Long-term protective effects of AAV9-mesencephalic astrocyte-derived neurotrophic factor gene transfer in parkinsonian rats

    Long-Term Protective Effects of AAV9-Mesencephalic Astrocyte-Derived Neurotrophic Factor Gene Transfer in Parkinsonian Rats Fei Hao, Chun Yang, Sha-Sha Chen, Yan-Yan Wang, Wei Zhou, Qiang Hao, Tao Lu, Barry Hoffer, Li-Ru Zhao, Wei-Ming Duan, Qun-Yuan Xu PII: DOI: Reference:

S0014-4886(17)30016-X doi:10.1016/j.expneurol.2017.01.008 YEXNR 12463

To appear in:

Experimental Neurology

Received date: Revised date: Accepted date:

14 September 2016 27 December 2016 17 January 2017

Please cite this article as: Hao, Fei, Yang, Chun, Chen, Sha-Sha, Wang, Yan-Yan, Zhou, Wei, Hao, Qiang, Lu, Tao, Hoffer, Barry, Zhao, Li-Ru, Duan, Wei-Ming, Xu, QunYuan, Long-Term Protective Effects of AAV9-Mesencephalic Astrocyte-Derived Neurotrophic Factor Gene Transfer in Parkinsonian Rats, Experimental Neurology (2017), doi:10.1016/j.expneurol.2017.01.008

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ACCEPTED MANUSCRIPT Title page Long-Term Protective Effects of AAV9-Mesencephalic Astrocyte-Derived Neurotrophic

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Factor Gene Transfer in Parkinsonian Rats

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Fei Hao1, Chun Yang1, Sha-Sha Chen1, Yan-Yan Wang2, Wei Zhou1, Qiang Hao1, Tao Lu1, Barry Hoffer3, Li-Ru Zhao4, Wei-Ming Duan1,5,6,* and Qun-Yuan Xu1,2,7,8,9,* 1

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Department of Anatomy, Capital Medical University, Beijing 100069, China Department of Neurobiology, Capital Medical University, Beijing 100069, China 3 Department of Neurosurgery, Case Western Reserve University, Cleveland, Ohio 44106, USA 4 Department of Neurosurgery, Upstate Medical University, Syracuse, New York 13210, USA 5 Center of Parkinson’s Disease, Beijing Institute for Brain Disorders, Beijing 100069, China 6 Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Cleveland, Ohio 44122, USA 7 Center of Neural Injury and Repair, Beijing Institute for Brain Disorders, Beijing 100069, China 8 Beijing Center of Neural Regeneration and Repair, Beijing 100069, China 9 Key Laboratory for Neurodegenerative Diseases of the Ministry of Education, Beijing 100069, China

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*Correspondence should be addressed to W.M.D. ([email protected]) and Q.Y.X. ([email protected]) Wei-Ming Duan Department of Anatomy, Capital Medical University No. 10 Xitoutiao, Youanmenwai, Fengtai district Beijing 100069, China Phone: +86-10-83950068 Fax: +86-10-83950067 Email: [email protected] Qun-Yuan Xu Department of Neurobiology, Capital Medical University No. 10 Xitoutiao, Youanmenwai, Fengtai district Beijing 100069, China Phone: +86-10-83911464 Fax: +86-10-83911464 Email: [email protected] 1

ACCEPTED MANUSCRIPT Abstract Intrastriatal injection of mesencephalic astrocyte-derived neurotrophic factor (MANF)

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protein has been shown to provide neuroprotective and neurorestorative effects in a 6-

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hydroxydopamine (6-OHDA) - lesioned rat model of Parkinson’s disease. Here, we

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used an adeno-associated virus serotype 9 (AAV9) vector to deliver the human MANF (hMANF) gene into the rat striatum 10 days after a 6-OHDA lesion to examine long-

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term effects of hMANF on nigral dopaminergic neurons and mechanisms underlying

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MANF neuroprotection. Intrastriatal injection of AAV9-hMANF vectors led to a robust and widespread expression of the hMANF gene in the injected striatum up to 24 weeks.

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Increased levels of hMANF protein were also detected in the ipsilateral substantia nigra.

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The hMANF gene transfer promoted the survival of nigral dopaminergic neurons,

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regeneration of striatal dopaminergic fibers and an upregulation of striatal dopamine levels, resulting in a long-term improvement of rotational behavior up to 16 weeks after viral injections. By using SH-SY5Y cells, we found that intra- and extracellular application of MANF protected cells against 6-OHDA-induced toxicity via inhibiting the endoplasmic reticulum stress and activating the PI3K/Akt/mTOR pathway. Our results suggest that AAV9-mediated hMANF gene delivery into the striatum exerts long-term neuroprotective and neuroregenerative effects on the nigrostriatal dopaminergic system in parkinsonian rats, and provide insights into mechanisms responsible for MANF neuroprotection. Keywords: Parkinson’s disease, Mesencephalic astrocyte-derived neurotrophic factor, 2

ACCEPTED MANUSCRIPT Gene transfer, Adeno-associated virus vectors, Dopaminergic neurons, Endoplasmic

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reticulum stress, PI3K/Akt/mTOR pathway Abbreviations: 6-OHDA, 6-hydroxydopamine; AAV, adeno-associated virus; ATF4,

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activating transcription factor 4; ATF6α, activating transcription factor 6α; Bip, binding

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immunoglobulin protein; CDNF, cerebral dopamine neurotrophic factor; CR3,

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complement receptor 3; CHOP, C/EBP-homologous protein; DA, dopaminergic; DOPAC, dihydroxyphenylacetic acid; ER, endoplasmic reticulum; eIF2α, eukaryotic initiation

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factor 2α; GFAP, glial fibrillary acidic protein; GDNF, glial cell line-derived neurotrophic

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factor; GFP, green fluorescent protein; HVA, homovanillic acid; hEPO, human

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erythropoietin; HRP, horseradish peroxidase; IRE1, inositol-requiring enzyme 1; IR,

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immunoreactive; LGP, lateral globus pallidus; MANF, mesencephalic astrocyte-derived neurotrophic factor; MHC, major histocompatibility antigen; mTOR, mammalian target of rapamycin; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease; PI3K, phosphoinositide-3-kinase; PDI, protein disulfide isomerase; PERK, protein kinase-like ER kinase; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; TH, tyrosine hydroxylase; UPR, unfolded protein response; XBP1s, spliced x-box binding protein 1

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ACCEPTED MANUSCRIPT Introduction

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Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secreted

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protein which has more selective neuroprotective effects on dopaminergic (DA)

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neurons than glial cell line-derived neurotrophic factor (GDNF) (Petrova et al., 2003). MANF is first identified from the culture medium of a rat mesencephalic

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type-1 astrocyte cell line (Petrova et al., 2003) and is one of numbers of cerebral

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dopamine neurotrophic factor (CDNF)/MANF family of neurotrophic factors (Lindholm and Saarma, 2010). MANF has been observed to be present in

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various parts of the developing and adult brain including the striatum and midbrain (Lindholm et al., 2008; Wang et al., 2014). Importantly, MANF was

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found to co-localize with DA neurons in the substantia nigra (SN) (Lindholm et al., 2008), and the mutation of MANF gene caused degeneration of DA axons in Drosophila melanogaster (Palgi et al., 2009), suggesting important roles of MANF in the development and function of DA neurons. It has been reported that endogenous MANF is up-regulated by endoplasmic reticulum (ER) stress inducers in vitro (Apostolou et al., 2008; Mizobuchi et al., 2007; Tadimalla et al., 2008; Yu et al., 2010) and by cerebral ischemia, myocardial ischemia or status epilepticus in vivo (Lindholm et al., 2008; Tadimalla et al., 2008; Yu et al., 2010).

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ACCEPTED MANUSCRIPT Although exact mechanisms responsible for MANF effects are still elusive, accumulating evidence has suggested that MANF plays an important role in

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regulating the ER stress and unfolded protein response (UPR) (Apostolou et al.,

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2008; Glembotski et al., 2012; Henderson et al., 2013; Lindahl et al., 2014;

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Parkash et al., 2009). In addition to secretion from cells like other neurotrophic

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factors, MANF has also been found to remain inside the cells and localize in the ER (Apostolou et al., 2008; Glembotski et al., 2012; Henderson et al., 2013;

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Matlik et al., 2015). MANF therefore possesses intra- and extracellular dual

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modes of action.

Intrastriatal administration of MANF protein has been shown to protect nigral

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DA neurons and restore the motor behavior in a 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson’s disease (PD) (Voutilainen et al., 2009). Since direct intracranial administration of protein is not feasible to develop into a longterm treatment, viral gene transfer of MANF into the brain has been examined in parkinsonian rats. By using lentiviral vectors, a combination of MANF and CDNF genes into the SN protected nigral DA neurons and improved the rotational behavior in 6-OHDA lesioned rats. (Cordero-Llana et al., 2014). By using adenoassociated virus serotype 2 (AAV2) vectors, delivery of CDNF gene into the striatum provided neuroprotective and neurorestorative effects in the same rat model of PD (Back et al., 2013; Ren et al., 2013). Recently, we developed an 5

ACCEPTED MANUSCRIPT efficient AAV serotype 9 (AAV9) - mediated gene delivery system which primarily transduced neuronal cells in the brain (Xue et al., 2010). By using this system,

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we showed that AAV9-mediated robust and stable expression of human

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erythropoietin (hEPO) gene in the striatum protected nigral DA neurons from 6-

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OHDA-induced neurodegeneration and led to behavioral improvement in

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parkinsonian rats (Xue et al., 2010). In the present study, we generated an AAV9mediated human MANF (hMANF) gene delivery system to apply in a rat model of

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PD. We attempted to address: 1) whether intrastriatal administration of AAV9-

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hMANF vectors could result in robust and stable expression of the hMANF gene;

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2) whether intrastriatal overexpression of the hMANF gene led to a long-term

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protection of nigral DA neurons and functional recovery in 6-OHDA lesioned rats; 3) mechanisms responsible for MANF neuroprotection. In addition, we used a DA cell line-SH-SY5Y cells and examined whether intra- or extracellular application of MANF protected cells against 6-OHDA-induced toxicity via inhibiting the ER stress and activating the PI3K/Akt/mTOR pathway.

