd

Medical Supplies) and frozen on dry ice. All spinal cord samples were stored at -80°C until needed,

and then cryosectioned.

For biodistribution, 20 m thick lumbar spinal cord sections were stained using streptavidin-

conjugated horseradish peroxidase. Motor neuron numbers were determined on 20 m thick lumbar

spinal cord sections stained with choline acetyl transferase (ChAT). Sections were washed in 0.1M

phosphate buffer (PB), pH 7.4 and then incubated in 3% H2O2, 50% ethanol to inactivate

endogenous peroxidase. Sections were subsequently washed in 0.1M PB, and incubated for 1 h at

room temperature in blocking solution (PB, 10% horse serum, 0.1% Triton X-100). Primary

antibody (goat anti-ChAT, 1:1000, Millipore) was diluted in the above blocking buffer and applied

onto sections for overnight incubation at room temperature. Sections were then washed at room

temperature in PB containing 0.1% Triton X-100 before incubation with biotinylated secondary

antibody and ABC reagents (Vector Elite Kit, Vector Laboratories; 6 l/ml avidin and 6 l/ml

biotin). Black immunoreactivity was revealed by a nickel-intensified diaminobenzidine reaction.

Sections were finally washed in PB and gradually dehydrated in ethanol and xylene before being

mounted in DePex mounting medium (VWR International). A total of 10 sections per mouse were

analyzed. Motor neuron numbers were compared using a two-tailed t-test. Jour

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Spinal cord lysate preparation

Following a 60 minute treatment of wildtype C57Bl6 and SOD1G93A mice with c29 or control

peptide delivered i.p, mice were sacrificed by cervical dislocation and spinal cords were dissected

immediately after sacrifice and snap-frozen. The lumbar spinal cords were thawed and dissected in

RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium

deoxycholate, 0.1% SDS, 1 mM EDTA, 1mM sodium orthovanadate, 1mM PMSF, 1µM BB94

(Betimastat), 15 mM sodium fluoride, 40 mM β-glycerophosphate, 0.5 mM 4-deoxypyridoxine, 1%

(v/v) Roche Complete-Mini inhibitor cocktail stock and 1mM DTT at pH 7.4. They were then lyzed

in 300µl of the same buffer using a Retch Tissue Lyser (Qiagen) for 3 min at 4°C. Lysates were

centrifuged at 16,000 x g and pellets discarded. Protein estimation of all lysates was done using the

Pierce BCA protein assay kit (Thermo Fisher). Western blots were performed as described below.

Motor neuron isolation and culture

Cultures of spinal motor neurons were prepared from embryonic day 13 wild-type C57BL/6J mice

and isolated by immunopanning as previously described (Wiese et al., 2010). Briefly, pregnant

dams were sacrificed by cervical dislocation and the uterus removed by Cesarean section and

placed in Neurobasal medium (NB; Gibco). Embryos were removed from the uterine sac and the

lumbar spinal cords were dissected on ice. Batches of 2 or 3 spinal cords were digested in 1 ml of

0.05% trypsin-ETDA (Gibco) for 10 min at 37°C in 15 ml tubes. To prevent further digestion, 0.14

mg/ml trypsin inhibitor (Sigma) solution was added, and the tissue was centrifuged at 100 g for 7

min. Spinal cords were trituated manually for 2 min with a polished glass pipette, after which the

suspension was filtered through a 100 μm sieve (Falcon). The remaining tissue was further

triturated ~8 times and filtered through another 100 μm sieve. The combined cell suspensions were

then immunopanned by being plated onto a 10 cm culture dish (Nunc) pre-coated with MLR-2

mouse anti-p75NTR antibody (Rogers et al., 2006). The dish was pre-coated with 8 ml of

immunopanning solution containing 1.5 μg/ml MLR-2 in 10 mM Tris-HCl, pH 9.5, for 2 hours and

washed with NB medium before the spinal cord suspension was applied. After 45 min incubation,

the dish was washed three times with NB medium and the remaining attached cells were isolated

from the plate by incubation for 1 min in 0.85 M KCl. The collected cell suspension was

centrifuged at 400 g for 5 min at room temperature. The supernatant was removed and the cell

pellet was resuspended in 1 ml of NB medium. Neurons were plated at a density of 3,000 live cells

per well in 4-well culture dishes (Greiner) pre-coated with 0.0015% poly-ornithine (overnight at

4°C) and 3.75 μg/ml laminin (for 30 min at 37°C). Cells were allowed to adhere for 1 h at 37°C,

after which 2 ml of motor neuron culture medium was added [NB supplemented with B27 (Life

Sciences), 500 μM glutamax (Life Sciences), 10% horse serum (Linaris), 25 μM β-mercaptoethanol Jour

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(Merck) and 1 ng/ml recombinant human BDNF (Peprotech)]. Cells were maintained at 37°C in a

humidified atmosphere with 5% CO2 for 24 h, after which the culture medium was replaced, live

cells counted (day 1), and the experimental treatments administered. Phase contrast images of motor

neurons were taken using an Olympus (6IX81) microscope.

Neuronal survival assays

In addition to BDNF, the following growth factors were used in primary neuronal culture assays at

concentrations indicated in individual experiments: purified mouse 2.5S NGF (Biosensis), proNGF

(SCIL proteins GmbH), proBDNF (Biosensis), recombinant rat cilliary neurotrophic factor (CNTF;

R&D Systems) and recombinant human glial-derived neurotrophic factor (GDNF; Peprotech). For

the neuronal survival assays, BDNF, CNTF or GDNF was added at the time of plating as indicated.

A concentration of 1ng/ml for each trophic factor has previously been determined to result in

optimal rates of survival (Wiese et al., 1999). In order to count the same field at each time point,

grids were etched onto the plates using an 18-gauge needle. Motor neurons were counted on day 1

(4 separate fields for each well) and the same fields were recounted on day 3. Cell survival was

expressed as a percentage compared to survival in BDNF alone on day 1.

For assays investigating the effects of the induction of p75NTR cleavage by the metalloproteinase

enhancer phorbol 12-myristate 13-acetate PMA (200nM), and blocking p75NTR cleavage with the

selective non-competitive metalloproteinase inhibitors TAPI-2 (200nM, extracellular cleavage) and

Compound E (20µM, intracellular cleavage ; Tian et al., 2002), the compounds were added to

established cultures on day 1 after the initial cell counts and cells were then counted again on day 3.

To assess the effects of c29 peptide on motor neuronal survival in limiting growth factor

concentrations, isolated spinal motor neurons were plated and treated with BDNF, as indicated, in

the presence of 1µM PTD-linked c29 or scrambled peptide, then cultured for 3 days with the

addition of fresh BDNF and peptide treatments on day 2. For growth factor-withdrawal assays,

following initial plating in 1ng/ml of BDNF, CNTF or GDNF, cultures were washed four times

with serum-free NB medium, after which the medium was replaced with medium containing

0.1 ng/ml BDNF, CNTF or GDNF and 1µM PTD-linked c29 or scrambled peptide, as indicated.

For assays investigating the effect of c29 on NGF-mediated motor neuron death, cultures were

grown in 1ng/ml BDNF, CNTF or GDNF for 1 day, before being treated with 100ng/ml 2.5S NGF

and 1µM PTD-linked c29 or scrambled peptide, as indicated. Motor neurons were counted on day 1

and, to assess the effects of various treatments, again on day 3.

Rates of survival were compared between conditions by ANOVA with Bonferonni post-hoc testing.

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Immortalized cell culture, pull-down experiments and western blotting,

Mycoplasma-free neuroblastoma x spinal cord (NSC-34) cells were maintained in Dulbecco’s

modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS and 1% penicillin/

streptomycin/glutamine solution (PSG; Cashman et al., 1992). For experiments, cells were grown to

60% confluency in medium comprising 1:1 DMEM:Ham’s F12, plus 1% N-2 supplement, 1% PSG,

1% modified Eagle’s medium non-essential amino acids, and 1µM retinoic acid (Sigma). The cells

were allowed to differentiate for up to 3 days, with the medium being replaced after 48 h.

