0001144204-13-053638.txt : 201310020001144204-13-053638.hdr.sgml : 2013100220131002161558ACCESSION NUMBER:0001144204-13-053638CONFORMED SUBMISSION TYPE:8-KPUBLIC DOCUMENT COUNT:32CONFORMED PERIOD OF REPORT:20131002ITEM INFORMATION:Other EventsITEM INFORMATION:Financial Statements and ExhibitsFILED AS OF DATE:20131002DATE AS OF CHANGE:20131002

FILER:

COMPANY DATA:COMPANY CONFORMED NAME:Amarantus Bioscience Holdings, Inc.CENTRAL INDEX KEY:0001424812STANDARD INDUSTRIAL CLASSIFICATION:PHARMACEUTICAL PREPARATIONS [2834]IRS NUMBER:000000000STATE OF INCORPORATION:NVFISCAL YEAR END:1231

FILING VALUES:FORM TYPE:8-KSEC ACT:1934 ActSEC FILE NUMBER:000-55016FILM NUMBER:131130447

BUSINESS ADDRESS:STREET 1:675 ALMANOR AVE.CITY:SUNNYVALESTATE:CAZIP:94085BUSINESS PHONE:(408) 737-2734

MAIL ADDRESS:STREET 1:675 ALMANOR AVE.CITY:SUNNYVALESTATE:CAZIP:94085

FORMER COMPANY:FORMER CONFORMED NAME:AMARANTUS BIOSCIENCE, INC.DATE OF NAME CHANGE:20130114

FORMER COMPANY:FORMER CONFORMED NAME:Amarantus BioSciences, Inc.DATE OF NAME CHANGE:20110527

FORMER COMPANY:FORMER CONFORMED NAME:Jumpkicks, Inc.DATE OF NAME CHANGE:20080123

8-K1v356539_8k.htm8-K

UNITED STATES

SECURITIES AND EXCHANGE COMMISSION

Washington, D.C. 20549

FORM 8-K

CURRENT REPORT

Pursuant to Section 13 OR 15(d) of TheSecurities Exchange Act of 1934

Date of Report (Date of earliest event reported):October 2, 2013

AMARANTUS BIOSCIENCE HOLDINGS, INC.

(Exact name of registrant as specified inits charter)

Nevada 333-148922 26-0690857

(State or other jurisdiction of

incorporation or organization)

(Commission File Number)

IRS Employer

Identification No.)

675 Almanor Ave

Sunnyvale, CA

94085

(Address of Principal Executive Offices) (Zip Code)

(408) 737-2734

(Registrants telephone number, includingarea code)

Check the appropriate box below if theForm 8-K filing is intended to simultaneously satisfy the filing obligation of the registrant under any of the following provisions:

Written communications pursuant to Rule 425 under the Securities Act

Soliciting material pursuant to Rule 14a-12 under the Exchange Act

Pre-commencement communications pursuant to Rule 14d-2(b) under the Exchange Act (17 CFR 240.14d-2(b))

Pre-commencement communications pursuant to Rule 13e-4(c) under the Exchange Act (17 CFR 240.13e-4(c))

Item 8.01Other Events.

On May 5, 2013, AmarantusBioScience Holdings, Inc. (the Company) submitted the final grant award progress report to the Michael J. Fox Foundationfor Parkinsons Research entitled Comparisons and Actions of MANF and GDNF in Rodent Models Parkinsons Disease. Acopy of the report is furnished as Exhibit 99.1 to this Current Report on Form 8-K.

The information disclosedunder this Item8.01, including Exhibit99.1 hereto, is being furnished and shall not be deemed filed forpurposes of Section18 of the Securities Exchange Act of 1934, as amended, nor shall it be incorporated by reference intoany registration statement or other document pursuant to the Securities Act of 1933, as amended, except as expressly set forthin such filing.

Item 9.01Financial Statements and Exhibits.

(d) Exhibits

Exhibit

No.

Description

99.1 Michael J. Fox Foundation for Parkinsons Research Grant Award Progress Report dated May 5, 2013

SIGNATURES

Pursuant to the requirementsof the Securities Exchange Act of 1934, the registrant has duly caused this report to be signed on its behalf by the undersignedthereunto duly authorized.

AMARANTUS BIOSCIENCE HOLDINGS, INC.