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ACCEPTED MANUSCRIPT Materials and methods

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In vivo study

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Animals

Adult female Sprague-Dawley rats (8-10 weeks old, 225-250 g) were

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obtained and housed under a 12 h light/dark cycle with ad libitum access to food

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and water in the Animal Core Facility of Capital Medical University (CMU), Beijing, China. All experiments associated with rats were performed following the

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National Institutes of Health Guide for the Care and Use of Laboratory Animals,

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and approved by the Animal Use and Care Committee of CMU. The number of

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animals used was the minimum required for statistical analysis, and all precautions were taken to minimize animal suffering. AAV9 vector production

The hMANF DNA was obtained from pCR3.1-hMANF vectors (kindly provided by Dr. Mart Saarma) and subcloned into an AAV expression plasmid. The expression of the hMANF gene was driven by the hybrid cytomegalovirus immediate early enhancer/chicken β-actin promoter. AAV9 vectors carrying either the hMANF or green fluorescent protein (GFP) gene were produced as previously described (Xue et al., 2010). Titers of AAV9-hMANF and AAV9-GFP 7

ACCEPTED MANUSCRIPT vectors were 1.02 × 1013 vg/ml and 1.0 × 1013 vg/ml, respectively.

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Intrastriatal injection of 6-OHDA and viral vectors

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Anesthetized rats (equithesin, 3 ml/kg, i.p.) received an injection of 15 μg of

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6-OHDA (5 μg/μl dissolved in 0.1% ascorbic acid, Sigma-Aldrich, St. Louis, MO,

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USA) into the right striatum according to the following stereotaxic coordinates: AP, +0.5 mm; ML, −2.8 mm; DV, −4.8 mm at a rate of 1 μl/min. The cannula was left

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in place for another 5 min before withdrawal. D-amphetamine (Sigma-Aldrich) -

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induced rotational asymmetry was performed 8 days later to select rats with more

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than 5 full body turns per min. One group of rats (denoted as 6-OHDA alone

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group) was sacrificed 10 days after 6-OHDA lesions (0 week), serving as a reference showing the extent of DA neuron degeneration at the time point of viral injections. Remaining rats were assigned into three groups based on rotational scores. Group 1 (6-OHDA-hMANF group): rats received an injection of 2 μl of AAV9-hMANF vectors (1013 vg/ml) into the right striatum (AP, 0 mm; ML, −2.8 mm; DV, −5 mm) at a rate of 0.2 μl/min; Group 2 (6-OHDA-GFP group) and Group 3 (6-OHDA-Saline group): rats received an injection of 2 μl of either AAV9GFP vectors (1013 vg/ml) or saline as the same coordinates as Group 1. Damphetamine-induced rotational behavior was tested 2, 4, 6, 8, 12 and 16 weeks after viral injection. Rats were sacrificed 6 weeks and 16 weeks after viral 8

ACCEPTED MANUSCRIPT injection respectively and brain tissues were prepared for immunohistochemistry, western

blot,

real-time

quantitative

PCR

and

high-performance

liquid

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chromatography (HPLC) analysis. Another study was performed in which naïve

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rats were injected with 2 μl of AAV9-hMANF vectors (hMANF group), AAV9-GFP

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vectors (GFP group) or saline (Saline group) into the right striatum to observe

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whether intrastriatal long-term overexpression of the hMANF gene affects the nigrostriatal DA pathway up to 24 weeks. The number of rats used in each part of

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the study was summarized in Table 1.

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Behavioral analysis

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Rotational behavior was examined as previous description (Chen et al., 2014; Xue et al., 2010). Briefly, rats were injected intraperitoneally with d-amphetamine (2.5 mg/kg, Sigma-Aldrich) followed by recording for 90 min using automated rotometer bowls (TSE systems, Chesterfield, MO, USA). Net rotational asymmetry score was expressed as the number of 360° turns per min. Rotation towards the lesioned side was considered to be positive. Immunohistochemistry Free floating brain sections containing the striatum (from AP+1.6 mm to AP0.92 mm) and SN (from AP-4.8 mm to AP-6.04 mm) from each rat were prepared 9

ACCEPTED MANUSCRIPT for immunohistochemistry as previously described (Chen et al., 2014; Xue et al., 2010). The avidin-biotin complex immunoperoxidase technique was used to

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visualize tyrosine hydroxylase (TH), MANF, major histocompatibility antigen

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(MHC) class II, complement receptor 3 (CR3) and glial fibrillary acidic protein

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(GFAP) immunoreactivity. Primary antibodies were rabbit anti-TH (1:300, sc-

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14007; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), rabbit anti-MANF (1:1000, ABN306; Millipore, Bedford, MA, USA), mouse anti-MHC class II (1:100,

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MCA46GA; AbD Serotec, Oxford, UK), mouse anti-CR3 (1:100, MCA275GA;

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AbD Serotec) and mouse anti-GFAP (1:400, MAB360; Millipore). Secondary

Burlingame,

CA,

USA).

For

double-

or

triple-label

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Laboratories,

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antibodies were biotinylated goat anti-rabbit or mouse IgG (1:200, Vector

immunofluorescent staining, primary antibodies were mouse anti-TH (1:400, MAB318; Millipore), rabbit anti-GFP (1:1000, AB3080; Millipore), rabbit antiMANF (1:1000, ABN306; Millipore), mouse anti-NeuN (1:100, MAB377; Millipore), chicken anti-NeuN (1:500, ABN91; Millipore), mouse anti-DARPP32 (1:2000, 611520; BD Biosciences, San Jose, CA) and chicken anti-GFAP (1:500, AB5541; Millipore). Secondary antibodies were Alexa Fluor-594-conjugated goat antimouse IgG, Alexa Fluor-488-conjugated-goat anti-rabbit IgG and Alexa Fluor647-conjugated goat anti-chicken IgY (all 1:200, Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA). Cell nuclei were counterstained with 10

ACCEPTED MANUSCRIPT 4’,6-diamidino-2-phenylindole (DAPI, 1:3000, Sigma-Aldrich). Primary antibodies were omitted as a negative control. Brain sections were examined using an

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Olympus IX51 microscope (Olympus, Inc., Tokyo, Japan) or a confocal laser-

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scanning microscope (Leica TCS SP8, Leica, Inc., Mannheim, Germany).

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Morphological assessment

TH-immunoreactive (IR) neurons and hMANF-IR neurons in the SN pars

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compacta (SNpc) were respectively counted using unbiased stereology by Stereo

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Investigator 8 software (MBF, Williston, VT, USA) in a double blind manner (Chen

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et al., 2014; Xue et al., 2010). The optical density and the loss area of TH-

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immunoreactivity in the striatum were analyzed with ImageJ software (NIH, Bethesda, MD, USA) (Chen et al., 2014; Xue et al., 2010). Striatal sections immunohistochemically processed for MHC class II, CR3 and GFAP were semiquantitatively evaluated as described previously (Yang et al., 2013). For the number of TH-IR neurons in the SNpc and the optical density of TH-IR fibers in the striatum, data were expressed as a percentage relative to the intact side. For the number of hMANF-IR cells in the SNpc, data were expressed as a percentage relative to the ipsilateral TH-IR cell counts. For the loss area of TH-IR fibers in the striatum, data were expressed as a percentage relative to the ipsilateral striatum. 11

ACCEPTED MANUSCRIPT Western blot for MANF

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Total protein from striatal and nigral tissues was extracted and immunoblot

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was performed as previously described (Xue et al., 2010). The primary antibody

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was rabbit anti-MANF (1:1000, ABN306; Millipore). GAPDH (1:3000, 5174; Cell Signaling Technology, Beverly, MA, USA) was used as an endogenous control.

IgG

(1:5000,

Jackson

Immunoresearch

Laboratories,

Inc.).

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anti-rabbit

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The secondary antibody was horseradish peroxidase (HRP) - conjugated goat

Immunoreactive bands were visualized by a chemiluminescence imaging system

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(Fusion Solo 4, Vilber Lourmat, France). Bands were analyzed using ImageJ

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software (NIH). Data were expressed as fold expression relative to the intact side. Real-time quantitative PCR for hMANF Total RNA from striatal and nigral tissues was extracted using TRIzol reagent (Sigma-Aldrich) according to manufacturer's instructions. Control tissues were always extracted separately from AAV9-hMANF injected tissues. A total of 1 μg RNA was converted to cDNA with the FastQuant reverse transcription kit with gDNase (TIANGEN Biotech Co., Ltd, Beijing, China). The reaction was performed using TaqMan® gene expression master mix with a final volume of 20 μl containing 1× TaqMan gene expression assay (rat GAPDH, Rn01775763_g1, Applied Biosystems, Foster City, CA, USA) or 250 nM TaqMan probe and 900 nM 12

ACCEPTED MANUSCRIPT each of forward and reverse primers for hMANF (Sangon Biotech, Shanghai, China) in a Bio-Rad IQ5 detection system (Bio-Rad, Hercules, CA, USA).

5’-CGACCTGAGCACAGTGGA-3’,

reverse

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primer:

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Sequences of primers and probe specific for hMANF were as follows: forward primer:

5’-

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GGGGCATATTTAGGCATC-3’, probe: 5’-FAM-AGAAGCTCCGAGTTAAA-MGB-3’.