For p75NTR shedding experiments, NSC-34 cells were cultured in poly-L-ornithine-coated 6-well

plates, starved for 1 h in serum-free DMEM, and treated with 200nM PMA, 200nM TAPI-2, or

20 µM Compound E, 50 ng/ml NGF, 5 ng/ml proNGF, 50 ng/ml BDNF or 5 ng/ml proBDNF for

6 h. To inhibit degradation of the p75NTR intracellular domain fragment, cells were treated with

1 µM epoxomicin (Sigma) 1 h prior to the addition of other compounds.

For experiments measuring phosphorylated TrkB, Akt, and Erk1/2, NSC-34 cells were

differentiated for 5 days before being serum starved for 4 h in serum-free DMEM. Cells were then

pretreated with 1µM c29 or scrambled control peptide for 1h prior to addition of 10ng/ml of BDNF

followed by treatments with growth factors as indicated for time intervals ranging from 1-90 min.

Following treatments, cells were immediately washed with ice cold PBS and lyzed and prepared for

SDS-PAGE analysis as described below.

Cells were lyzed using chilled lysis buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2

mM EDTA, 1% NP-40, 1% Triton X-100, 10% glycerol, 1mM phenylmethanesulfonyl fluoride,

1mM sodium orthovanadate, 1µM batimastat (BB-94), and 1% Roche protease inhibitor cocktail

(Ceni et al., 2010).

HEK293 cells used for pull-down assays were routinely cultured in DMEM supplemented with

10% FBS, in 5% CO2 at 37 °C. For experiments, cells were transfected with TrkB plasmid DNA

using FuGENE 6 transfection reagent (Roche Applied Science), lyzed 48 h after transfection and

the lysates used for c29 pull-down experiments as described below.

To perform pull-down assays, biotinylated c29 or LC1 control peptide was bound to Dynabeads

MyOne Streptavidin T1 (Invitrogen) before incubation with transfected HEK293 or NSC-34 cell

lysates in the presence of 2% BSA. Nonspecific binding was removed by washing the beads five

times in Tris-buffered saline (TBS) supplemented with 0.05% Tween 20, and the proteins were

eluted by boiling samples in reducing sample buffer (20 mM dithioerythritol, 2.5% SDS).

Cell lysates, lumbar spinal cord lysates or pulled-down proteins were electrophoresed through 4-

12% Bis-Tris buffered SDS gels (Life Sciences), transferred onto a PVDF or nitrocellulose Jour

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membrane and probed using the following antibodies: rabbit anti-p75NTR intracellular domain

#9992 (1:5000), rabbit anti-TrkB (1:500; Biosensis), phospho-TrkB (Ser478; 1:250; Biosensis),

mouse pan anti-Akt (1:2000; Cell Signaling Technologies), XP™ rabbit mAb rabbit anti-

phosphorylated serine 473 (Ser 473) Akt (1:2000; Cell Signaling Technologies), XP™ rabbit mAb

phospho-p44/42 MAP kinase (Erk1/2; Thr202/Tyr204) (1:2000; Cell Signaling Technologies),

rabbit phospho-p44/42 MAP kinase (Erk1/2; Thr202/Tyr204) (1:1000; Millipore), rabbit anti-pan

p44/42 Erk1/2 (1:1000; Cell Signaling Technologies), rabbit cleaved caspase-3 (Asp175; 1:1000;

Cell Signaling Technologies) and rabbit caspase-3 (8G10; 1:1000; Cell Signaling Technologies),

CREB (48H2) and phospho-(Ser133) CREB (87G3) (1:1000; Cell Signaling Technologies), and

mouse anti-beta-III tubulin (1:2000; Promega). The membranes were blocked for 1 h at room

temperature in 0.1% Tween-20, and 0.02% NaN3 in TBS, pH 8.0, and either 4% skim milk powder

for p75NTR detection, 3% BSA for phosphorylated Erk1/2 and Akt, or 1% BSA for phosphorylated

TrkB, before being incubated overnight with primary antibodies at 4C. Membranes were then

washed in TBS-Tween 20 (TBST), and incubated for 1 h with LICOR donkey anti-rabbit 680 or

donkey anti-mouse 800 secondary antibody (1:50000; Invitrogen) in TBS at room temperature after

which they were washed and imaged using the Gel Doc XR imaging system (Bio- Rad).

Alternatively, blots were probed with appropriate species-specific HRP-conjugated secondary

antibodies (Jackson ImmunoResearch) and visualized with either Supersignal West Femto

Sensitivity Substrate (Pierce) or Clarity™ Western ECL substrate (Bio-Rad) using a Fujifilm

ImageQuant™ LAS-4000 (Fuji Corporation). For stripping and re-probing, western blot

membranes were treated with 10 ml of Restore™ PLUS Western blot stripping buffer (Thermo

Fisher) for 30 min at room temperature on a orbital shaker, washed three times with TBST and

blocked with either 5% skim milk powder or 3% BSA in TBST for 1 h. The membranes were then

incubated with appropriate primary and secondary antibodies as described above.

Statistical analyses

N represents the number of individual animals; n represents the number of individual cell culture

experiments, each with 4 replicates. Stereology, rotarod and weight measurements, p75ECD

analysis and, for key experiments, the motor neuron culture counts were conducted by researchers

blinded to the treatment conditions. Densitometry analysis of relative protein levels on western

blots was performed using Image Studio Digits (Licor), Image Lab (Bio-Rad) and Fiji (Image J,

NIH). All representative graphs measuring relative densitometry of proteins on western blots were

calculated from three independent experiments, with bands normalised to loading controls (tubulin

or actin) probed and exposed at the same time. Statistical analysis was performed using GraphPad

Prism (GraphPad Software) as described in each method. p < 0.05 was considered statistically

significant.

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Results

c29 prevents the death of axotomized ulnar motor neurons and delays disease progression of

SOD1G93A-induced motor neuron disease.

It is clear that a balance between Trk receptor-mediated survival and p75NTR-mediated cell death-

signaling pathways regulate motor neuron survival both during development and following injury

or disease. In particular, the role of these competing neurotrophin signaling pathways has been well

studied in motor neurons following axotomy and in degenerative conditions including amyotrophic

lateral sclerosis. Therefore, to determine if c29 was capable of inhibiting motor neuron cell death in

vivo, we first used the injury paradigm of ulnar nerve axotomy model in which 50% of the

ipsilateral motor neurons typically die, and where both down-regulation of p75NTR by antisense

oligonucleotides (Lowry et al., 2001) and BDNF application (Yuan et al., 2000) have been shown to

promote motor neuron survival. Following ulnar nerve axotomy of newborn rat pups, c29 or control

peptide was applied to the cut nerve stump in gel foam. After 5 days, the number of live motor

neurons was determined by stereological counts following histological processing of the spinal

cords. Axotomy resulted in a loss of ~45% of ChAT-positive motor neurons in the ipsilateral

cervical enlargement (C7-C8) in the control peptide treated axotomized animals compared to the

contralateral side (Fig. 1A,B). In contrast, the number of surviving motor neurons in the spinal cord

of animals treated with c29 was significantly higher, with the average neuronal loss being less than

15% that of the contralateral side (Fig. 1A,B).