Date: October 2, 2013 By: /s/ Gerald E. Commissiong

Name: Gerald E. Commissiong

Title: Chief Executive Officer

EX-99.12v356539_ex99-1.htmEXHIBIT 99.1

Michael J. Fox Foundation for ParkinsonsResearch

Grant Award Progress Report

Comparisons and Actions of MANF and GDNFin

Rodent Models Parkinsons Disease

MJFF Program: Neurotrophic Factors Program

Final Report: Phases 1 and 2

5th May 2013

Principal Investigator: J.W. CommissiongPhD, CSO

Amarantus Biosocience Holdings Inc.

This report is based on the results of experiments carried outby Amarantus Bioscience with support from the Michael J. Fox Foundation, under the supervision of John W. Commissiong, the companys chief scientific officer. Amarantus engaged the Swiss Consulting Firm NeuroAssets (CEO: David A. Lowe, PhD, assisted by Roman Urfer, PhD) to perform an independent review of the results, and prepare a written report based on the data.

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EXECUTIVE SUMMARY

The objectives of this study were (1) to confirm MANFs activity in the 6-OHDA model of Parkinsons disease (PD),(2) to evaluate striatal and nigral administration of MANF, (3) to administer MANF in neuroprotection and neuroregeneration protocols,(4) to asses different dose levels of MANF, (5) to compare MANF with GDNF under identical experimental conditions, (6) to applyan array of behavioral, structural and functional measures, and (7) to measure diffusion of MANF after convection enhanced delivery.

MANF displayed strong neuroprotective activity when administered to the striatum as evidenced by normalized ipsilateral rotationalbehavior evoked by amphetamine and protection of TH+ cell bodies in the substantia nigra.

MANF prevented the striatal 6-OHDA-induced decrease of striatal dopaminergic terminals when administered to the substantianigra.

MANFs activity is dependent on its location of administration and MANFs effects manifest themselves distal tothe administration site. Striatal administration of MANF protects nigral cell bodies while nigral administration of MANF protectsstriatal dopaminergic fiber densities.

MANF may display effects contralateral to the growth factor administration site.

MANF could be delivered to the striatum by convection enhanced delivery and MANF diffusion and distribution volumes could bemeasured by immunohistochemistry.

Continued MANF development for the treatment of PD is warranted based on the results of this present study, the known mechanismof action and published literature, and will involve the following elements:

oRodent PD studies to investigate mechanistic hypotheses on site-specific pathways activated by MANF, distal action of MANF(including activation of contralateral circuits), protection from 6-OHDA-induced apoptosis and reactive oxygen species formation,and kinetics of dopamine levels in the striatum of freely behaving animals.

oNon-human primate PK study to optimize MANF dose, site of administration, and dosing regimen to optimally cover target tissues.

oNon-human primate pharmacodynamic study in a model of Parkinsons disease as preclinical proof-of-concept and in supportof the human clinical study design.

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Table of Contents

1 Introduction 5

1.1 MANF biology and structure in relation to Parkinsons disease therapy 5

1.2 In vivo activities of MANF with focus on PD 8

1.3 Grant background and objectives 10

1.3.1 Grant framework 10

1.3.2 Study timelines 10

1.3.3 Study objectives 10

2 Methods 12

2.1 MANF and GDNF protein source and characterization 12

2.2 Animal housing 13

2.3 Administration of 6-OHDA, MANF and GDNF 13

2.3.1 Phase 1: Striatal administration of 6-OHDA, MANF and GDNF 13

2.3.2 Phase 2: Striatal administration of 6-OHDA, nigral administration of MANF and GDNF 14

2.4 Amphetamine-induced rotational behavior 14

2.5 Transcardiac perfusion and tissue collection 15

2.6 Quantification of TH+ cells in the substantia nigra (Phase 1) 16

2.7 Embedding and sectioning of rat brains (Phase 2) 16

2.8 Quantification of TH+ neurons in the substantia nigra by stereology (Phase 2) 16

2.9 Quantification of dopaminergic terminals in the striatum (Phase 2) 17

2.10 Determination of striatal levels of dopamine, DOPAC and HVA 18

2.11 MANF striatal diffusion by convection enhanced delivery (CED) 19

2.12 Statistical analyses 21

3 Results 22

3.1 Overall study design 22

3.2 Phase 1: Striatal administration of growth factors 23

3.2.1 Neuroprotection protocol 24

3.2.2 Neuroregeneration protocol 27

3.3 Phase 2: Nigral administration of growth factors 31

3.3.1 Neuroprotection protocol 31

3.3.2 Neuroregeneration protocol 40

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3.4 Diffusion of MANF with convection-enhanced delivery 47