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Cycling conditions were 2 min at 50 °C, 10 min at 95 °C, then 40 cycles of 15 sec

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at 95 °C and 1 min at 60 °C. A 2-∆Ct method was used to present data.

of

DA,

dihydroxyphenylacetic

acid

(DOPAC)

and

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Concentrations

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HPLC analysis

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homovanillic acid (HVA) from striatal tissues were determined by HPLC with electrochemical detection as described previously (Yu et al., 2016). Briefly, striatal tissues were homogenized in 0.4 M perchloric acid and then centrifuged at 12,000 ×g for 20 min. A total of 20 μl samples were injected into the C18 reversed phase column and the mobile phase consisted of 0.07 M sodium acetate, 0.05 M citric acid, 0.1 mM sodium EDTA, 0.2 mM sodium 1octanesulfonate and 10% methanol at pH value of 4.1 in a 5600A CoulArray detector system (ESA, Brighton, MA, USA). The flow rate was 1 ml/min and the temperature was set to 25 °C. A CoulArray® data analysis system (ESA) was used for data collection and analysis. Levels of DA, DOPAC and HVA were 13

ACCEPTED MANUSCRIPT calculated as nmol/g of tissue weight. Data were expressed as a percentage

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relative to the intact side.

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In vitro study

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Cell culture and drug treatments

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SH-SY5Y cells (ATCC, Manassas, VA, USA) were maintained as previously

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described (Chen et al., 2014). An appropriate dose of 6-OHDA to injure cells was determined by MTT assay (Chen et al., 2014). To determine whether

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recombinant human MANF (rhMANF) protein (R&D systems, Minneapolis, MN,

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USA) protects cells, cells were co-incubated with 6-OHDA and rhMANF protein at

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serial concentrations (0.25 ng/ml, 2.5 ng/ml, 25 ng/ml, 250 ng/ml and 2500 ng/ml). Moreover, SH-SY5Y cells were treated with 6-OHDA for different time points (1 h, 3 h, 6 h, 9 h, 12 h and 24 h) or in the absence or presence of rhMANF protein at the indicated time point. Cells were also pre-treated with the PI3K inhibitor, wortmannin (wort, 1 μM), for 1 h followed by exposure to 6-OHDA in the absence or presence of rhMANF protein. Plasmid transfection and drug treatments pAAV-hMANF and pAAV-GFP plasmids were respectively extracted using HiSpeed plasmid maxi kit (Qiagen, Valencia, CA, USA) according to 14

ACCEPTED MANUSCRIPT manufacturer’s instructions. SH-SY5Y cells were transfected with pAAV-hMANF or pAAV-GFP plasmid using lipofectamine-2000 reagent (Invitrogen, Carlsbad,

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CA, USA) according to manufacturer's instructions. After 24 or 48 h, cells were

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prepared for western blot analysis and immunofluorescent staining. Transfected

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cells were also treated with 6-OHDA for MTT assay and western blot analysis.

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Western blot

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Total protein extraction was prepared as previously described (Hao et al.,

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2015). Nuclear protein extraction was performed using NE-PER nuclear and

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cytoplasmic extraction kit according to manufacturer's instructions (Thermo

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Fisher Scientific, Rockford, IL, USA). Primary antibodies were rabbit antiphospho-eukaryotic initiation factor 2α (p-eIF2α; 3398), rabbit anti-eIF2α (5324), rabbit anti-activating transcription factor 4 (ATF4; 11815), rabbit anti-C/EBPhomologous protein (CHOP; 5554), rabbit anti-spliced x-box binding protein 1 (XBP1s; 12782), rabbit anti-phospho-Akt (p-Akt; 4060), rabbit anti-Akt (4691) (all 1:1000, all from Cell Signaling Technology), rabbit anti-binding immunoglobulin protein (Bip; 1:2000, ab108613; Abcam, Cambridge, UK), mouse anti-activating transcription factor 6α (ATF6α; 1:400, ab122897; Abcam), rabbit anti-phosphomTOR (p-mTOR; 1:1000, ab109268; Abcam) and rabbit anti-mTOR (1:1000, ab32028; Abcam). GAPDH (1:3000, 5174; Cell Signaling Technology) was used 15

ACCEPTED MANUSCRIPT as an endogenous control for total protein and TATA binding protein (TBP; 1:500, ab125009; Abcam) for nuclear protein. Secondary antibodies were HRP-

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conjugated goat anti-rabbit or mouse IgG (all 1:5000, Jackson Immunoresearch

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Laboratories, Inc.). Bands were analyzed using ImageJ software (NIH). Data

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were presented as fold expression relative to control (untreated) cells.

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Immunofluorescent staining

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Cells were fixed for immunofluorescent staining as previously described

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(Chen et al., 2014). Primary antibodies were rabbit anti-GFP (1:1000, AB3080;

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Millipore), rabbit anti-MANF (1:2000, ABN306; Millipore) and mouse anti-protein

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disulfide isomerase (PDI, 1:50, ab2792; Abcam). Secondary antibodies were Alexa Fluor-488-conjugated goat anti-rabbit IgG and Alexa Fluor-594-conjugated goat anti-mouse IgG (all 1:200, Jackson Immunoresearch Laboratories, Inc.). Primary antibodies were omitted as a negative control. Cells were examined using a confocal laser-scanning microscope (Leica TCS SP8). Statistical analysis One- and two-factor analysis of variance (ANOVA) followed by Tukey's post hoc test were used for statistical analyses. In all in vitro experiments, data were obtained from at least three independent experiments. At least three replicas per 16

ACCEPTED MANUSCRIPT group were used in each experiment. One-factor ANOVA was used in all in vitro experiments. Statistical analyses were performed using GraphPad Prism 6.0

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(GraphPad Software, Inc., La Jolla, CA, USA). Data were presented as mean ±

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standard error of the mean (SEM) and p < 0.05 was considered statistically

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significant.

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Results

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AAV9-mediated hMANF gene delivery induces long-term expression of hMANF in

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the striatum and SN

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We successfully generated an AAV9-hMANF vector, and an AAV9-GFP

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vector was used as control (Fig. 1a and b). Robust hMANF or GFP transgene expression was observed in the injected striatum and was distributed throughout a large area of the striatum both 6 weeks and 16 weeks after viral injection (Fig. 1c-f). The limited immunostaining of hMANF was found in the overlying cerebral cortex and the corpus callosum along the needle track (Fig. 1c and d). Double fluorescent immunostaining showed that almost all MANF-IR or GFP-expressing cells in the striatum were co-labeled with NeuN (Fig. 1g and h), not with GFAP (Supplemental Fig. 1). In addition, MANF immunoreactivity and GFP expression were also found in the ipsilateral SN. A number of MANF-IR cells were found in the SNpc (8.15 ± 0.33% of ipsilateral TH-IR cell counts at 6 weeks and 10.89 ± 17

ACCEPTED MANUSCRIPT 1% at 16 weeks) and co-labeled with TH in 6-OHDA-hMANF rats (Fig. 1i and k). In contrast, only a few GFP expressing cells were found in the SNpc and also co-

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labeled with TH in 6-OHDA-GFP rats (Fig. 1j and l). Moreover, MANF-IR or GFP

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labeled fibers were detected in the ipsilateral SN pars reticulata (SNpr) at 6

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weeks and 16 weeks (Fig. 1i-l).

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Western blot analysis showed that significant levels of MANF protein were

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present in the injected striatum relative to the contralateral striatum in 6-OHDAhMANF rats at 6 weeks (30-fold) and 16 weeks (27-fold) (Fig. 2a and b). Levels

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of MANF protein were also increased by more than 3 fold in the ipsilateral SN relative to the contralateral SN at both 6 weeks and 16 weeks (Fig. 2a and b).

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There was no significant difference in MANF protein levels between 6 weeks and 16 weeks in the ipsilateral striatum and SN of the 6-OHDA-hMANF group (twofactor ANOVA, F(1, 12) = 0.52 and 0.14, p > 0.05). Quantitative PCR showed that significant levels of hMANF mRNA were detected in the injected striatum of rats in the 6-OHDA-hMANF group at 6 weeks (2-∆CT = 0.067 ± 0.009; Fig. 2c) and 16 weeks (0.057 ± 0.006; Fig. 2d). Signals from hMANF mRNA were also detected in ipsilateral nigral tissues of rats in the 6-OHDA-hMANF group at 6 weeks (0.00016 ± 0.00002; Fig. 2c) and 16 weeks (0.00011 ± 0.00003; Fig. 2d). There was no significant difference in hMANF mRNA levels between 6 weeks and 16 weeks in the ipsilateral striatum and SN of the 6-OHDA-hMANF group (two-factor 18

ACCEPTED MANUSCRIPT ANOVA, F(1, 24) = 0.93 and 0.016, p > 0.05). No hMANF mRNA was detected in

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nigral tissues in the other two groups (Fig. 2c and d).

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the contralateral striatum and SN in the 6-OHDA-hMANF group or in striatal and

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Intrastriatal delivery of AAV9-hMANF improves amphetamine-induced rotational

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asymmetry

A two-factor ANOVA displayed significant differences in net rotational scores

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between 6-OHDA-hMANF, 6-OHDA-GFP and 6-OHDA-Saline groups (F(2, 301) =

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100.6, p < 0.05; Fig. 3a). There was no significant difference in rotational scores

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between three groups at 0 week (one-factor ANOVA, F(2, 43) = 1.88, p > 0.05; Fig.