As PTD-fused peptides and proteins given to mice systemically have previously been shown to

cross the blood brain barrier (Schwarze et al., 2000), we confirmed that biotinylated-c29 peptide

reached the spinal neurons after intraperitoneal injection of wildtype mice. In mice sacrificed one

hour after 5 mg/kg peptide injection, immunostaining for biotin was observed in grey matter of the

spinal cord in c29-treated animals but not in controls (non-biotinylated peptide-treated) animals

(Fig. 2A,B). Next, we tested the effect of systemic c29 treatment in the SOD1G93A mouse model of

amyotrophic lateral sclerosis. c29 was administered chronically (5 mg/kg/day) to the mice from 9

weeks of age via a subcutaneous osmotic minipump, and the course of disease was monitored. No

significant difference between c29-treated mice and vehicle-treated animals was observed in terms

of lifespan (Fig. 2C). However, c29-treated mice had a delayed disease onset, taking significantly

longer to lose 10% of their body weight (p<0.01; median SOD1+ vehicle: 31 days; SOD1+c29: 42

days; Fig. 2D) and c29-treated animals maintained their performance on the rotarod between 100

and 125 days, the time frame during which the performance of the vehicle-treated animals declined

dramatically (Fig. 2E). Furthermore, at this time, the number of motor neurons in the spinal cord of

c29-treated SOD1G93A animals was significantly higher than that in vehicle-treated animals (Fig. Jour

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2F,G), and fewer cells appeared atrophic (Fig. 2G).

p75NTR cleavage associates with neurotrophin-induced cell death in vivo and in vitro.

As upregulation of p75NTR expression, proteolysis and death signaling are associated with motor

neuron degeneration, we asked whether the reduced neuronal loss was associated with altered

p75NTR cleavage/expression by measuring the levels of p75NTR ECD present in the urine of

SOD1G93A animals over the course of the disease. In SOD1G93A animals, the level of p75NTR ECD

present in urine was significantly higher than in C57BL/6J (wildtype) healthy controls

presymptomatically from 60 days of age and throughout disease progression (Fig. 3A). However, in

SOD1G93A animals treated with c29, the level of p75NTR ECD present in the urine between 80 and

120 days of age was significantly lower than that found in the urine of vehicle-treated animals (Fig.

3B). Together, these results indicate that c29 can potentiate the survival of injured or dying motor

neurons in vivo, which corresponds to lowered p75NTR expression/cleavage.

To determine whether cleavage of p75NTR is important for neurotrophin-mediated motor neuron

death, we asked whether neurotrophins stimulate the cleavage of p75NTR using the motor neuron-

like NSC-34 cell line. As previously reported for other cell types, treatment of cultures with PMA,

which activates metalloproteases (Ni et al., 2001), stimulated the cleavage of p75NTR, resulting

predominantly in the generation of its intracellular domain fragment. This cleavage could be

prevented with the metalloprotease inhibitor TAPI-2, whereas treatment of cells with the -secretase

inhibitor Compound E together with PMA resulted in accumulation of the C-terminal p75NTR

fragment (Fig. 3C; Jung et al., 2003; Kanning et al., 2003; Zampieri et al., 2005). Treatment of

NSC-34 cells with BDNF or proBDNF produced predominantly intracellular domain fragments of

p75NTR whereas either NGF or proNGF treatment predominantly resulted in generation of the C-

terminal fragment (Fig. 3C).

The ability of NGF and the proneurotrophins to induce p75NTR-mediated cell death in the presence

of low concentrations of the survival factor BDNF was then tested. Treatment of E13 TrkB-

dependent motor neuron cultures with NGF (Fig. 4A,D) or proNGF (Fig. 4C), resulted in

significant neuronal death over 48 h, whereas proBDNF treatment had no effect (Fig. 4C). NGF-

induced death was prevented by treatment with the p75NTR cleavage inhibitor TAPI-2 (Fig. 4A)

Together these results suggest that cleavage of p75NTR resulting in accumulation of the C-terminal

fragment is associated with death signaling in motor neurons.

c29 promotes survival of BDNF-maintained neurons

We next asked whether c29 inhibits the (pro)NGF-induced death of motor neurons. Co-treatment of

motor neuron cultures with c29 at the time of proNGF or NGF addition resulted in significantly Jour

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reduced cell death of cultures grown in BDNF (Fig. 4B-D). The scrambled control peptide had no

significant effect on either cell death or survival, and c29 had no effect on proBDNF-treated motor

neurons (Fig. 4C).

To determine whether c29 was acting by affecting p75NTR cleavage, NSC-34 cells were treated with

NGF or proNGF together with c29 or scrambled control peptide. No difference in the rate of p75NTR

cleavage was observed in cells treated with the peptides (Fig. 4E), indicating that c29 was more

likely to be affecting the balance of survival signaling downstream of p75NTR activation.

To determine whether c29 was acting to prevent p75NTR-mediated death signaling or promote TrkB-

mediated signaling, neurons cultured in CNTF or GDNF instead of BDNF were treated with NGF,

which also resulted in a 40-50% reduction in survival, similar to that induced in BDNF conditions.

The cleavage inhibitor TAPI-2 significantly inhibited NGF-induced cell death in CNTF- and

GDNF-supported cultures (Fig. 4F,G). However, c29 did not inhibit the NGF-induced death of

CNTF- or GDNF-dependent neurons (Fig. 4F,G). These results demonstrate that NGF-induced

death signaling relies on a cleaved form of p75NTR, that its death signaling is independent of TrkB

signaling, and, finally, that c29 does not appear to act in a dominant-negative manner in vitro to

inhibit p75NTR signaling, but instead acts by promoting neuronal survival only in the presence of

BDNF.

c29 protects motor neurons from BDNF, but not CNTF or GDNF withdrawal.

We next tested whether c29 could potentiate BDNF survival signaling in conditions of growth

factor withdrawal. Neurons plated and maintained in reduced BDNF concentrations (0.1 or

0.01 ng/ml) and treated with c29 had increased rates of survival compared to control peptide-treated

cultures (Fig. 5A). Similarly, in motor neuron cultures undergoing BDNF withdrawal (cultures

changed to 0.1 ng/ml BDNF-containing medium following initial plating in 1ng/ml BDNF),

treatment with c29 resulted in significantly increased neuronal survival compared to that of cultures

treated with control peptide, with survival at levels greater than or equal to that of cultures grown in

10-fold higher BDNF concentrations (Fig. 5B). c29 treatment had no survival effect in the absence

of any exogenous growth factor (Fig. 5A), nor any effect on the survival of motor neurons

undergoing CNTF (Fig. 5C) or GDNF withdrawal (Fig. 5D). The scrambled control peptide again

had no significant effect on either cell death or survival. These results indicate that c29 has no

intrinsic cell survival-inducing properties, but rather that coincident BDNF-TrkB receptor-mediated

signaling is required for c29 to promote neuronal survival in these conditions.

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c29 does not regulate p75NTR cleavage but interacts with and promotes TrkB survival signals.

To determine whether the c29 sequence was able to interact with TrkB (in addition to TrkA;

Matusica et al., 2013) and thereby potentially affect activation of TrkB signals, we performed pull-

down experiments. Biotinylated c29 peptide, but not a control biotinylated peptide (LC1), was able

to pull down overexpressed TrkB from lysates of transfected HEK293 cells (Fig. 6A), as well as

endogenously expressed TrkB from lysates of NSC-34 cells (Fig. 6B). These results indicate that

the 29-amino acid intracellular juxtamembrane sequence is sufficient for p75NTR to interact with

TrkB.

