A brain-targeted, modified neurosin (kallikrein-6) reduces α-synuclein accumulation in a mouse model of multiple system atrophy
- Brian Spencer†1Email author,
- Elvira Valera†1,
- Edward Rockenstein1,
- Margarita Trejo-Morales1,
- Anthony Adame1 and
- Eliezer Masliah1, 2
© Spencer et al. 2015
Received: 26 May 2015
Accepted: 8 September 2015
Published: 23 September 2015
Multiple system atrophy (MSA) is a progressive, neurodegenerative disease characterized by parkinsonism, resistance to dopamine therapy, ataxia, autonomic dysfunction, and pathological accumulation of α-synuclein (α-syn) in oligodendrocytes. Neurosin (kallikrein-6) is a serine protease capable of cleaving α-syn in the CNS, and we have previously shown that lentiviral (LV) vector delivery of neurosin into the brain of a mouse model of dementia with Lewy body/ Parkinson’s disease reduces the accumulation of α-syn and improves neuronal synaptic integrity.
In this study, we investigated the ability of a modified, systemically delivered neurosin to reduce the levels of α-syn in oligodendrocytes and reduce the cell-to-cell spread of α-syn to glial cells in a mouse model of MSA (MBP-α-syn). We engineered a viral vector that expresses a neurosin genetically modified for increased half-life (R80Q mutation) that also contains a brain-targeting sequence (apoB) for delivery into the CNS. Peripheral administration of the LV-neurosin-apoB to the MBP-α-syn tg model resulted in accumulation of neurosin-apoB in the CNS, reduced accumulation of α-syn in oligodendrocytes and astrocytes, improved myelin sheath formation in the corpus callosum and behavioral improvements.
Thus, the modified, brain-targeted neurosin may warrant further investigation as potential therapy for MSA.
The synucleinopathies are a heterogeneous group of neurodegenerative disorders that affect 5 million people worldwide and includes Parkinson’s disease (PD), dementia with Lewy bodies (DLB), neurodegeneration with brain iron accumulation, pure autonomic failure (PAF) and multiple system atrophy (MSA) (Reviewed in ). Multiple system atrophy is a rapidly progressive, neurological condition characterized by parkinsonism resistant to dopamine therapy, ataxia, autonomic dysfunction, and pathological accumulation of α-synuclein (α-syn) [2–4]. This disorder differs from other synucleinopathies in that α-syn accumulates not only within neurons and astrocytes, but also within oligodendrocytes in the form of glial cytoplasmic inclusions . This intracellular accumulation of toxic α-syn species leads to degeneration of oligodendroglial cells, loss of trophic support to neurons and subsequent neurodegeneration.
The mechanisms through which α-syn leads to neurotoxicity are not completely clear, however recent evidence supports a role for oligomerization [6, 7]. Increasing evidence supports the notion that α-syn, which is primarily generated by neurons, can be toxic once released to the extracellular environment [6, 8, 9]. Extracellular aggregated α-syn can then propagate to other neurons and glial cells in a prion-like fashion [10, 11]. Although it had been previously suggested that the sole source of oligodendroglial α-syn was through endocytosis, a recent report showed α-syn mRNA in MSA oligodendrocytes suggesting that the origin of oligodendroglial α-syn might be both of endogenous nature and the result of propagation from neurons and/or other oligodendroglial cells . Furthermore, propagation and accumulation of α-syn within astrocytes could lead to activation of these cells and subsequent neuroinflammation [13–15]. Therefore, the development of therapeutic interventions for MSA and related neurodegenerative disorders has been focused simultaneously on reducing α-syn accumulation, increasing α-syn clearance and preventing α-syn propagation.
Neurosin, (human kallikrein 6, KLK6, Zyme, Protease M), is a serine protease capable of cleaving α-syn [16–19]. This enzyme is found to be expressed throughout the body in many tissues  including the CNS in the choroid plexus and in oligodendrocytes and astroglial cells  of healthy individuals , as well as neurons and microglia of the hippocampus of Alzheimer’s disease patients [16, 22]. Down-regulation of neurosin is associated with accumulation of α-syn in patients with DLB/PD [23–25] as well in animal models of DLB/PD , whereas over-expression of neurosin in the brain via lentiviral (LV) vector reduces the accumulation of α-syn and improves neuronal synaptic integrity in an α-syn tg mouse model of DLB/PD .