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3a). Net rotational scores for rats in the 6-OHDA-hMANF group were significantly reduced following the time (8.79 ± 0.58 turns/min at 0 week to 1.3 ± 0.48 turns/min at 16 weeks) when compared to that in 6-OHDA-GFP (10.69 ± 0.9 to 8.32 ± 1.04 turns/min) and 6-OHDA-Saline (9.77 ± 0.65 to 6.62 ± 0.92 turns/min) groups 2, 4, 6, 8, 12 and 16 weeks after viral injection (one-factor ANOVA followed by Tukey's post hoc test, F(2,

43)

= 12.42, 12.6, 18.27, 20, 18.77 and

22.31, *p < 0.05; Fig. 3a). In the 6-OHDA-hMANF group, rotational scores were significantly lower at 6, 8, 12 and 16 weeks than that at 0 week (one-factor ANOVA followed by Tukey's post hoc test, F(6, 119) = 12.91, #p < 0.05; Fig. 3a). Intrastriatal delivery of AAV9-hMANF promotes regeneration of striatal TH-IR 19

ACCEPTED MANUSCRIPT fibers and survival of nigral TH-IR neurons

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TH-IR fiber density in the injected striatum was 44.12 ± 2.68% of the

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contralateral striatum at 0 week (Fig. 3b and p). TH-IR fiber densities in the

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injected striatum were both significantly greater in the 6-OHDA-hMANF group at 6 weeks (42.21 ± 3.03%) and 16 weeks (65.51 ± 2.93%) than that in 6-OHDA-

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GFP (29.39 ± 2.77% and 37.88 ± 4.64%) and 6-OHDA-Saline (29.62 ± 2.24%

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and 38.19 ± 3.73%) groups (one-factor ANOVA followed by Tukey's post hoc test, F(2, 13) = 6.91 and F(2, 12) = 17.13, *p < 0.05, ***p < 0.001; Fig. 3d-f, j-l and p). TH-

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IR fiber density in the injected striatum was significantly greater at 16 weeks than that at 0 week and 6 weeks in the 6-OHDA-hMANF group (one-factor ANOVA

3b, f, l and p).

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followed by Tukey's post hoc test, F(2, 13) = 16.08, ##p < 0.01, ###p < 0.001; Fig.

The loss area of TH immunoreactivity in the injected striatum was significantly reduced in the 6-OHDA-hMANF group (58.31 ± 2.75% at 6 weeks and 26.67 ± 6.28% at 16 weeks) when compared to that in 6-OHDA-GFP (78.87 ± 2.64% and 68.31 ± 1.7%) and 6-OHDA-Saline (78.39 ± 1.21% and 67.68 ± 1.8%) groups (one-factor ANOVA followed by Tukey's post hoc test, F(2, 13) = 27.5 and F(2,

12)

= 37.53, ***p < 0.001; Fig. 3d-f, j-l and q). The loss area of TH

immunoreactivity in the injected striatum was significantly less at 16 weeks than 20

ACCEPTED MANUSCRIPT that at 0 week (62.56 ± 2.1%) and 6 weeks in the 6-OHDA-hMANF group (onefactor ANOVA followed by Tukey's post hoc test, F(2, 13) = 24.34, ###p < 0.001; Fig.

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3b, f, l and q).

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The number of TH-IR cells in the ipsilateral SNpc was 67.53 ± 3.24% of the contralateral SNpc at 0 week (Fig. 3c and r). The number of TH-IR cells in the

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ipsilateral SNpc was both significantly greater in the 6-OHDA-hMANF group at 6

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weeks (66.88 ± 1.3%) and 16 weeks (65.37 ± 2.92%) than that in 6-OHDA-GFP (33 ± 1.71% and 29.6 ± 1.49%) and 6-OHDA-Saline (33.8 ± 1.44% and 30.24 ±

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2.16%) groups (one-factor ANOVA followed by Tukey's post hoc test, F(2, 13) = 151.1 and F(2, 12) = 81.45, ***p < 0.001; Fig. 3g-i, m-o and r). In 6-OHDA-GFP and

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6-OHDA-Saline groups, the number of TH-IR cells in the ipsilateral SNpc was both less at 6 weeks and 16 weeks than that at 0 week (one-factor ANOVA followed by Tukey's post hoc test, F(2, 13) = 85.46 and 76.89, ###p < 0.001; Fig. 3c, g, h, m, n and r).

Intrastriatal delivery of AAV9-hMANF increases concentrations of striatal DA, DOPAC and HVA and reduces the ratio of DOPAC/DA A two-factor ANOVA showed significant differences in concentrations of striatal DA, DOPAC and HVA and the ratio of DOPAC/DA between groups (F(2, 47) = 27.59, 17.91, 20.71 and 4.72, p < 0.05; Fig. 4a-d and Table S1). 21

ACCEPTED MANUSCRIPT Concentrations of DA, DOPAC and HVA in the ipsilateral striatum were all significantly greater in the 6-OHDA-hMANF group than that in 6-OHDA-GFP and

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6-OHDA-Saline groups at 6 weeks and 16 weeks (one-factor ANOVA followed by

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Tukey's post hoc test, DA: F(2, 16) = 11.1 and 36.08, DOPAC: F(2, 16) = 6.31 and

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17.56, HVA: F(2, 16) = 6.42 and 23.8, *p < 0.05, **p < 0.01, ***p < 0.001; Fig. 4a-c

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and Supplemental Table 1). The DOPAC/DA ratio (indicator of DA metabolism) was lower in the 6-OHDA-hMANF group than that in 6-OHDA-GFP and 6-OHDA-

44)

= 4.72, *p < 0.05; Fig. 4d and Supplemental Table 1). There was no

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F(2,

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Saline groups at 16 weeks (two-factor ANOVA followed by Tukey's post hoc test,

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significant difference in the (DOPAC+HVA)/DA ratio (indicator of DA turnover)

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between three groups at 6 weeks and 16 weeks (two-factor ANOVA, F(2, 44) = 4.28, p > 0.05; Fig. 4e and Supplemental Table 1). Striatal DA concentrations were greater at 6 weeks (41.25 ± 2.85%) and 16 weeks (43.57 ± 2.74%) than that at 0 week (29.87 ± 3.02%) in the 6-OHDA-hMANF group (one-factor ANOVA followed by Tukey's post hoc test, F(2, 17) = 6.21, #p < 0.05; Fig. 4a). Intrastriatal long-term overexpression of hMANF gene does not affect the nigrostriatal DA system and does not induce obvious immune responses in 6OHDA-untreated naïve rats A two-factor ANOVA showed that there were no differences in d22

ACCEPTED MANUSCRIPT amphetamine-induced rotational scores or body weight between Saline, GFP and hMANF groups from 0 week to 24 weeks (F(2, 147) = 5.31 and 2.59, p > 0.05; Fig.

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5a and b). TH-IR fiber density in the striatum and the number of TH-IR cells in the

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SNpc did not differ in all treatment groups (one-factor ANOVA, F(3, 12) = 0.83 and

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2.51, p > 0.05; Fig. 5c and d). Moreover, there were also no differences in

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concentrations of DA, DOPAC and HVA or in ratios of DOPAC/DA and (DOPAC+HVA)/DA in the striatum in all treatment groups (one-factor ANOVA, F(3, = 0.62, 0.46, 1.04, 0.21, 0.42, p > 0.05; Fig. 5e). Intrastriatal injection of AAV9-

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12)

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hMANF vectors did not result in aberrant sprouting of TH-IR fibers in the

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ipsilateral striatum, lateral globus pallidus (LGP) and SNpr, corresponding to

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areas containing hMANF immunoreactivity (Fig. 5f-q). In addition, there were only modest levels of MHC II-IR cells, activated CR3-IR microglia/macrophages and GFAP-IR astrocytes around the needle track in the injected striatum (Fig. 5r-t). Rating scores for MHC class II, CR3 and GFAP in the injected striatum were 0.75, 1 and 0, respectively. Intracellular overexpression of MANF protects SH-SY5Y cells via inhibiting 6OHDA-induced ER stress To determine whether intracellular overexpression of MANF protected SHSY5Y cells, cells were transfected with pAAV-hMANF plasmids followed by 623

ACCEPTED MANUSCRIPT OHDA treatment. Western blot analysis showed that levels of intracellular MANF protein were significantly increased 24 and 48 h after transfection (F(2, 6) = 84.72,

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***p < 0.001 versus the control group; Fig. 6a). Immunofluorescent staining

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showed that the fluorescence intensity of MANF was significantly greater in

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pAAV-hMANF transfected cells than that in non-transfected cells (Fig. 6b).

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Intracellularly overexpressed MANF was primarily detected in the perinuclear area, whereas GFP expression was visible throughout the cell body (Fig. 6b).

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Double fluorescent immunostaining showed that intracellularly overexpressed

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MANF was co-labeled with PDI, an ER-resident marker protein (Fig. 6c). MTT

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assay showed that the cell viability was significantly increased in the 6-

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OHDA+pAAV-hMANF group (65.15 ± 2%) when compared to that in 6OHDA+pAAV-GFP (45.74 ± 3.7%) and 6-OHDA (43.88 ± 3.2%) groups (F(3, 20) = 388.8, ##p < 0.01; Fig. 6d). Western blot analysis showed that intracellular overexpression of MANF decreased levels of ER stress-associated proteins, including p-eIF2α, ATF4, CHOP, XBP1s, Bip and ATF6α (N), which were all increased by 6-OHDA treatment (F(5, 12) = 296.7, 126, 184.3, 136.8, 47.85 and 18.88, #p < 0.05; Fig. 6e, f and Supplemental Fig. 2). Extracellular rhMANF protein protects SH-SY5Y cells against 6-OHDA-induced toxicity via the PI3K/Akt/mTOR pathway

24

ACCEPTED MANUSCRIPT MTT assay showed that when rhMANF protein was added to the medium, the cell viability was significantly increased at any tested concentrations,

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including 0.25 ng/ml (68.09 ± 1.77%), 2.5 ng/ml (73.62 ± 1.4%), 25 ng/ml (75.48

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± 1.58%), 250 ng/ml (80.19 ± 0.56%) and 2500 ng/ml (74.06 ± 2.04%), when

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compared to that in 6-OHDA-treated alone cells (52.12 ± 1.99%; F(6, 35) = 89.32,

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***p < 0.001; Fig. 7a). This neuroprotective effect of rhMANF reached the highest level at the concentration of 250 ng/ml. However, western blot analysis showed

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that treatment with rhMANF protein did not decrease levels of any tested proteins

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associated with the UPR, including p-eIF2α, ATF4, CHOP, XBP1s, Bip and

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ATF6α (N) (F(3, 8) = 53.3, 21.53, 134.6, 21.03, 31.49 and 13.12, p > 0.05; Fig. 7c).