We next investigated whether c29 was potentiating survival signals by examining the activation of

Erk1/2, and Akt in NSC-34 cells treated with BDNF for various periods of time (0-90 min). We

found that c29 treatment resulted in a higher level of peak activation at all times measured for both

Erk1/2 (Fig. 6C) and Akt (Fig. 6D). As Erk1/2 is regulated not only by BDNF (McCarty and

Feinstein, 1999), but also by CNTF (Frebel and Wiese, 2006) and GDNF (Chiariello et al., 1998;

Trupp et al., 1999) following activation of their specific receptors, we asked whether this response

was specific for TrkB-mediated signaling. Analysis of activated Erk1/2 in NSC-34 cells treated

with these growth factors for 15 minutes demonstrated that although BDNF-induced

phosphorylation of Erk1/2 was significantly enhanced in the presence of c29, c29 treatment of

CNTF- or GDNF-supported cultures had no effect on the level of phosphorylated Erk1/2, which

was equivalent to that of cultures treated with scrambled control peptide (Fig. 6E). These results

indicate that, in conditions where growth factors are limited, c29 enhances survival signaling and

thus neuronal survival in a BDNF/TrkB-dependent manner.

Finally, to establish whether c29 could activate BDNF-mediated survival signaling within the spinal

cords of SOD1G93A mice, 16 week-old SOD1G93A animals were treated acutely with c29 or control

peptide. SOD1G93A mice treated with the control peptide had very low levels of phosphorylated

TrkB (Fig. 7A) Erk1/2 and Akt (Fig. 7B) and CREB (Fig. 7C), in comparison to the levels in

lysates from wildtype animals. However, the activity of these survival-signaling proteins was

significantly increased by acute c29 treatment in comparison to that in control peptide-treated

animals (Fig. 7A-C). Conversely levels of cleaved (activated) caspase 3 in SOD1 spinal cord

lysates were reduced by c29 treatment compared to the control treatment (Fig. 7D). Together these

findings indicate that c29 can enhance neuronal survival in a BDNF/TrkB-dependent manner in

vitro and in diseased motor neurons in vivo.

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Discussion

In this study we demonstrated that the c29 peptide, derived from the intracellular juxtamembrane

domain of p75NTR, can promote neuronal survival in motor neurons in vitro, as well as following

axotomy and in disease conditions in vivo. Although c29 treatment reduced the amount of the ECD

fragment of p75NTR in vivo, and motor neuron cell death induced by neurotrophins in vitro was

accompanied by increased cleavage of p75NTR, c29 did not directly affect p75NTR cleavage in vitro.

Rather, it interacted with TrkB and acted in a BDNF-dependent manner to promote survival

signaling, without directly inhibiting NGF-induced p75NTR death signaling.

NGF triggers an active motor neuron death pathway mediated by the p75NTR C-terminal

fragment.

p75NTR is well known to mediate neuronal death in conditions of injury and disease, typically

triggered by p75NTR ligands that do not bind to co-expressed Trk receptors (Ibanez and Simi, 2012),

i.e. in motor neurons, NGF or proNGF elicit p75NTR- mediated death signals (Henderson et al.,

1998; Sedel et al., 1999; Wiese et al., 1999; Sendtner et al., 2000). There is also emerging evidence

that regulated intramembrane proteolysis may be important for p75NTR functions, including

induction of cell death signaling pathways and promotion of cell survival in vitro, although reports

of ligand activation of p75NTR cleavage are inconsistent (Kenchappa et al., 2006; Skeldal et al.,

2011; Vicario et al., 2015). Our in vitro experiments measuring cleavage of endogenous p75NTR in

response to exogenous ligand application established that cell death ligands increase the production

of the C-terminal fragment, which is associated with increased death signaling (Coulson et al.,

1999; Underwood et al., 2008; Vicario et al., 2015), whereas cell survival ligands result in great

production of the intracellular domain fragment, consistent with Trk-mediated cleavage of p75NTR

facilitating survival via this fragment (Ceni et al., 2010; Kommaddi et al., 2011; Matusica et al.,

2013).

In vitro, motor neuron death was induced by activating p75NTR by either NGF or proNGF in the

presence of BDNF survival signaling, consistent with previous reports that survival or death is

regulated by the strength of competing signaling pathways (Song et al., 2010). However, NGF-

induced cell death ensued even in the absence of Trk activity, i.e. in cultures maintained in GDNF

or CNTF, indicating that NGF triggers an active cell death pathway which is not simply due to a

passive loss of growth factor support due to sequestration of p75NTR away from a BDNF-TrkB high

affinity complex by NGF, as previously suggested (Davies et al., 1993; Sendtner et al., 2000; Vesa

et al., 2000). In addition, ligand-dependent death mediated by p75NTR in any of the growth factor

conditions tested was blocked by inhibiting p75NTR cleavage, indicating that cleavage and ligand

were equally important in inducing death signaling. Jour

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c29 promotes motor neuron survival in vivo

Our experiments aimed to test the effect of the c29 peptide in vivo in conditions in which p75NTR

death signaling were active, namely following axotomy and in the SOD1G93A model of amyotrophic

lateral sclerosis. Notably, both of these conditions trigger increased cleavage of p75NTR (DiStefano

et al., 1993; Perlson et al., 2009), and we observed increased p75NTR ECD levels with SOD1G93A

disease progression, as previously observed in human amyotrophic lateral sclerosis (Shepheard et

al., 2014). As a result of c29 treatment, we observed a significant improvement in motor neuron

survival after axotomy and in SOD1G93A animals, and in the latter model, reduced levels of p75NTR

ECD and a coincident improvement in rotarod performance and a delay in disease-induced weight

loss, suggesting that the functional outcomes could be the result of reduced p75NTR activation and

cell death signaling. However, the observed reduction in the level of p75NTR ECD may simply

reflect a slower disease progression. Although p75NTR expression and the generation of the cleavage

fragment are upregulated in motor neurons coincident with axonal degeneration, p75NTR is also

upregulated in oligodendrocytes, Schwann cells and microglia following nerve injury (reviewed in

Meeker and Williams, 2014). Therefore, it is not clear from our experiments whether the p75NTR

ECD is derived from motor neurons and/or other cell types and whether it reflects basal levels of

p75NTR cleavage accompanying broader expression, or increased cleavage specifically reflecting

activation of p75NTR degenerative processes. The reduction in the level of p75NTR ECD afforded by

c29 treatment is therefore difficult to interpret in isolation.

Although we cannot rule out that possibility that c29 acted to prevent p75NTR death signaling in

vivo, our in vitro experiments strongly indicate that it did not directly affect the rate of p75NTR

cleavage or block p75NTR death signaling. Firstly, no effect on the rate of NGF or proNGF-induced

cleavage of p75NTR was observed following treatment of cultures with c29, nor did c29 treatment

affect the rate of NGF-induced death of neurons cultured in the absence of BDNF, even though

p75NTR cleavage inhibitors promoted neuronal survival in the same conditions. Rather, our data

suggest that c29 treatment, by keeping motor neurons alive or healthier for longer (as discussed

below), indirectly affected other disease mechanisms, including reducing the upregulation/cleavage

of p75NTR. Therefore, the specific process being measured by quantifying p75NTR ECD levels

remains unclear. Nonetheless, as p75NTR ECD levels increased with disease progression and c29

treatment resulted in reduced steady-state levels of p75NTR ECD in mid disease, we suggest further

validation of this fragment as a biomarker of motor neuron disease severity is warranted.

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c29 promotes motor neuron survival in vitro but requires coincident TrkB activation

Although we hypothesized that c29 might act to block p75NTR death signaling, our results

consistently demonstrated that c29 only promotes motor neuron survival when coincident with

TrkB activation in vitro. Specifically: (i) c29 overcame NGF-induced death signaling in neurons

cultured in BDNF but not in neurons cultured in CNTF; (ii) in low concentrations of BDNF that

would not otherwise support motor neuron survival, co-treatment of cultures with c29 promoted

rates of survival at or above that of cultures in 10-fold higher BDNF concentrations, but had no

effect on cultures grown in low concentrations of CNTF or GDNF; and (iii) c29 treatment

facilitated both increased activation of and longer-lived survival signaling in motor neuron-like

NSC-34 cells, but only in cultures grown in the presence of BDNF. This conclusion is consistent

with our in vivo findings.