4 Discussion 50

5 Conclusions and Outlook 56

6 References 57

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1 Introduction

1.1 MANF biology and structure in relation to Parkinsonsdisease therapy

Parkinsons disease (PD) is a neurodegenerative disordercharacterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. The lack of dopamine causesthe classical motor symptoms of bradykinesia, rigidity and resting tremors. Current PD therapy is strictly symptomatic and is focusedon dopamine replacement strategies. PD therapeutic agents include various formulations of L-DOPA, dopamine receptor agonists, monoamineoxidase (MAO) B inhibitors and catechol O-methyltransferase (COMT) inhibitors. Much of the scientific and clinical efforts on thediscovery and development of new compounds and agents for treatment of PD symptoms attempt to address motor dysfunction includingdyskinesia and gait disorders, hallucinations / psychosis, depression / anxiety, autonomic failure but few aim to achieve diseasemodification or neuroprotection (Meissner et al., 2011). The intended application of mesencephalic, astrocyte-derived neurotrophicfactor (MANF) falls into this latter category offering the possibility of a neuroprotective (i.e., halting disease progression)and a neuroregenerative (i.e., reversal of neurodegeneration) treatment of PD.

MANF (Petrova et al., 2003) and cerebral dopamine neurotrophicfactor (CDNF) (Lindholm et al., 2007) form a distinct family of evolutionary conserved trophic factors with a unique domain organization.MANF was initially purified from conditioned media from an immortalized ventral mesencephalic astrocytic cell line (Petrova etal., 2003). Biochemical analyses combined with bioinformatics revealed that the MANF protein is encoded by the gene for human arginine-richprotein (ARP) also known as human arginine-rich, mutated in early stage tumors (ARMET) (Shridhar et al., 1996).

The three-dimensional structures of MANF and CDNF were solvedby NMR (Hoseki et al., 2010, Hellman et al., 2011) and X-ray crystallography (Parkash et al., 2009) and offer important insightinto the function of these growth factors. The NMR-structure of mature MANF identified two distinct domains joined by a linker.The N-terminal domain (N-domain) of MANF is entirely helical and contains three disulfide bonds. A cluster of positively chargedresidues in the p and 310 helices of the structure are conserved amongMANF homologues and may indicate functionally important residues. A weak but significant structural similarity to the N-domainwas found with saposin-like proteins (i.e., Saposin D). Saposins are required for the degradation of plasma membrane-derived glycosphingolipidsin the lysosome. However, the charged surface of MANF suggests that interacting molecules and the biological function may differconsiderably between MANF and saposins but the similarity between the two may indicate a function of MANF at intracellular orextracellular membranes.

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The C-terminal domain (C-domain) of MANF is also entirely helicaland contains one disulfide bond between conserved cysteines in the CXXC motif between a-helices5 and 6. The CXXC motif is a consensus sequence of proteins of the thiol-protein oxidoreductase superfamily. No enzymatic oxidoreductaseactivity has been detected for MANF so far (Mizobuchi et al, 2007, data not shown). The MANF C-domain is structurally similarto SAP-domains (SAF-A/B, Acinus, PLAS) and most similar to the SAP-domain of Ku70, a cytoplasmic protein with anti-apoptotic activity(Hellman et al., 2011). Based on structural considerations it is thus conceivable that MANF displays functions related to apoptosis.

MANF expression is widespread in the nervous system and in non-neuronaltissues (Lindholm et al., 2008). mRNA levels in human brain tissues was highest in the cerebral frontal cortex, optic nerve, cerebellum,dentate nucleus and pons. High levels were also detected in medulla, cerebellum white matter, cerebral pedunculi, colliculi, corpuscallosum and hippocampus. Low levels of mRNA were detected in many additional brain tissues, including the substantia nigra. MANFprotein expression in the substantia nigra was only partially co-localized with tyrosine hydroxylase (TH) (Lindholm et al., 2008).MANF is thus expressed in potential target tissues relevant to the treatment of PD.