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We therefore speculate that rhMANF protein may protect cells via other pathways. Considering that the PI3K/Akt/mTOR pathway plays a key role in promoting cell survival, we next examined whether rhMANF protein protected cells via this pathway. Western blot analysis showed that levels of p-Akt and p-mTOR in the 6OHDA+rhMANF group were significantly greater than that in the 6-OHDA group (F(3, 8) = 98.51 and 39.89, ##p < 0.01, ###p < 0.001; Fig. 7d). Moreover, when treated with wort, a PI3K inhibitor, levels of p-Akt and p-mTOR were significantly decreased in the 6-OHDA+rhMANF+wort group when compared to that in the 6OHDA+rhMANF group (F(3, 8) = 108.6 and 64.44, ###p < 0.001, **p < 0.01; Fig. 7e). More importantly, the cell viability was significantly lower in the 625

ACCEPTED MANUSCRIPT OHDA+rhMANF+wort group (57.43 ± 1.67%) than that in the 6-OHDA+rhMANF group (78.4 ± 1.53%; F(5,

30)

= 393.3, ###p < 0.001; Fig. 7b). These results

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suggest that rhMANF protein protects cells against 6-OHDA-induced toxicity via

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the PI3K/Akt/mTOR pathway.

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Discussion

We report for the first time that AAV9-mediated hMANF gene transfer into

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the rat striatum leads to long-term neuroprotection of nigral DA neurons and

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correction of rotational asymmetry in parkinsonian rats. Intracellular MANF

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protects cells via inhibiting 6-OHDA-induced ER stress and extracellular MANF

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protects cells via activating the PI3K/Akt/mTOR pathway. When compared to previous studies of MANF and CDNF gene transfer using viral vectors (Back et al., 2013; Cordero-Llana et al., 2014; Ren et al., 2013), our gene delivery system possesses the following advantages: 1. It can cause robust and long-term transgene expression in the injected area; 2. Gene transduction has neuronal preference, which enables transgene products to be axonal transported to the SN from the injected striatum, leading to local neuroprotection of DA neurons; 3. AAV9-hMANF vectors can also be axonal transported to the SN, eliciting a continuous production of the hMANF gene locally; 4. Striatal hMANF gene transfer exerts direct effects on axonal outgrowth of DA neurons in the striatum; 5. 26

ACCEPTED MANUSCRIPT Gene transduction can only result in modest levels of inflammatory and immune

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responses, suggesting that our gene delivery system is relatively safe.

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Our AAV9-mediated hMANF gene delivery system is very efficient. The

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widespread and stable expression of the hMANF gene is a prerequisite for a long-term cellular and behavioral improvement in parkinsonian rats. Our results

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showed that AAV9 vectors mediated robust and stable expression of the hMANF

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gene in the striatum and the vast majority of striatal transduced cells were medium spiny neurons as confirmed by MANF/NeuN/DARPP32 labeling

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(Supplemental Fig. 1). This preference for neural transduction is consistent with our previous study (Xue et al., 2010). In addition to the striatum, we also

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observed hMANF immunoreactivity in the ipsilateral SN. In the SNpr, MANF-IR fibers were found, suggesting that MANF was anterogradely transported to the SN from the injected striatum. In the SNpc, MANF-IR cells were found and colabeled with TH-IR DA neurons. By using primers and probes specific for the hMANF gene, we detected the hMANF mRNA in the ipsilateral SN. These results suggest that both MANF protein and AAV9-hMANF vectors are retrogradely transported along the nigrostriatal pathway. This finding supports previous reports showing that AAV9 vectors could be taken up by axon terminals and retrogradely transported to cell bodies (Cearley and Wolfe, 2006; Masamizu et al., 2011). In addition, mRNA and protein levels of the hMANF gene in the ipsilateral striatum 27

ACCEPTED MANUSCRIPT and SN remain the same levels at 6 weeks and 16 weeks, suggesting that there

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is no a waning and loss of the hMANF expression over time.

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Robust and long-term expression of the hMANF gene in the rat brain may

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raise safety concerns because that an excessive amount of hMANF protein may be generated, and hMANF is a xenogeneic protein for rats. Numerous reports

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have documented that continuous overexpression of the GDNF gene has side

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effects including aberrant sprouting in areas outside of the striatum, downregulation of TH activity in the intact striatum and a loss of body weight

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(Georgievska et al., 2002, 2004; Manfredsson et al., 2009; Rosenblad et al., 2003). However, our data showed that intrastriatal long-term overexpression of

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the hMANF gene have no overt side effects, including aberrant sprouting in areas corresponding to hMANF immunoreactivity, down-regulation of TH activity in the intact striatum, induction of the rotational behavior and a loss of body weight. Moreover, there were only modest levels of inflammatory and immune responses observed in the AAV9-hMANF-injected striatum at 24 weeks. These findings are consistent with our previous observations that levels of inflammatory and immune responses substantially subside in the injected striatum 24 weeks after intrastriatal injection of AAV9-hEPO, although encoded hEPO protein is also xenogeneic for rats (Yang et al., 2013). Recent several studies show that MANF possesses anti-inflammation and immune modulation properties (Chen et al., 28

ACCEPTED MANUSCRIPT 2015; Neves et al., 2016; Zhu et al., 2016), which may contribute to an explanation of reduced host response to AAV9-hMANF transduction. We

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therefore conclude that our AAV9-hMANF gene delivery system is efficacious and

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safe.

Our study has demonstrated that intrastriatal injection of AAV9-hMANF vectors

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leads to both neuroprotection and neuroregeneration of the nigrostriatal DA

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pathway in 6-OHDA-lesioned parkinsonian rats. Intrastriatal administration of 6OHDA is a partial lesion model of PD, which is characterized by a progressive

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axonal degeneration of nigral DA neurons (Kirik et al., 1998; Przedborski et al., 1995; Sauer and Oertel, 1994). This model is widely used to examine effects of

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treatments on both neuroprotection of DA neurons during the acute phase of axonal loss and atrophy of cell bodies, and neuroregeneration of DA neurons during the chronic phase of protracted neurodegeneration (Bjorklund et al., 1997; Chen et al., 2014; Lee et al., 1996). Our data showed that intrastriatal injection of 15 μg of 6-OHDA led to a 33% loss of nigral TH-IR DA neurons 10 days after lesion. At this time-point, we injected AAV9-hMANF into the same side of the striatum to examine effects of AAV9-hMANF gene delivery on neuroprotection and neuroregeneration in the nigrostriatal DA pathway. It has been shown that this time frame falls within the therapeutic window for MANF and CDNF (Lindholm et al., 2007; Voutilainen et al., 2011; Voutilainen et al., 2009). As nigral 29

ACCEPTED MANUSCRIPT DA neurons underwent a protracted and retrograde degeneration, the loss of nigral TH-IR DA cells was further increased from 33% at 10 days to 67% in both

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6-OHDA-GFP and 6-OHDA-Saline groups 8 weeks after lesion. In contrast,

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intrastriatal injection of AAV9-hMANF prevented progressive reduction in nigral

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TH-IR neurons and loss of striatal TH-IR fibers and the same number of nigral

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TH-IR cells were maintained up to 16 weeks following viral injection. It is speculated that rescued DA neurons include cells that temporarily lose DA

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phenotype but are not fully destroyed after 6-OHDA lesion. A previous study

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showed that axonal regeneration of DA neurons occurred at the first 4-5 months

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after intrastriatal 6-OHDA lesion (Kirik et al., 2000). Our data showed that striatal

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TH-IR fiber density was significantly greater and the loss area of striatal TH-IR fibers was significantly lower at 16 weeks after AAV9-hMANF injection than that at 0 week, while the number of nigral TH-IR neurons remained the same levels at 0 week, 6 weeks and 16 weeks. These results suggest that AAV9-mediated hMANF gene transfer into the striatum promotes the regeneration of DA fibers, probably by facilitating regrowth of DA fibers and axonal branching from remaining axons. The neuroregeneration of striatal DA fibers may have clinical significance because this may increase the sensitivity to PD medications. Detailed morphological studies are needed to examine how MANF facilitates axonal regeneration of DA fibers. In good agreement with a previous study 30

ACCEPTED MANUSCRIPT showing that MANF possesses neuroprotective and neurorestorative effects on nigral DA neurons in a rat model of PD (Voutilainen et al., 2009), our study

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provides novel supporting evidence that intrastriatal delivery of the hMANF gene

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by AAV9 vectors exerts long-term cellular and functional benefits on the

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nigrostriatal DA system in 6-OHDA-lesioned rats. Moreover, it is very important to

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also examine effects of our AAV9-hMANF system in other animal models of PD,

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such as an α-synuclein model (Decressac et al., 2011; Lo Bianco et al., 2004). A recent study reported that intrastriatal overexpression of MANF mediated by

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lentiviruses did not improve rotational behavior or protect nigral DA neurons against 6-OHDA-induced degeneration in parkinsonian rats (Cordero-Llana et al.,

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2014). These results are contradictory to our results and others (Voutilainen et al., 2009). Several facts may contribute to this discrepancy. Firstly, Cordero-Llana and colleagues claimed that their 6-OHDA lesion led to an up to 90% loss of nigral DA neurons which was too severe to be rescued (Cordero-Llana et al., 2014). Neuroprotective strategies in PD therapy have a potential opportunity to provide clinically meaningful benefits in the earlier stage of the disease when the nigrostriatal system is hypofunctional but not completely destroyed (Kordower et al., 2013). In animal studies, when neurotrophic factors are applied into the striatum, therapeutic proteins need to be axonal transported into the distal area, the SN, to exert their neuroprotective effects. If the nigrostriatal DA pathway is 31

ACCEPTED MANUSCRIPT subjected to severe damage, nigral DA neurons cannot be rescued due to poor axonal transport and/or cell death. Secondly, the fail of neuroprotection may

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result from low local levels of MANF. The titer of lentivirus was relatively low

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(8×108 TU/ml) and intrastriatal injection of lentivirus only led to a local

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transduction. A previous study showed that MANF exerted its beneficial effects in

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a dose-dependent manner (Voutilainen et al., 2009).