In the axotomy paradigm, an exogenous supply of BDNF is reported to promote neuronal survival

(Sendtner et al., 1992; Boyd and Gordon, 2001; Murray et al., 2010). Thus, given that at least low

endogenous levels of neurotrophins are present at the axotomized cell bodies, c29 could have acted

by enhancing the survival signaling of locally produced BDNF. Similarly, previous work has

indicated that BDNF is upregulated in neurons, glia and immune cells within the spinal cord

following injury (Li et al., 2006; Li et al., 2009; Xin et al., 2012), which may have allowed c29 to

act in motor neurons. Indeed, acute treatment of SOD1G93A animals with c29 resulted in enhanced

phosphorylation of TrkB, Akt, Erk1/2 and CREB within the spinal cord coincident with reduced

levels of cleaved caspase-3, which was not induced by control peptide treatment. Our current results

therefore provide strong evidence to support the idea that c29 enhances BDNF-induced TrkB

survival signaling both in vitro and in vivo which in turn blocks death signals.

Role of the p75NTR juxtamembrane (c29) region in Trk signaling

One characterized role of p75NTR is its ability to increase ligand-binding affinity for the TrkA

receptor and increase neuronal survival (Hempstead et al., 1991; Lee et al., 1994; Horton et al.,

1997; Esposito et al., 2001), and it is widely accepted that p75NTR also interacts with other Trk

receptors with equivalent effect (Hempstead et al., 1991; Davies et al., 1993; Huber and Chao,

1995; Bibel et al., 1999; Vesa et al., 2000; Ceni et al., 2010; Kommaddi et al., 2011). Although the

precise mechanism responsible for this outcome remains elusive (Barker, 2007), the transmembrane

and juxtamembrane regions of p75NTR have been implicated in this interaction (Hempstead et al.,

1991; Bibel et al., 1999; Esposito et al., 2001; Iacaruso et al., 2011), and more recent studies also

suggest that cleavage of p75NTR plays an important role in this process (Ceni et al., 2010;

Kommaddi et al., 2011; Matusica et al., 2013). Our observation that a soluble peptide mimicking

the juxtamembrane region of p75NTR is able to enhance the function of TrkA (Matusica et al., 2013) Jour

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and TrkB (herein) accords with the previous studies. In particular, the high conservation of the

juxtamembrane region of p75NTR supports the idea that this domain is essential for p75NTR function

(Hutson and Bothwell, 2001). We have previously shown its importance in p75NTR-mediated death

signaling when attached to the membrane as well as demonstrating that the function of this domain

differs following intramembranous cleavage (Coulson et al., 2000; Matusica et al., 2013). i.e.

cleavage of p75NTR to its intracellular domain fragment is a requirement for enhancing NGF affinity

for TrkA in vitro, and this can be reproduced using the c29 peptide (Matusica et al., 2013).

Proposed mechanism of c29 action

As the current findings using c29 in vitro mirror our recent work in TrkA-dependent neurons

(Matusica et al., 2013), we propose that c29 interacts with all Trk receptors to facilitate ligand

binding. Increased numbers of ligand-bound TrkB receptors per cell allow activation of

significantly more receptors than are normally activated in equivalent concentrations of

neurotrophins (unless the concentrations were already saturating). Thus the threshold for BDNF

survival/trophic signaling per neuron would be reached sooner and/or be higher in the presence of

c29. In the context of our experiments, the resulting increased TrkB survival signaling was

sufficient to promote survival, including overriding coincident p75NTR-mediated death signals.

Although recent work has suggested that TrkB expression causes a more severe SOD1 disease

phenotype (Zhai et al., 2011), it appears that this deleterious effect is mediated by the truncated

TrkB isoform T1 (Yanpallewar et al., 2012). TrkB.T1 lacks the intracellular kinase domain and,

unlike full-length TrkB, is upregulated in motor neurons in SOD1 animals (Zhang and Huang,

2006). Thus the motor neuron survival-promoting effect of c29 in SOD1 animals can be explained

by selective enhancement of full-length TrkB trophic signaling, which can counteract p75NTR-

induced death (Casaccia-Bonnefil et al., 1996; Yoon et al., 1998; Friedman, 2000).

Alternatively or additionally, c29 could also have acted in vivo to promote motor neuron survival

via TrkC (or TrkA), which is upregulated in the longer-lived motor neurons in motor neuron disease

patients (Duberley et al., 1997; Nishio et al., 1998). Unlike BDNF, exogenous delivery of the TrkC

ligand NT-3 has shown promise in slowing disease progression and maintaining motor neuron–

muscle connectivity in a motor neuron disease mouse model (specifically the pmn mouse; (Haase et

al., 1998). Given that c29 acts in a similar fashion in vitro to promote both TrkA and TrkB signals,

it would be surprising if c29 did not affect TrkC in the same manner. Thus, the ability of c29 to

promote motor neuron survival in vivo can be explained by our in vitro findings that Trk receptor

function (TrkB and/or TrkC) is enhanced in neurons in conditions of low-level neurotrophin

exposure or activation of p75NTR death signaling. Even though c29 treatment did not alter the

lifespan of the aggressive SOD1G93A model mice, it remains to be determined whether earlier Jour

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treatment (e.g. prior to when p75NTR cleavage is first evident), a different dose, a more biostable

mimetic, or a targeted treatment route of c29, either alone or in combination with other candidate

therapeutics, would provide any additional benefit to animals undergoing motor neuron

degeneration.

In summary, our results show that regulated intramembrane proteolysis of p75NTR plays an

important role in (pro)NGF-mediated motor neuron death, which can be induced even in the

presence of coincident survival signals. We also demonstrate that a peptide mimetic of the p75NTR

intracellular domain (c29), that interacts with TrkB receptors, potentiates the survival of motor

neurons in a BDNF- and TrkB-dependent manner, under conditions of growth factor withdrawal as

well as active ligand killing, by redressing the neurotrophin signaling imbalance in favor of trophic

support.

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References:

Barker, P. A. (2007). High Affinity Not in the Vicinity? Neuron 53, 1-4.

Bibel, M., Hoppe, E. and Barde, Y. A. (1999). Biochemical and functional interactions between

the neurotrophin receptors trk and p75NTR. EMBO J 18, 616-622.

Blochl, A. and Blochl, R. (2007). A cell-biological model of p75NTR signaling. J Neurochem 102,

289-305.

Boyd, J. G. and Gordon, T. (2001). The neurotrophin receptors, trkB and p75, differentially

regulate motor axonal regeneration. J Neurobiol 49, 314-325.

Branco, F., Cardenas, D. D. and Svircev, J. N. (2007). Spinal cord injury: a comprehensive

review. Phys Med Rehabil Clin N Am 18, 651-679, v.

Casaccia-Bonnefil, P., Carter, B. D., Dobrowsky, R. T. and Chao, M. V. (1996). Death of

oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature

383, 716-719.

Cashman, N. R., Durham, H. D., Blusztajn, J. K., Oda, K., Tabira, T., Shaw, I. T., Dahrouge,

S. and Antel, J. P. (1992). Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble

developing motor neurons. Dev Dyn 194, 209-221.

Ceni, C., Kommaddi, R. P., Thomas, R., Vereker, E., Liu, X., McPherson, P. S., Ritter, B. and

Barker, P. A. (2010). The p75NTR intracellular domain generated by neurotrophin-induced receptor

cleavage potentiates Trk signaling. J Cell Sci 123, 2299-2307.

Cheema, S. S., Barrett, G. L. and Bartlett, P. F. (1996). Reducing p75 nerve growth factor

receptor levels using antisense oligonucleotides prevents the loss of axotomized sensory neurons in

the dorsal root ganglia of newborn rats. J Neurosci Res 46, 239-245.