MANF in vivo biology has been studied in Drosophila(Palgi et al., 2009) and zebrafish (Chen et al., 2012). DmMANF was required for the development of the Drosophila nervoussystem. Maternal and zygotic DmMANF null mutants led to a complete loss of dopaminergic neurites and a drastic reduction of dopaminelevels. These events were followed by a degeneration of axonal bundles in the embryonic central nervous system with subsequentcell death. MANF is widely expressed during embryonic development and in adult organs of zebrafish (Chen et al., 2012). In thebrain, MANF-positive cells were located close to TH-positive cells in preoptic, ventral and dorsal thalamic regions and only fewMANF-containing cells were found to co-express TH. Knockdown of MANF expression during development with antisense oligonucleotidesresulted in no apparent phenotype. However, the level of dopamine was reduced by about 50% and the expression of the two TH genes,th1 and th2, was reduced in some brain regions. MANF is thus involved in the development of the dopaminergic systemin Drosophila and zebrafish. Since developmental processes are sometimes re-activated in response to neuronal injury itis conceivable that MANF could have regenerative activity in PD.

The regulation of expression and secretion of MANF was extensivelystudied in the context of the cellular stress response. MANF expression is induced by the unfolded protein response (UPR) (Apostolouet al., 2008). The MANF promoter contains an ER stress response element, ERSE-II, that is activated by known ER stressors liketunicamycin and thapsigargin (Apostolou et al., 2008; Tadimalla et al., 2009). Consequently, induction of MANF expression by ERstressors was demonstrated in several independent studies (NIH3T3 cells / tunicamycin, thapsigargin, Mizobuchi et al., 2007; U2OS,293, SHST5Y cells / tunicamycin, thapsigargin, Apostolou et al., 2008; Primary cultured neurons / tunicamycin, Yu et al., 2010;Cardiac myocytes, HeLa cells / tunicamycin, thapsigargin, DTT, Glembotski et al., 2012; Neuro2a cells / thapsigargin, Oh-hashiet al., 2012). It is thus well established that ER stress induces MANF expression in many different cell types.

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The endoplasmic reticulum (ER) is a major site of protein synthesis.ER quality control mechanisms monitor protein folding and prevent the transport and secretion of immature proteins. When ER stressoverwhelms the capacity of the quality control system, unfolded or misfolded proteins accumulate in the ER. ER stress sensor proteins,PERK, IRE1 and ATF6 activate an intracellular signal transduction pathway called the unfolded protein response (UPR). The UPRincreases the expression of several target genes to restore ER homeostasis. The functions of UPR target genes vary broadly andinclude protein folding helpers (i.e., chaperones) and proteins involved in glycosylation, oxidative stress response, proteintrafficking, lipid biosynthesis and ER-associated degradation. Aspects of ER stress and the UPR have been linked to the developmentof several neurodegenerative disorders (Lindholm et al., 2006). In the context of PD, it is noteworthy that a prominent featureof this disease is the presence of intraneuronal cytoplasmic inclusion bodies, known as Lewy bodies. Studies of families withrare autosomal recessive PD identified several genes coding for mutated proteins that could be causative for PD. Among them, aggregatedalpha-synuclein is found in Lewy bodies. In the transgenic mouse line A53TaS aggregatedalpha-synuclein was associated with abnormal UPR that could promote neuronal death (Colla et al., 2012). It is thus conceivablethat a growth factor such as MANF whose expression is induced by ER stress and the UPR could counteract degenerative mechanismscaused by protein aggregation.

The question arises whether MANF could have activities thatmanifest themselves intracellularly that would not require secretion of MANF and the subsequent activation of a receptor-mediatedsignaling pathway. Knock-down of MANF expression by siRNA rendered HeLa cells more sensitive to cell death induced by ER stress.Moreover, overexpression of MANF in U2OS cells protected cells from ER-stress induced cell death (Apostolou et al., 2008). Knock-downof MANF expression by micro-RNA increased cell death of cardiomyocytes after simulated ischemia / reperfusion while overexpressionof MANF protected these cells from serum-deprivation induced caspase-3 activation and ischemia-induced cell death (Tadimalla etal., 2008). Overexpression or microinjection of MANF and the C-terminal domain of MANF prevented apoptotic cell death of sympatheticneurons (Hellman et al., 2011). Both siRNA-mediated knockdown and overexpression of MANF will affect ER-resident MANF as well assecreted MANF. The observed effects could thus be explained by extracellular MANF binding to a receptor or by ER-resident MANFperforming an intracellular function. Hence, intracellular and possibly exogenous MANF protein protects cells from stress inducedby ischemia, serum-deprivation and more specifically, ER stress.