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Protective effects of MANF on DA neurons may be by both intra- and extracellular dual modes of action. In our study, 6-OHDA treatment resulted in

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an upregulation of endogenous MANF in SH-SY5Y cells with the similar pattern as chaperone Bip (Supplemental Fig. 2), suggesting that endogenous MANF

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may play an important role in counteracting the ER stress. Numerous studies have suggested that MANF may exert its effects by regulating the ER stress and UPR (Apostolou et al., 2008; Glembotski et al., 2012; Henderson et al., 2013; Lindahl et al., 2014; Parkash et al., 2009). Our data showed that intracellularly overexpressed MANF localized in the ER lumen and inhibited the activation of ER stress-associated three pathways, including protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and ATF6α pathway. Importantly, the cell viability was significantly increased in MANF-overexpressed cells following 6OHDA treatment. These results suggest that intracellular application of MANF protects cells from 6-OHDA toxicity at least partially via alleviating the ER stress. 32

ACCEPTED MANUSCRIPT However, protective mechanisms of extracellular MANF seem to be different from intracellular MANF, because that extracellular rhMANF protein did not inhibit 6-

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OHDA-induced ER stress although the cell viability was significantly increased.

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Our data showed that extracellular MANF protected cells against 6-OHDA-

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induced toxicity via activating the PI3K/Akt/mTOR pathway. It has been reported

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that extracellular application of MANF cannot enter the cells (Hellman et al., 2011). Extracellular MANF therefore probably activates the PI3K/Akt/mTOR

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pathway via the receptor mediation. Moreover, PKC and NFκB pathways have

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also been reported to be involved in cytoprotective effects of MANF (Chen et al.,

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2015; Yang et al., 2014). Nevertheless, whether these pro-survival pathways are

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related to MANF-mediated ER effects needs to be further explored. In conclusion, AAV9-mediated hMANF gene transfer into the striatum exerts long-term neuroprotective and neuroregenerative effects on nigral DA neurons against 6-OHDA-induced neurodegeneration in parkinsonian rats. Robust and stable expression of the hMANF gene seems not to have overt side effects. Activation of the PI3K/Akt/mTOR pathway and inhibition of the ER stress may constitute mechanisms underlying MANF neuroprotection for DA cells against 6OHDA-induced toxicity. Our study supports the development of clinical trials on MANF-based gene therapy for PD.

33

ACCEPTED MANUSCRIPT Conflict of interest

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All authors have declared that no conflict of interest exists.

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Acknowledgments

This study was supported by grants from the National Key Basic Research

China

(81271388,

QYX),

Beijing

Natural

Science

Foundation

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of

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Program of China (2011CB504100, WMD), National Natural Science Foundation

(KZ201110025022, WMD) and Capital Medical University Natural Science

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Foundation (2016ZR08, CY). We thank Mart Saarma (University of Helsinki) for

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kindly providing the plasmid pCR3.1-hMANF and Huan-Ying Zhao (Capital

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Medical University) for her excellent technical help in primer and probe design.

34

ACCEPTED MANUSCRIPT References

PT

Apostolou, A., Shen, Y., Liang, Y., Luo, J., Fang, S., 2008. Armet, a UPR-

RI

upregulated protein, inhibits cell proliferation and ER stress-induced cell

SC

death. Exp. Cell Res. 314, 2454-2467.

Back, S., Peranen, J., Galli, E., Pulkkila, P., Lonka-Nevalaita, L., Tamminen, T.,

NU

Voutilainen, M.H., Raasmaja, A., Saarma, M., Mannisto, P.T., Tuominen, R.K.,

MA

2013. Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease. Brain Behav. 3, 75-88.

TE

D

Bjorklund, A., Rosenblad, C., Winkler, C., Kirik, D., 1997. Studies on neuroprotective and regenerative effects of GDNF in a partial lesion model of

AC CE P

Parkinson's disease. Neurobiol. Dis. 4, 186-200. Cearley, C.N., Wolfe, J.H., 2006. Transduction characteristics of adenoassociated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. Mol. Ther. 13, 528-537. Chen, L., Feng, L., Wang, X., Du, J., Chen, Y., Yang, W., Zhou, C., Cheng, L., Shen, Y., Fang, S., Li, J., Shen, Y., 2015. Mesencephalic astrocyte-derived neurotrophic factor is involved in inflammation by negatively regulating the NF-kappaB pathway. Sci. Rep. 5, 8133. Chen, S.S., Yang, C., Hao, F., Li, C., Lu, T., Zhao, L.R., Duan, W.M., 2014. Intrastriatal GDNF gene transfer by inducible lentivirus vectors protects 35

ACCEPTED MANUSCRIPT dopaminergic neurons in a rat model of parkinsonism. Exp. Neurol. 261, 8796.

PT

Cordero-Llana, O., Houghton, B.C., Rinaldi, F., Taylor, H., Yanez-Munoz, R.J.,

RI

Uney, J.B., Wong, L.F., Caldwell, M.A., 2014. Enhanced Efficacy of the

SC

CDNF/MANF Family by Combined Intranigral Overexpression in the 6-

NU

OHDA Rat Model of Parkinson's Disease. Mol. Ther. 23, 244-254. Decressac, M., Ulusoy, A., Mattsson, B., Georgievska, B., Romero-Ramos, M.,

MA

Kirik, D., Bjorklund, A., 2011. GDNF fails to exert neuroprotection in a rat

D

alpha-synuclein model of Parkinson's disease. Brain 134, 2302-2311.

TE

Georgievska, B., Kirik, D., Bjorklund, A., 2002. Aberrant sprouting and

AC CE P

downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by long-lasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer. Exp. Neurol. 177, 461-474.

Georgievska, B., Kirik, D., Bjorklund, A., 2004. Overexpression of glial cell linederived neurotrophic factor using a lentiviral vector induces time-and dosedependent downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system. J. Neurosci. 24, 6437-6445. Glembotski, C.C., Thuerauf, D.J., Huang, C., Vekich, J.A., Gottlieb, R.A., Doroudgar, S., 2012. Mesencephalic astrocyte-derived neurotrophic factor 36

ACCEPTED MANUSCRIPT protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion. J. Biol. Chem. 287, 25893-

PT

25904.

RI

Hao, Q., An, J.Q., Hao, F., Yang, C., Lu, T., Qu, T.Y., Zhao, L.R., Duan, W.M.,

Differentiation

of

Adult

Human

Bone

Marrow-Derived

NU

Multilineage

SC

2015. Inducible Lentivirus-Mediated Expression of the Oct4 Gene Affects

Mesenchymal Stem Cells. Cell Reprogram. 17, 347-359.

MA

Hellman, M., Arumae, U., Yu, L.Y., Lindholm, P., Peranen, J., Saarma, M., Permi,

D

P., 2011. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a

TE

unique mechanism to rescue apoptotic neurons. J. Biol. Chem. 286, 2675-

AC CE P

2680.

Henderson, M.J., Richie, C.T., Airavaara, M., Wang, Y., Harvey, B.K., 2013. Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors. J. Biol. Chem. 288, 4209-4225. Kirik, D., Rosenblad, C., Bjorklund, A., 1998. Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat. Exp. Neurol. 152, 259-277. Kirik, D., Rosenblad, C., Bjorklund, A., Mandel, R.J., 2000. Long-term rAAV37

ACCEPTED MANUSCRIPT mediated gene transfer of GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the

PT

lesioned nigrostriatal system. J. Neurosci. 20, 4686-4700.

RI

Kordower, J.H., Olanow, C.W., Dodiya, H.B., Chu, Y., Beach, T.G., Adler, C.H.,

SC

Halliday, G.M., Bartus, R.T., 2013. Disease duration and the integrity of the

NU

nigrostriatal system in Parkinson's disease. Brain 136, 2419-2431. Lee, C.S., Sauer, H., Bjorklund, A., 1996. Dopaminergic neuronal degeneration

MA

and motor impairments following axon terminal lesion by instrastriatal 6-

D

hydroxydopamine in the rat. Neuroscience 72, 641-653.

TE

Lindahl, M., Danilova, T., Palm, E., Lindholm, P., Voikar, V., Hakonen, E., Ustinov,

AC CE P

J., Andressoo, J.O., Harvey, B.K., Otonkoski, T., Rossi, J., Saarma, M., 2014. MANF is indispensable for the proliferation and survival of pancreatic beta cells. Cell Rep. 7, 366-375. Lindholm, P., Peranen, J., Andressoo, J.O., Kalkkinen, N., Kokaia, Z., Lindvall, O., Timmusk, T., Saarma, M., 2008. MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain. Mol. Cell Neurosci. 39, 356-371. Lindholm, P., Saarma, M., 2010. Novel CDNF/MANF family of neurotrophic factors. Dev. Neurobiol. 70, 360-371. Lindholm, P., Voutilainen, M.H., Lauren, J., Peranen, J., Leppanen, V.M., 38

ACCEPTED MANUSCRIPT Andressoo, J.O., Lindahl, M., Janhunen, S., Kalkkinen, N., Timmusk, T., Tuominen, R.K., Saarma, M., 2007. Novel neurotrophic factor CDNF

PT

protects and rescues midbrain dopamine neurons in vivo. Nature 448, 73-77.