Chiariello, M., Visconti, R., Carlomagno, F., Melillo, R. M., Bucci, C., de Franciscis, V., Fox,

G. M., Jing, S., Coso, O. A., Gutkind, J. S. et al. (1998). Signalling of the Ret receptor tyrosine

kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the

ERKs and JNKs pathways induced by Ret. Oncogene 16, 2435-2445.

Coulson, E. J., Reid, K. and Bartlett, P. F. (1999). Signaling of neuronal cell death by the

p75NTR neurotrophin receptor. Molr Neurobiol 20, 29-44.

Coulson, E. J., Reid, K., Baca, M., Shipham, K. A., Hulett, S. M., Kilpatrick, T. J. and

Bartlett, P. F. (2000). Chopper, a new death domain of the p75 neurotrophin receptor that mediates

rapid neuronal cell death. J Biol Chem 275, 30537-30545.

Davies, A. M., Lee, K. F. and Jaenisch, R. (1993). p75-deficient trigeminal sensory neurons have

an altered response to NGF but not to other neurotrophins. Neuron 11, 565-574.

Dechant, G. and Barde, Y. A. (2002). The neurotrophin receptor p75NTR: novel functions and

implications for diseases of the nervous system. Nat Neurosci 5, 1131-1136.

DiStefano, P. S., Chelsea, D. M., Schick, C. M. and McKelvy, J. F. (1993). Involvement of a

metalloprotease in low-affinity nerve growth factor receptor truncation: inhibition of truncation in

vitro and in vivo. J Neurosci 13, 2405-2414.

Domeniconi, M., Hempstead, B. L. and Chao, M. V. (2006). Pro-NGF secreted by astrocytes

promotes motor neuron cell death. Mol Cell Neurosci.

Duberley, R. M., Johnson, I. P., Anand, P., Leigh, P. N. and Cairns, N. J. (1997). Neurotrophin-

3-like immunoreactivity and Trk C expression in human spinal motoneurones in amyotrophic

lateral sclerosis. J Neurol Sci 148, 33-40.

Ernfors, P. (2001). Local and target-derived actions of neurotrophins during peripheral nervous

system development. Cell Mol Life Sci 58, 1036-1044.

Esposito, D., Patel, P., Stephens, R. M., Perez, P., Chao, M. V., Kaplan, D. R. and Hempstead,

B. L. (2001). The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate

high affinity binding to nerve growth factor. J Biol Chem 276, 32687-32695.

Frebel, K. and Wiese, S. (2006). Signalling molecules essential for neuronal survival and

differentiation. Biochem Soc Trans 34, 1287-1290.

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Friedman, W. J. (2000). Neurotrophins induce death of hippocampal neurons via the p75 receptor.

J Neurosci 20, 6340-6346.

Haase, G., Pettmann, B., Vigne, E., Castelnau-Ptakhine, L., Schmalbruch, H. and Kahn, A. (1998). Adenovirus-mediated transfer of the neurotrophin-3 gene into skeletal muscle of pmn mice:

therapeutic effects and mechanisms of action. J Neurol Sci 160 Suppl 1, S97-105.

Hempstead, B. L., Martin-Zanca, D., Kaplan, D. R., Parada, L. F. and Chao, M. V. (1991).

High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity

NGF receptor. Nature 350, 678-683.

Henderson, C. E., Yamamoto, Y., Livet, J., Arce, V., Garces, A. and deLapeyriere, O. (1998).

Role of neurotrophic factors in motoneuron development. J Physiol Paris 92, 279-281.

Henriques, A., Pitzer, C. and Schneider, A. (2010). Neurotrophic growth factors for the treatment

of amyotrophic lateral sclerosis: where do we stand? Front Neurosci 4, 32.

Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G. and Dowdy, S. F. (2001). Synthetic

protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 61,

474-477.

Horton, A., Laramee, G., Wyatt, S., Shih, A., Winslow, J. and Davies, A. M. (1997). NGF

binding to p75 enhances the sensitivity of sensory and sympathetic neurons to NGF at different

stages of development. Mol Cell Neurosci 10, 162-172.

Huber, L. J. and Chao, M. V. (1995). A potential interaction of p75 and trkA NGF receptors

revealed by affinity crosslinking and immunoprecipitation. J Neurosci Res 40, 557-563.

Hutson, L. D. and Bothwell, M. (2001). Expression and function of Xenopus laevis p75NTR

suggest evolution of developmental regulatory mechanisms. J Neurobiol 49, 79-98.

Iacaruso, M. F., Galli, S., Marti, M., Villalta, J. I., Estrin, D., Jares-Erijman, E. A. and

Pietrasanta, L. I. (2011). Structural Model for p75NTR-TrkA Intracellular Domain Interaction: A

Combined FRET and Bioinformatics Study. J Mol Biol. 414, 681-98

Ibanez, C. F. and Simi, A. (2012). p75 neurotrophin receptor signaling in nervous system injury

and degeneration: paradox and opportunity. Trends Neurosci 35, 431-440.

Jung, K. M., Tan, S., Landman, N., Petrova, K., Murray, S., Lewis, R., Kim, P. K., Kim, D. S.,

Ryu, S. H., Chao, M. V. et al. (2003). Regulated intramembrane proteolysis of the p75

neurotrophin receptor modulates its association with the TrkA receptor. J Biol Chem 278, 42161-

42169.

Kanning, K. C., Hudson, M., Amieux, P. S., Wiley, J. C., Bothwell, M. and Schecterson, L. C. (2003). Proteolytic processing of the p75 neurotrophin receptor and two homologs generates C-

terminal fragments with signaling capability. J Neurosci 23, 5425-5436.

Kaplan, D. R. and Miller, F. D. (2000). Neurotrophin signal transduction in the nervous system.

Curr Opin Neurobiol 10, 381-391.

Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L.

and Carter, B. D. (2006). Ligand-dependent cleavage of the P75 neurotrophin receptor is

necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons. Neuron 50, 219-

232.

Kommaddi, R. P., Thomas, R., Ceni, C., Daigneault, K. and Barker, P. A. (2011). Trk-

dependent ADAM17 activation facilitates neurotrophin survival signaling. FASEB J 25, 2061-2070.

Lee, K. F., Davies, A. M. and Jaenisch, R. (1994). p75-deficient embryonic dorsal root sensory

and neonatal sympathetic neurons display a decreased sensitivity to NGF. Development 120, 1027-

1033.

Li, F., Li, L., Song, X. Y., Zhong, J. H., Luo, X. G., Xian, C. J. and Zhou, X. F. (2009).

Preconditioning selective ventral root injury promotes plasticity of ascending sensory neurons in the

injured spinal cord of adult rats--possible roles of brain-derived neurotrophic factor, TrkB and p75

neurotrophin receptor. Eur J Neurosci 30, 1280-1296.

Li, L., Xian, C. J., Zhong, J. H. and Zhou, X. F. (2006). Upregulation of brain-derived

neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats. Neurotox

Res 9, 269-283. Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Lowry, K. S., Murray, S. S., McLean, C. A., Talman, P., Mathers, S., Lopes, E. C. and

Cheema, S. S. (2001). A potential role for the p75 low-affinity neurotrophin receptor in spinal

motor neuron degeneration in murine and human amyotrophic lateral sclerosis. Amyotroph Lateral

Scler Other Motor Neuron Disord 2, 127-134.

Matusica, D., Skeldal, S., Sykes, A. M., Palstra, N., Sharma, A. and Coulson, E. J. (2013). An

intracellular domain fragment of the p75 neurotrophin receptor (p75NTR) enhances tropomyosin

receptor kinase A (TrkA) receptor function. J Biol Chem 288, 11144-11154.

McCarty, J. H. and Feinstein, S. C. (1999). The TrkB receptor tyrosine kinase regulates cellular

proliferation via signal transduction pathways involving SHC, PLC, and CBL. J Recept Signal

Transduct Res 19, 953-974.