Apoptosis induced by ER stress or other mechanisms may playa role in the progress of nigral dopaminergic neurodegeneration in PD. Therefore, anti-apoptotic activities of MANF could be importantfor its therapeutic potential in PD. Recombinant MANF selectively increased the survival of dopaminergic (i.e., TH+)neurons (Petrova et al., 2003) in mixed neuronal cultures. Recombinant MANF decreased caspase-3 activation in a dose-dependentmanner in cardiomyocytes that were serum-starved (Tadimalla et al., 2009). Recombinant ARMET fully protected primary mixed corticaland hippocampal neuronal cultures exposed to tunicamycin using quantification of TUNEL-positive cells as a marker of apoptosis(Yu et al., 2010). MANF thus seems to have anti-apoptotic activity when administered as an exogenous protein.

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MANF increased the frequency of spontaneous and miniature gamma-aminobutyricacid (GABA)-receptor mediated inhibitory postsynaptic currents (IPSCs) without changing the mean amplitudes in mechanically dissociateddopaminergic neurons (Zhou et al., 2006). In enzymatically dissociated neurons, MANF had no effect on currents induced by exogenousGABA.

1.2 In vivo activities of MANF with focus on PD

MANF was identified as a mesencephalic astrocyte-derived neurotrophicfactor and displays a spectrum of cellular activities that could translate to neuroprotective or restorative effects in PD. Therefore,MANF was tested in the 6-hydroxy dopamine (6-OHDA) model of PD using differing administration protocols. A single intrastriatalinjection of MANF was administered either 6 hours pre-6-OHDA (i.e., neuroprotection protocol) or 4 weeks after 6-OHDA (i.e., neuroregenererationprotocol) and outcomes on amphetamine-induced rotational behavior and TH+ cells in the substantia nigra and TH+fibres in the striatum were evaluated (Voutilainen et al., 2009). In the neuroprotection protocol, MANF (10 g) reduced the6-OHDA-induced deficit in rotational behavior two weeks after administration by about 80% and this effect was sustained at thefour week time-point (90% reduction). The TH+ neurons in the substantia nigra were protected (70%) by MANF (10 mg)but the effect on TH+ fibers in the striatum was modest (10% protection). In the neuroregeneration protocol, MANF displayeda time dependent decrease of amphetamine-induced rotational behavior that led to a cumulative reduction of about 50% at week 12compared to vehicle treated animals. This effect was observed at the same dose level of MANF (10 g). TH+ cells wereprotected to some degree (25%) but the effects in this protocol were substantially smaller than the ones observed in the neuroprotectionprotocol. GDNF (Glia cell line-derived neurotrophic factor) was profiled using an identical treatment regimen and even though thisgrowth factor displayed similar activities as MANF it was generally less active under the same conditions.

A second study investigated MANF activities in the rat 6-OHDAmodel in which the growth factor was applied by an osmotic mini-pump starting two weeks after 6-OHDA for two weeks with a two-siteintrastriatal infusion. The total amounts of administered MANF were 21, 42 and 63 g (Voutilainen et al., 2011). These amountswere considerably higher than the active dose in the previous study (i.e., 10 mg) andthis difference could be of importance given the U-shaped dose-response curve of MANF. MANF did not display significant effectson amphetamine-induced rotational behavior or provide protection of TH+ cells and fibers in the substantia nigra andstriatum, respectively. However, the vehicle control displayed very low cumulative rotation numbers indicating rapid spontaneousrecovery. Moreover, GDNF was not different from vehicle even though in a parallel experiment in which GDNF and CDNF were investigatedin an otherwise identical protocol GDNF showed a clear trend towards reduction of rotational behavior. Hence, conclusions on MANFactivity after chronic intrastriatal infusion cannot be based on this study.