RI

Lo Bianco, C., Deglon, N., Pralong, W., Aebischer, P., 2004. Lentiviral nigral

SC

delivery of GDNF does not prevent neurodegeneration in a genetic rat model

NU

of Parkinson's disease. Neurobiol. Dis. 17, 283-289. Manfredsson, F.P., Burger, C., Rising, A.C., Zuobi-Hasona, K., Sullivan, L.F.,

MA

Lewin, A.S., Huang, J., Piercefield, E., Muzyczka, N., Mandel, R.J., 2009.

D

Tight Long-term dynamic doxycycline responsive nigrostriatal GDNF using a

TE

single rAAV vector. Mol. Ther. 17, 1857-1867.

AC CE P

Masamizu, Y., Okada, T., Kawasaki, K., Ishibashi, H., Yuasa, S., Takeda, S., Hasegawa, I., Nakahara, K., 2011. Local and Retrograde Gene Transfer into Primate Neuronal Pathways Via Adeno-Associated Virus Serotype 8 and 9. Neuroscience 193, 249-258. Matlik, K., Yu, L.Y., Eesmaa, A., Hellman, M., Lindholm, P., Peranen, J., Galli, E., Anttila, J., Saarma, M., Permi, P., Airavaara, M., Arumae, U., 2015. Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity. Cell Death Dis. 6, e2032. Mizobuchi, N., Hoseki, J., Kubota, H., Toyokuni, S., Nozaki, J., Naitoh, M., Koizumi, A., Nagata, K., 2007. ARMET is a soluble ER protein induced by 39

ACCEPTED MANUSCRIPT the unfolded protein response via ERSE-II element. Cell Struct. Funct. 32, 41-50.

PT

Neves, J., Zhu, J., Sousa-Victor, P., Konjikusic, M., Riley, R., Chew, S., Qi, Y.,

RI

Jasper, H., Lamba, D.A., 2016. Immune modulation by MANF promotes

SC

tissue repair and regenerative success in the retina. Science 353, aaf3646.

NU

Palgi, M., Lindstrom, R., Peranen, J., Piepponen, T.P., Saarma, M., Heino, T.I., 2009. Evidence that DmMANF is an invertebrate neurotrophic factor

MA

supporting dopaminergic neurons. Proc. Natl. Acad. Sci. U. S. A. 106, 2429-

D

2434.

TE

Parkash, V., Lindholm, P., Peranen, J., Kalkkinen, N., Oksanen, E., Saarma, M.,

AC CE P

Leppanen, V.M., Goldman, A., 2009. The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional. Protein Eng. Des. Sel. 22, 233-241. Petrova, P., Raibekas, A., Pevsner, J., Vigo, N., Anafi, M., Moore, M.K., Peaire, A.E., Shridhar, V., Smith, D.I., Kelly, J., Durocher, Y., Commissiong, J.W., 2003. MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J. Mol. Neurosci. 20, 173-188. Przedborski, S., Levivier, M., Jiang, H., Ferreira, M., Jackson-Lewis, V., Donaldson, D., Togasaki, D.M., 1995. Dose-dependent lesions of the dopaminergic nigrostriatal pathway induced by intrastriatal injection of 640

ACCEPTED MANUSCRIPT hydroxydopamine. Neuroscience 67, 631-647. Ren, X., Zhang, T., Gong, X., Hu, G., Ding, W., Wang, X., 2013. AAV2-mediated

PT

striatum delivery of human CDNF prevents the deterioration of midbrain

RI

dopamine neurons in a 6-hydroxydopamine induced parkinsonian rat model.

C.,

Georgievska,

B.,

Kirik,

D.,

2003.

Long-term

striatal

NU

Rosenblad,

SC

Exp. Neurol. 248, 148-156.

overexpression of GDNF selectively downregulates tyrosine hydroxylase in

MA

the intact nigrostriatal dopamine system. Eur. J. Neurosci. 17, 260-270.

neurons

following

intrastriatal

terminal

lesions

with

6-

TE

dopamine

D

Sauer, H., Oertel, W.H., 1994. Progressive degeneration of nigrostriatal

AC CE P

hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59, 401-415. Tadimalla, A., Belmont, P.J., Thuerauf, D.J., Glassy, M.S., Martindale, J.J., Gude, N., Sussman, M.A., Glembotski, C.C., 2008. Mesencephalic astrocytederived neurotrophic factor is an ischemia-inducible secreted endoplasmic reticulum stress response protein in the heart. Circ. Res. 103, 1249-1258. Voutilainen, M.H., Back, S., Peranen, J., Lindholm, P., Raasmaja, A., Mannisto, P.T., Saarma, M., Tuominen, R.K., 2011. Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease. Exp. Neurol. 228, 99-108. 41

ACCEPTED MANUSCRIPT Voutilainen, M.H., Back, S., Porsti, E., Toppinen, L., Lindgren, L., Lindholm, P., Peranen, J., Saarma, M., Tuominen, R.K., 2009. Mesencephalic astrocyte-

PT

derived neurotrophic factor is neurorestorative in rat model of Parkinson's

RI

disease. J. Neurosci. 29, 9651-9659.

SC

Wang, H., Ke, Z., Alimov, A., Xu, M., Frank, J.A., Fang, S., Luo, J., 2014.

NU

Spatiotemporal Expression of MANF in the Developing Rat Brain. PLoS One 9, e90433.

MA

Xue, Y.Q., Ma, B.F., Zhao, L.R., Tatom, J.B., Li, B., Jiang, L.X., Klein, R.L., Duan,

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W.M., 2010. AAV9-mediated erythropoietin gene delivery into the brain

TE

protects nigral dopaminergic neurons in a rat model of Parkinson's disease.

AC CE P

Gene Ther. 17, 83-94.

Yang, C., Yang, W.H., Chen, S.S., Ma, B.F., Li, B., Lu, T., Qu, T.Y., Klein, R.L., Zhao, L.R., Duan, W.M., 2013. Pre-immunization with an intramuscular injection of AAV9-human erythropoietin vectors reduces the vector-mediated transduction following re-administration in rat brain. PLoS One 8, e63876. Yang, S., Huang, S., Gaertig, M.A., Li, X.J., Li, S., 2014. Age-Dependent Decrease in Chaperone Activity Impairs MANF Expression, Leading to Purkinje Cell Degeneration in Inducible SCA17 Mice. Neuron 81, 349-365. Yu, Y., Wang, K., Deng, J., Sun, M., Jia, J., Wang, X., 2016. Electroacupuncture Produces the Sustained Motor Improvement in 6-Hydroxydopamine42

ACCEPTED MANUSCRIPT Lesioned Mice. PLoS One 11, e0149111. Yu, Y.Q., Liu, L.C., Wang, F.C., Liang, Y., Cha, D.Q., Zhang, J.J., Shen, Y.J.,

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Wang, H.P., Fang, S., Shen, Y.X., 2010. Induction profile of MANF/ARMET

RI

by cerebral ischemia and its implication for neuron protection. J. Cereb.

SC

Blood Flow Metab. 30, 79-91.

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Zhu, W., Li, J., Liu, Y., Xie, K., Wang, L., Fang, J., 2016. Mesencephalic astrocyte-derived neurotrophic factor attenuates inflammatory responses in

of

p38-MAPKs

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phosphorylation

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lipopolysaccharide-induced neural stem cells by regulating NF-kappaB and

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Immunotoxicol. 38, 205-213.

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pathways.

Immunopharmacol.

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nd

2 str injection

6-OHDA alone

6-OHDA

6-OHDA-hMANF

6-OHDA

AAV9-hMANF

6-OHDA-GFP

6-OHDA

AAV9-GFP

6-OHDA-Saline

6-OHDA

Saline

6-OHDA

AAV9-hMANF

6-OHDA

AAV9-GFP

6-OHDA

Saline

Time nd (Post-2 str injection)

IHC or IF

HPLC

WB

qRT-PCR

0 week

6

6

6 weeks

6

7

3

3

6

3

3

6

3

3

5

7

3

3

5

6

3

3

5

6

3

3

4

4

4

4

4

4

4

4

5

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1 str injection

GFP

AAV9-GFP

Saline

Saline

Naïve

No-injection

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AAV9-hMANF

24 weeks

st

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hMANF

16 weeks

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5

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st

Group

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Table 1. Summary of rats used in each part of the study

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Notes and abbreviations: The interval time between the 1 and 2 striatal injection was 10 days. The values shown were the number of rats used in each part of the study. 6-OHDA, 6-hydroxydopamine; AAV9, adenoassociated virus serotype 9; hMANF, human mesencephalic astrocyte-derived neurotrophic factor; GFP, green fluorescent protein; Str, striatum.