Meeker, R. and Williams, K. (2014). Dynamic nature of the p75 neurotrophin receptor in response

to injury and disease. J Neuroimmune Pharmacol 9, 615-628.

Murray, L. M., Talbot, K. and Gillingwater, T. H. (2010). Review: neuromuscular synaptic

vulnerability in motor neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy.

Neuropathol Appl Neurobiol 36, 133-156.

Murray, S. S., Bartlett, P. F., Lopes, E. C., Coulson, E. J., Greferath, U. and Cheema, S. S. (2003). Low-affinity neurotrophin receptor with targeted mutation of exon 3 is capable of mediating

the death of axotomized neurons. Clin Exp Pharm & Physiol 30, 217-222.

Ni, C. Y., Murphy, M. P., Golde, T. E. and Carpenter, G. (2001). -Secretase cleavage and

nuclear localization of ErbB-4 receptor tyrosine kinase. Science 294, 2179-2181.

Nishio, T., Sunohara, N. and Furukawa, S. (1998). Neutrophin switching in spinal motoneurons

of amyotrophic lateral sclerosis. Neuroreport 9, 1661-1665.

Pehar, M., Cassina, P., Vargas, M. R., Castellanos, R., Viera, L., Beckman, J. S., Estevez, A.

G. and Barbeito, L. (2004). Astrocytic production of nerve growth factor in motor neuron

apoptosis: implications for amyotrophic lateral sclerosis. J Neurochem 89, 464-473.

Perlson, E., Jeong, G. B., Ross, J. L., Dixit, R., Wallace, K. E., Kalb, R. G. and Holzbaur, E. L. (2009). A switch in retrograde signaling from survival to stress in rapid-onset neurodegeneration. J

Neurosci 29, 9903-9917.

Rogers, M. L., Atmosukarto, I., Berhanu, D. A., Matusica, D., Macardle, P. and Rush, R. A. (2006). Functional monoclonal antibodies to p75 neurotrophin receptor raised in knockout mice. J

Neurosci Methods 158, 109-120.

Roux, P. P. and Barker, P. A. (2002). Neurotrophin signaling through the p75 neurotrophin

receptor. Prog Neurobiol 67, 203-233.

Schwarze, S. R., Hruska, K. A. and Dowdy, S. F. (2000). Protein transduction: unrestricted

delivery into all cells? Trends Cell Biol 10, 290-295.

Sedel, F., Bechade, C. and Triller, A. (1999). Nerve growth factor (NGF) induces motoneuron

apoptosis in rat embryonic spinal cord in vitro. Eur J Neurosci 11, 3904-3912.

Seeburger, J. L., Tarras, S., Natter, H. and Springer, J. E. (1993). Spinal cord motoneurons

express p75NGFR and p145trkB mRNA in amyotrophic lateral sclerosis. Brain research 621, 111-

115.

Sendtner, M., Holtmann, B., Kolbeck, R., Thoenen, H. and Barde, Y. A. (1992). Brain-derived

neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature

360, 757-759.

Sendtner, M., Pei, G., Beck, M., Schweizer, U. and Wiese, S. (2000). Developmental motoneuron

cell death and neurotrophic factors. Cell Tissue Res 301, 71-84.

Shepheard, S. R., Chataway, T., Schultz, D. W., Rush, R. A. and Rogers, M. L. (2014). The

extracellular domain of neurotrophin receptor p75 as a candidate biomarker for amyotrophic lateral

sclerosis. PloS One 9, e87398.

Skeldal, S., Matusica, D., Nykjaer, A. and Coulson, E. J. (2011). Proteolytic processing of the

p75 neurotrophin receptor: A prerequisite for signalling?: Neuronal life, growth and death

signalling are crucially regulated by intra-membrane proteolysis and trafficking of p75NTR.

Bioessays. 33, 614-25 Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Song, W., Volosin, M., Cragnolini, A. B., Hempstead, B. L. and Friedman, W. J. (2010).

ProNGF induces PTEN via p75NTR to suppress Trk-mediated survival signaling in brain neurons.

J Neurosci 30, 15608-15615.

Tian, G., Sobotka-Briner, C. D., Zysk, J., Liu, X., Birr, C., Sylvester, M. A., Edwards, P. D.,

Scott, C. D. and Greenberg, B. D. (2002). Linear non-competitive inhibition of solubilized human

gamma-secretase by pepstatin A methylester, L685458, sulfonamides, and benzodiazepines. J Biol

Chem 277, 31499-31505.

Trupp, M., Scott, R., Whittemore, S. R. and Ibanez, C. F. (1999). Ret-dependent and -

independent mechanisms of glial cell line-derived neurotrophic factor signaling in neuronal cells. J

Biol Chem 274, 20885-20894.

Underwood, C. K. and Coulson, E. J. (2007). The p75 neurotrophin receptor. Int J Biochem Cell

Biol. 40, 1664-1668

Underwood, C. K., Reid, K., May, L. M., Bartlett, P. F. and Coulson, E. J. (2008).

Palmitoylation of the C-terminal fragment of p75NTR regulates death signaling and is required for

subsequent cleavage by gamma-secretase. Mol Cell Neurosci 37, 346-358.

Vesa, J., Kruttgen, A. and Shooter, E. M. (2000). p75 reduces TrkB tyrosine autophosphorylation

in response to brain-derived neurotrophic factor and neurotrophin 4/5. J Biol Chem 275, 24414-

24420.

Vicario, A., Kisiswa, L., Tann, J. Y., Kelly, C. E. and Ibanez, C. F. (2015). Neuron-type-specific

signaling by the p75NTR death receptor regulated by differential proteolytic cleavage. J Cell Sci.

128, 1507-17

Wiese, S., Metzger, F., Holtmann, B. and Sendtner, M. (1999). The role of p75NTR in

modulating neurotrophin survival effects in developing motoneurons. Eur J Neurosci 11, 1668-

1676.

Wiese, S., Beck, M., Karch, C. and Sendtner, M. (2004). Signalling mechanisms for survival of

lesioned motoneurons. Acta Neurochirurgica 89, 21-35.

Wiese, S., Herrmann, T., Drepper, C., Jablonka, S., Funk, N., Klausmeyer, A., Rogers, M. L.,

Rush, R. and Sendtner, M. (2010). Isolation and enrichment of embryonic mouse motoneurons

from the lumbar spinal cord of individual mouse embryos. Nat Protoc 5, 31-38.

Xin, J., Mesnard, N. A., Beahrs, T., Wainwright, D. A., Serpe, C. J., Alexander, T. D.,

Sanders, V. M. and Jones, K. J. (2012). CD4+ T cell-mediated neuroprotection is independent of

T cell-derived BDNF in a mouse facial nerve axotomy model. Brain Behav Immun 26, 886-890.

Yanpallewar, S. U., Barrick, C. A., Buckley, H., Becker, J. and Tessarollo, L. (2012). Deletion

of the BDNF truncated receptor TrkB.T1 delays disease onset in a mouse model of amyotrophic

lateral sclerosis. PloS One 7, e39946.

Yoon, S. O., Casaccia-Bonnefil, P., Carter, B. and Chao, M. V. (1998). Competitive signaling

between TrkA and p75 nerve growth factor receptors determines cell survival. J Neurosci 18, 3273-

3281.

Yuan, Q., Wu, W., So, K. F., Cheung, A. L., Prevette, D. M. and Oppenheim, R. W. (2000).

Effects of neurotrophic factors on motoneuron survival following axonal injury in newborn rats.

Neuroreport 11, 2237-2241.

Yuen, E. C. and Mobley, W. C. (1995). Therapeutic applications of neurotrophic factors in

disorders of motor neurons and peripheral nerves. Mol Med Today 1, 278-286.