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The profiling of GDNF in the 6-OHDA model of Parkinsonsdisease has been the subject of many publications and study designs included neuroprotection and neuroregeneration protocols inwhich the growth factor was administered either to the striatum (Rosenblad et al., 1998; Kirik et al., 2000; Lindholm et al., 2007;Vouitilainen et al., 2009) or the substantia nigra (Sauer et al., 1995; Winkler et al., 1996; Kearns et al., 1996; Lapchak et al,1997; Kirik et al., 2000). The functional and structural readouts in these studies included amphetamine-induced rotational behavior,assessments of the structural integrity and function of dopaminergic terminals and fibers (i.e., striatal TH+ fibers,dopaminergic terminal densities, dopamine levels) and the survival of dopaminergic neurons in the substantia nigra (i.e., TH+nigral cell bodies). The reported activities of GDNF in these experimental systems and treatment paradigms are described in theresult section of this report. However, GDNF did display distinct sets of neuroprotective and neuroregenerative activities dependingon the location of GDNF injection (i.e., striatal versus nigral) and it is thus of interest to profile GDNF side-by-side with thenovel neurotrophic growth factor MANF to understand commonalities and distinct features of these neurotrophic factors.

In conclusion, MANF displays a promising profile of cellularactivities relevant to disease mechanisms of PD. Consequently, MANF in vivo activity was demonstrated in the 6-OHDA modelof PD. However, there remain significant uncertainties as to the effects of MANF on behavior, cellular markers and biochemicalread-outs in models of PD. Therefore, this present study was designed to further investigate MANF in the 6-OHDA model, to provideadditional evidence and independent confirmation of MANFs activities and to add further experimental support for the developmentof MANF for treatment of PD. Moreover, an additional object for this study was to understand how MANF and GDNF compare under thesame experimental conditions.

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1.3 Grant background and objectives

1.3.1Grant framework

MJFF Program: Neurotrophic Factors Program

Award start date: April 4, 2010

Award duration: 1 year (Extended by agreement with MJFF)

Project title: Comparison and actions of MANF and GDNF in rodent models of

Parkinsons disease

Principal investigator: John W. Commissiong, PhD

Organization: Amarantus Bioscience Holdings Inc., c/o The Parkinsons Institute,

675 Almanor Ave., Sunnyvale, CA 94085, USA.

1.3.2 Study timelines

Award start date was April 4, 2010.

Completion of Phase 1 experiments in October 2010.

Written report of Phase 1 results submitted to MJFF on October10, 2010.

Oral presentation to MJFF in New York on November 9, 2011.

Follow-up teleconference between Jamie Ebeling (MJFF) and JohnCommissiong (AMBS) was held on December 4, 2011. A change of scope of the study was agreed on by MJFF and consequently densitometryand stereology methods were included in the Phase 2 of the grant.

Given delays in reporting data from the Phase 2 of this study,AMBS management decided to contract Drs. Lowe and Urfer of NeuroAssets, a Swiss-based consulting company, to collate and assistDr. John Commissiong in writing this final report of Phase 1 and 2 of the study. The final report of this study was submitted toMJFF in May 2013.

1.3.3 Study objectives

(1) Confirm the activity of MANF in a well established modelof PD (i.e., intrastriatal administration of 6-OHDA).

(2) Compare the activity of MANF with GDNF under identical experimentalconditions.

(3) Assess and compare the activities of MANF and GDNF aftersingle injections into the striatum versus the substantia nigra.

(4) Assess and compare the activities of MANF and GDNF whenapplied prior to 6-OHDA (i.e., neuroprotection) or weeks after the administration of the toxin (i.e., neuroregeneration).

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(5) Assess activities of different dose levels of MANF.

(6) Measure the diffusion of MANF by convection enhanced deliveryafter administration to the striatum.

(7) Apply an array of well established and accepted read-outsincluding behavioral (i.e, amphetamine-induced rotations), functional (i.e., dopamine and dopamine metabolites levels) and structural(i.e., TH+ cell counts in the substantia nigra; TH+ terminal densities in the striatum) measures.

The study was conducted in two phases. In Phase 1, the growthfactors were applied to the striatum and in Phase 2 to the substantia nigra. The detailed study design is described in section3.1.