44

ACCEPTED MANUSCRIPT Figure Legends

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Fig. 1. Transgene expression in 6-hydroxydopamine (6-OHDA) lesioned rat brain. (a) Structure of an adeno-associated virus serotype 9 (AAV9) - human mesencephalic

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astrocyte-derived neurotrophic factor (hMANF) vector. (b) Structure of an AAV9-green

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fluorescence protein (GFP) vector. (c-f) Representative immunostained sections

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showing the expression of hMANF (c,d) or GFP (e,f) in the injected striatum 6 weeks and 16 weeks after viral injection. (g,h) Representative immunofluorescent stained

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sections showing co-localization of MANF-immunoreactive (g) or GFP-expressing (h)

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cells (green) with NeuN (red) in the injected striatum. (i-l) Representative

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immunofluorescent stained sections showing co-localization of MANF-immunoreactive

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or GFP-expressing cells (green) with tyrosine hydroxylase (TH) (red) in the ipsilateral substantia nigra pars compacta (SNpc), and MANF-immunoreactive or GFP-expressing fibers (green) in the ipsilateral SN pars reticulata (SNpr). Scale bar = 1 mm for c and d; 500 μm for e and f; 100 μm for g-l; 10 μm for high magnification images in g-l. Fig. 2. Levels of human mesencephalic astrocyte-derived neurotrophic factor (hMANF) protein and mRNA in striatal and nigral tissues of 6-hydroxydopamine (6-OHDA) lesioned rats. (a,b) Western blot showing the fold expression of MANF protein in the injected striatum and ipsilateral substantia nigra (SN) relative to the contralateral striatum and SN 6 weeks (a) and 16 weeks (b) after intrastriatal viral injection. N=3 per group. (c,d) Quantitative PCR showing levels of hMANF mRNA in the striatum and SN 6 weeks (c) and 16 weeks (d) after intrastriatal viral injection. N=3 per 45

ACCEPTED MANUSCRIPT group. L, left; R, right.

mesencephalic

astrocyte-derived

neurotrophic

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Fig. 3. Intrastriatal delivery of adeno-associated virus serotype 9 (AAV9) - human factor

(hMANF)

improves

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rotational asymmetry and promotes regeneration of striatal tyrosine hydroxylase

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(TH) - immunoreactive fibers and survival of nigral TH-immunoreactive neurons

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in 6-hydroxydopamine (6-OHDA) lesioned rats. (a) The diagram summarizes scores of net rotational asymmetry in 6-OHDA-Saline (n=17), 6-OHDA-GFP (n=17) and 6-

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OHDA-hMANF (n=18) groups 0, 2, 4, 6, 8, 12 and 16 weeks after viral injection. A one-

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factor ANOVA followed by Tukey's post hoc test was used to make group comparisons.

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*p < 0.05 versus the other two groups; #p < 0.05 versus 0 week in the 6-OHDA-hMANF

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group. (b-o) Representative immunostained sections showing TH-immunoreactivity in the striatum and substantia nigra (SN) in the 6-OHDA alone group (0 week) (b,c) and in 6-OHDA-Saline, 6-OHDA-GFP and 6-OHDA-hMANF groups 6 weeks (d-i) and 16 weeks (j-o) after viral injection. Scale bar = 1 mm for b, also for d-f and j-l; 1 mm for c, also for g-i and m-o. (p) Percentages of TH-immunoreactive fiber densities in the ipsilateral striatum relative to the contralateral striatum. (q) Percentages of the loss area of TH-immunoreactive fibers in the ipsilateral striatum. (r) Percentages of THimmunoreactive cell counts in the ipsilateral SN pars compacta (SNpc) relative to the contralateral SNpc. For p-r, a one-factor ANOVA followed by Tukey's post hoc test was used to make group comparisons. *p < 0.05, ***p < 0.001 versus the other two groups; ##p < 0.01, ###p < 0.001 versus indicated groups. N=5-6 per group. 46

ACCEPTED MANUSCRIPT Fig. 4. Intrastriatal delivery of adeno-associated virus serotype 9 (AAV9) - human mesencephalic

astrocyte-derived

neurotrophic

factor

(hMANF)

increases

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concentrations of striatal DA, DOPAC and HVA and reduces the ratio of

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DOPAC/DA in 6-hydroxydopamine (6-OHDA) lesioned rats. (a-e) Percentages of DA

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(a), DOPAC (b) and HVA (c) and ratios of DOPAC/DA (d) and (DOPAC+HVA)/DA (e) in ipsilateral striatal tissues relative to contralateral striatal tissues of rats in the 6-OHDA

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alone group (0 week) and in 6-OHDA-Saline, 6-OHDA-GFP and 6-OHDA-hMANF

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groups 6 weeks and 16 weeks after viral injection. A one-factor ANOVA followed by Tukey's post hoc test was used to make group comparisons. *p < 0.05, **p < 0.01, ***p

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< 0.001 versus the other two groups; #p < 0.05, ##p < 0.01 versus indicated groups.

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homovanillic acid.

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N=6-7 per group. DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA,

Fig. 5. Intrastriatal long-term overexpression of human mesencephalic astrocytederived neurotrophic factor (hMANF) gene in 6-hydroxydopamine (6-OHDA) untreated naïve rats does not affect the nigrostriatal dopaminergic system and does not induce obvious immune responses 24 weeks after viral injection. (a) The diagram showing scores of net rotational behavior of rats in Saline, GFP and hMANF groups 0, 4, 8, 12, 16, 20 and 24 weeks after viral injection. N=8 per group. (b) The diagram showing the body weight of rats in Saline, GFP and hMANF groups 0, 4, 8, 12, 16, 20 and 24 weeks after viral injection. N=8 per group. (c) Percentages of tyrosine hydroxylase (TH)-immunoreactive fiber densities in the ipsilateral striatum relative to 47

ACCEPTED MANUSCRIPT the contralateral striatum. N=4 per group. (d) Percentages of TH-immunoreactive cell counts in the ipsilateral substantia nigra pars compacta (SNpc) relative to the SNpc.

N=4

per

group.

(e)

Percentages

of

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contralateral

dopamine

(DA),

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dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and ratios of

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DOPAC/DA and (DOPAC+HVA)/DA in ipsilateral striatal tissues relative to contralateral striatal tissues. N=4 per group. (f-q) Representative immunostained sections showing

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hMANF and TH immunoreactivity in the adjacent striatal (f,g), lateral globus pallidus

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(LGP) (j,k) and nigral (n,o) sections. High magnification images showing inset areas (g,k,o) in the left (non-injected side) and right (injected side) striatum (h,i), LGP (l,m)

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and SN (p,q). Scale bar = 1 mm for o, also for f,g,j,k,n; 100 μm for q, also for h,i,l,m,p.

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(r-t) Representative immunostained sections showing major histocompatibility antigen

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(MHC) class II (r), complement receptor 3 (CR3) (s) and glial fibrillary acidic protein (GFAP) (t) immunoreactivity in the needle track (white arrow) of the injected striatum. Scale bar = 100 μm for t, also for r,s. Fig.

6.

Intracellular

neurotrophic

factor

overexpression

(MANF)

of

protects

mesencephalic

SH-SY5Y

cells

astrocyte-derived via

inhibiting

6-

hydroxydopamine (6-OHDA) - induced endoplasmic reticulum (ER) stress. (a) Western blot showing levels of intracellular MANF protein 24 and 48 h after transfection with

pAAV-hMANF

plasmid.

(b)

Immunofluorescent

staining

showing

the

fluorescence intensity of MANF or GFP (green) in non-transfected cells or cells transfected with pAAV-hMANF or pAAV-GFP plasmid. Scale bar = 500 μm. (c) Double 48

ACCEPTED MANUSCRIPT immunofluorescent staining showing co-localization of intracellular MANF with protein disulfide isomerase (PDI) in cells transfected with pAAV-hMANF plasmid. Scale bar =

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10 μm. (d) MTT assay showing the cell viability in 6-OHDA-treated cells with or without

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pAAV-hMANF or pAAV-GFP transfection. (e) Representative immunoreactive bands of

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p-eIF2α, ATF4, CHOP, XBP1s, Bip and ATF6α (N) in 6-OHDA-treated cells with or without pAAV-hMANF or pAAV-GFP transfection. (f) Quantitative analysis showing the

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fold expression of p-eIF2α, ATF4, CHOP, XBP1s, Bip and ATF6α (N) in 6-OHDA-treated

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cells with or without pAAV-hMANF or pAAV-GFP transfection relative to untreated control cells. A one-factor ANOVA followed by Tukey's post hoc test was used to make

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0.01 versus indicated groups.

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group comparisons. *p < 0.05, ***p < 0.001 versus the control group; #p < 0.05, ##p <

Fig. 7. Extracellular recombinant human mesencephalic astrocyte-derived neurotrophic factor (rhMANF) protein protects SH-SY5Y cells against 6hydroxydopamine (6-OHDA) - induced toxicity via the PI3K/Akt/mTOR pathway. (a) MTT assay showing the cell viability in cells treated with 6-OHDA in the absence or presence of rhMANF protein. (b) MTT assay showing the cell viability in cells treated with 6-OHDA and rhMANF protein in the absence or presence of PI3K inhibitor wortmannin (wort). (c) Western blot showing the fold expression of p-eIF2α, ATF4, CHOP, XBP1s, Bip and ATF6α (N) in cells treated with 6-OHDA in the absence or presence of rhMANF protein relative to untreated control cells. (d) Western blot showing the fold expression of p-Akt and p-mTOR in cells treated with 6-OHDA in the 49

ACCEPTED MANUSCRIPT absence or presence of rhMANF protein relative to untreated control cells. (e) Western blot showing the fold expression of p-Akt and p-mTOR in cells treated with 6-OHDA and

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rhMANF protein in the absence or presence of wort relative to untreated control cells. A

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one-factor ANOVA followed by Tukey's post hoc test was used to make group

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comparisons. For a, ***p < 0.001 versus the 6-OHDA-treated alone group. For b-e, *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group; ##p < 0.01, ###p < 0.001

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versus indicated groups. ns, no significance.

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ACCEPTED MANUSCRIPT Highlights:  Intrastriatal delivery of AAV9-hMANF leads to robust striatal transduction.  Intrastriatal delivery of AAV9-hMANF reduces rotational scores of parkinsonian rats.

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 Intrastriatal delivery of AAV9-hMANF promotes the survival of nigral DA neurons.

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 Intracellular overexpression of MANF protects SH-SY5Y cells via inhibiting ER stress.

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 Extracellular rhMANF protein protects SH-SY5Y cells via the PI3K/Akt/mTOR pathway.

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