Zampieri, N., Xu, C., Neubert, T. A. and Chao, M. V. (2005). Cleavage of p75 neurotrophin

receptor by alpha -secretase and gamma -secretase requires specific receptor domains. J Biol Chem.

Zhai, J., Zhou, W., Li, J., Hayworth, C. R., Zhang, L., Misawa, H., Klein, R., Scherer, S. S.,

Balice-Gordon, R. J. and Kalb, R. G. (2011). The in vivo contribution of motor neuron TrkB

receptors to mutant SOD1 motor neuron disease. Hum Mol Genet 20, 4116-4131.

Zhang, J. and Huang, E. J. (2006). Dynamic expression of neurotrophic factor receptors in

postnatal spinal motoneurons and in mouse model of ALS. J Neurobiol 66, 882-895.

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Figures

Figure 1. c29 prevents the death of motor neurons after axotomy

(A) Percentage cervical motor neuron number, relative to contralateral motor neuron number, 5

days after unilateral axotomy of the ulnar nerve of newborn rats. c29 but not vehicle peptides

resulted in significant protection from axotomy-induced motor neuron loss (N = 6 animals per

group, mean ± s.d.; *** p < 0.001; two-tailed t-test). (B) Representative micrographs showing

Nissl-stained cervical motor neurons within in the ventral horn of rat pups following unilateral ulnar

nerve axotomy and treatment with control peptide (PTD) or c29 as indicated. Scale bar represents

100µm.

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Figure 2 c29 prevents the death of motor neurons in SOD1G93A mice

Immunostaining for biotin in spinal cords of animals 1 h after i.p. injection of 5 mg/kg non-

biotinylated scrambled peptide (A) or biotinylated c29 peptide (representative of N=3 animals per

treatment) Scale bar represents 250µm (B). Rate of survival (C), average body weight (D), and

rotarod performance (E) of SOD1G93A mice treated via subcutaneous pumps with either vehicle

(black) or ~5mg/kg/day c29 (red) from ~63 days of age until death (N = 8 animals per treatment

group, mean ± s.e.m; *p<0.05, one-way ANOVA with Sidak’s multiple comparison post-hoc test).

(F) Average number of motor neuron cell bodies per coronal section of the lumbar spinal cord of

116 day old mice treated with c29 or vehicle (mean ± s.e.m, N = 4 animals per group, p<0.01; two-

tailed t-test). (G) Representative images of spinal motor neurons in 112-week old SOD1G93A mice

treated with c29 or vehicle. Black arrows: atrophic neurons, red arrows : non-atrophic neurons.

Scale bar represents 50µm.

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Figure 3. Treatment with c29 decreases the amount of endogenous p75NTR cleavage in SOD1G93A

mice

(A) Relative levels of urinary p75NTR ECD in wildtype and SOD1G93A mice over the course of the

disease, showing the increase of p75NTR ECD from disease onset to end stage. (The number of urine

samples from 20 SOD1 animals and 13 wildtype animals recovered per time point are indicated in

each bar). (B) Relative levels of urinary p75NTR ECD levels in SOD1G93A mice treated with saline

(black bars) compared to mice treated with c29 via a subcutaneous osmotic minipump from 63 days

of age (red bars). Levels of urinary p75NTR ECD were standardized to urinary creatinine. (C)

Western blot analysis of NSC-34 cells treated with PMA, TAPI-2, Compound E, NGF (50 ng/ml),

proNGF (5 ng/ml), BDNF (50 ng/ml) or proBDNF (5ng/ml) for 6 h showing the generation of

p75NTR C-terminal fragment (CTF) and intracellular domain fragment (ICD). (The number of urine

samples collected from 6 c29-treated and 6 saline-treated SOD1G93A animals during the time

periods are indicated in each bar. * p < 0.05; ** p < 0.01, *** p < 0.001; one-way ANOVA with

Bonferroni's multiple comparison post-hoc test).

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Figure 4. c29 promotes survival of BDNF-maintained neurons.

Percentage of primary E13 motor neurons surviving after 3 days in culture in 1 ng/ml BDNF and

50 ng/ml NGF, with or without the addition of TAPI-2 (A) or c29 or scrambled (SC) control

peptides (B). (C) Percentage of motor neurons surviving after 3 days in culture in BDNF and

proNGF or proBDNF with or without the addition of c29 or scrambled peptides (D) DIC

micrographs of motor neurons cultured for 3 days in the presence of 1 ng/ml BDNF or 1 ng/ml

BDNF and 50 ng/ml NGF with or without c29 treatment. Scale bar = 50 µm. (E) Representative

western blot of NSC-34 cell lysates 6 h after treatment with PMA and TAPI-2, Compound E, NGF

or proNGF, with or without c29 or scrambled peptide (SC). Percentage of primary E13 motor

neurons surviving after 3 days in culture in CNTF (F) or GDNF (G) and NGF with or without co-

treatment with TAPI-2, c29 or scrambled peptide (n = 3 experiments per condition; mean ± s.e.m;

** p < 0.01, *** p < 0.001; one-way ANOVA). Jour

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Figure 5. c29 protects motor neurons from BDNF, but not CNTF or GDNF withdrawal.

(A) Percentage of primary E13 motor neurons surviving after 3 days in culture in various

concentrations of BDNF. Percentage of motor neurons surviving 24 h after (B) BDNF, (C) CNTF

or (D) GDNF withdrawal, when treated with 1µM c29 or scrambled control peptide (SC). (n = 3

experiments; mean ± s.e.m; **p < 0.01, *** p < 0.001; one-way ANOVA).

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Figure 6. c29 interacts with TrkB and promotes Trk survival signals.

Western blots (WB) probed with an anti-Trk antibody of protein pulled down from (A) HEK293

cells overexpressing TrkB receptors, or (B) NSC-34 cells that express endogenous TrkB, using as a Jour

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bait streptavidin-linked beads conjugated to biotinylated c29 peptide or beads conjugated to the

biotinylated LC1 peptide of the extracellular juxtamembrane domain of p75NTR (control).

Quantification and representative western blots of the levels of activated (p-) or pan Erk1/2 (C) or

Akt (D) in NSC-34 cell lysates after treatment with 1ng/ml BDNF for the indicated period in the

presence of c29 or scrambled peptide (SC). (NT: no treatment n = 3 experiments, mean ± s.e.m *

p < 0.05; ** p < 0.001; two-way ANOVA with Bonferroni's multiple comparison post-hoc test

relative to SC) (E) Quantification and representative western blots of the levels of activated and pan

Erk1/2 15 minutes after treatment with 1ng/ml BDNF, CNTF or GDNF in the presence of c29 or

scrambled peptide (n = 5 experiments, mean ± s.e.m; *** p < 0.001; one-way ANOVA relative to

SC).

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Figure 7. Treatment with c29 enhances TrkB-mediated signaling.

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Representative western blots (and quantification) of spinal cord lysates from positive control

(wildtype; WT) mice and 16-week old SOD1G93A (SOD1) mice acutely treated with c29 or

scrambled peptide (SC). Blots are probed for (A) phosphorylated TrkB (Ser 478; arrow), pan Trk,

(B) phosphorylated (p-)Akt (Ser473) and pan Akt , phosphorylated Erk1/2 (p40/p44) and pan

Erk1/2, (C) p-CREB (Ser 133; arrow) or pan CREB or (D) cleaved (cl-) caspase 3 (arrow). The

loading control for total protein levels is tubulin or actin. Relative densitometry quantification of

western blot bands indicate that the relative levels of phosphorylated Akt, Erk1/2 and TrkB are

higher in mice treated with c29 whereas levels of activated caspase 3 in SOD1G93A mice are reduced

by c29 treatment (N= 4 wildtype and 4 c29-treated SOD1G93A animals, and 3 SC-treated SOD1G93A

animals; * p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA with Bonferroni's multiple

comparison post-hoc test).

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