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2 Methods

2.1 MANF and GDNF protein source and characterization

The gene of a human MANF variant (R155P; Petrova et al., 2003)with a C-terminal 6xHis tag and an enterokinase cleavage site was inserted into the kanamycin resistant expression vector pJexpress411. E. coli BL21(DE3) cells transformed with the resulting expression vector were grown in a 5 liter fermenter in fortifiedLB medium containing a phosphate buffer and glucose. Cells were grown at 37C until the glucose was nearly exhausted at whichtime a glucose feed was started. The glucose concentration was maintained at or below 1 g/l. MANF expression was induced by additionof Isopropyl -D-1-thiogalactopyranoside(IPTG) to a final concentration of 1 mM and cells were harvested 4 h postinduction using a continuous flow centrifuge. Cell paste was stored at -80 C.

The chromatography system and the columns for MANF purificationwere sanitized by soaking in 0.5 M NaOH, rinsed with low endotoxin water, and equilibrated in buffers prepared with low endotoxinwater. One hundred grams of cells from the fermentor run were resuspended in 1 liter of 20 mM NaH2PO4, 0.25 M NaCl, pH 8 witha hand held homogenizer and passed through a microfluidizer three times at approximately 15,000 psi. The lysate was clarifiedby centrifugation and filtration. The clarified lysate was applied to a 35 ml IMAC fast flow (FF) column (2.6 cm by 6.3 cm) equilibratedin Buffer NA (20 mM NaH2PO4, 5 mM imidazole, 0.5 M NaCl, pH 8). The column was washed with 2 column volumes(CV) Buffer NA, 2 CV Buffer NA containing 2 M urea and 1 % Triton X-100, 2 CV Buffer NA, and Buffer NA containing 25 mM imidazole.The protein was eluted with Buffer NA containing 200 mM imidazole and the column was purged with Buffer NB (20 mM NaH2PO4,500 mM imidazole, 0.5 M NaCl, pH 8). Fractions were collected in sterile 125 ml capacity PETG bottles. The pooled fractions containingMANF were dialyzed against 2 liters of 20 mM NaH2PO4, 50 mM NaCl, 0.1 % Tween 20, pH 7.5 at room temperature.The dialysate was diluted to OD280 ~2 with 20 mM NaH2PO4, 50 mM NaCl, 0.1 % Tween 20, pH 7.5, CaCl2 wasadded to 2 mM, and 160 units enterokinase (EKMax, Invitrogen) was added (to approximately 1 unit/ml). Digestion proceeded at roomtemperature for 8 hours and terminated by addition of EDTA to 5 mM final concentration. The solution was stored at 4C overnight.The pH of the EK-digested MANF was adjusted to 6 with HCl and filtered through a 0.22 m cellulose acetate filter (Corning).The entire 80 ml of EK-digested MANF preparation was applied to a sanitized prepacked 5 mL SP HP HiTrap column (GE Healthcare)equilibrated in Buffer SA (10 mM NaH2PO4, pH 6) containing 50 mM NaCl, followed by a wash with several CVof Buffer SA containing 50 mM NaCl. Bound proteins were eluted by an initial step to 150 mM NaCl, followed by a continuous gradientto 0.6 M NaCl, and a final step to 1 M NaCl. Fractions containing MANF were combined and stored at 4C. This pool was combinedwith the pool from a previous purification run using a similar protocol and was dialyzed against 10 mM Na citrate, 150 mM NaCl,pH 6.0. The dialysate was passed through a Mustang E filter and concentrated using a sanitized Amicon Ultra-15 (10 kDa molecularweight cut off) to an OD280 of 10. The endotoxin level of this protein preparation was less than 10 EU per mg of protein usinga Pyrosate LAL clot assay kit (Cape Cod Associates). The biological activity of MANF was verified in a dopaminergic cellculture assay (Takeshima et al., 1994; Takeshima et al, 1996).

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Recombinant human GDNF was obtained from Peprotech (Catalog# 450-10). The mature sequence of human GDNF with a methionine residue at the N-terminal with no tags was expressed in E.coli.The purity of the recombinant material was reported as greater than 98% by SDS-PAGE and HPLC analyses. The biological activityof GDNF was tested in a rat C6 cell proliferation assay with an ED50 of 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