Neurosin is expressed as a catalytically inactive pre-pro protein and is activated through autocatalytic proteolysis. Upon expression and secretion, auto-activation occurs first via cleavage of the pre-pro neurosin at Q19 followed by cleavage at K21, which produces the mature neurosin enzyme. The mature neurosin can then auto-proteolytically inactivate itself with cleavage at R80. This amino acid, when altered to Glutamine (Q), prevents the auto-inactivation generating a longer-acting enzyme . In vitro proteolytic reactions with neurosin show that autocatalytic cleavage can begin as early as 10 min after incubation, and full cleavage to the inactive form occurs by 240 min incubation .
Since we have previously shown that stereotaxic injection of LV-neurosin into the hippocampus reduced the local accumulation of neuronal α-syn in a mouse model of DLB , for this study we sought to determine if gene therapy with this vector would also reduce the neurodegenerative process in a mouse model of MSA [27, 28]. However, given that the synucleinopathy in MSA is more disseminated throughout the CNS than PD, a more systemic approach is needed.
For this reason, we engineered the recombinant neurosin for systemic delivery with the R80Q mutation to allow for longer half-life in order that the active form of the enzyme would reach the BBB, transcytose the endothelial cells, and reach the site of α-syn accumulation before it gets degraded. In addition, we fused the 38 amino acid LDL-R binding domain from apoB; which we have previously shown to transport active enzymes from the blood to the brain [29–32]. In this instance we used a lentiviral vector to deliver the gene for NR-R80Q-apoB to the liver for systemic delivery. The liver acts as a depot organ to produce and secrete the active enzyme to the blood stream. Cells expressing large amounts of the LDL-R, including hepatocytes and Kupffer cells of the liver, will clear the majority of the recombinant protein from the blood. Binding to the LDL-R of these cells will result in clearance by ESCRT-III-mediated endocytosis leading to lysosomal degradation . In contrast, at the BBB, the LDL-R will bind the recombinant protein and rather than target for degradation, will transcytose the protein to the neuronal side for release .
In this study, we show that the brain-targeted neurosin accumulated throughout the CNS in neurons, astrocytes and microglia, and resulted in reduced accumulation of α-syn in oligodendrocytes and astrocytes with possible clearance via microglia. It also ameliorated myelin degeneration, neuropathology and behavioral deficits. Thus, the modified, brain-targeted neurosin may warrant further investigation as potential therapy for MSA.
Modifications to neurosin increase its stability without decreasing its ability to degrade α-syn
We have previously shown that neurosin, when expressed from a lentiviral vector is able to degrade α-syn and promote the survival of neuronal cells in culture when challenged with exogenous α-syn . This vector was delivered by direct stereotaxic injection to an α-syn tg mouse model of DLB/PD. While this delivery route was efficient at delivering the enzyme to a localized area of the mouse brain, a more amenable route of delivery for the human brain might involve broad blood-based systemic delivery across the blood-brain barrier (BBB). For this purpose, we engineered neurosin with a novel BBB transport tag derived from the LDL-R binding domain of Apolipoprotein B, which we designated apoB [25, 29, 30, 34].
Neurosin-R80Q-apoB transits to the CNS
Immunohistochemical analysis showed higher levels of immunoreactivity in the neocortex, hippocampus and the striatum in the non-tg and MBP-α-syn tg mice injected with LV-NR-R80Q-apoB compared to mice injected with LV-Control (Fig. 2d-e). In addition to detecting the NR-R80Q-apoB in the brain, following LV-NR-R80Q-apoB systemic injection, higher levels of neurosin immunoreactivity were detected in the blood and CSF of both non-tg and MBP-α-syn tg mice compared to mice injected with LV-Control (Fig. 2f-h). Specifically, a 2-fold increase in neurosin was detected in both the blood (Fig. 2g) and CSF (Fig. 2h) in animals that received the systemic LV-NR-R80Q-apoB injection.
Neurosin-R80Q-apoB delivery to the CNS reduces α-syn accumulation in MBP-α-syn transgenic mouse model
Next, we analyzed if the reduced number of α-syn-containing oligodendrocytes in the animals treated with LV-NR-R80Q-apoB was related to proximity of these cells to cells containing the neurosin-apoB and/or to effects at the mRNA level. Double labeling/co-localization studies showed a reduction of α-syn in the MBP-α-syn tg mouse in the areas adjacent to where cells contained high levels of V5-tagged neurosin in mice that were treated with the LV-NR-R80Q-apoB vector (Additional file 2: Figure S2). In contrast, real-time PCR analysis of the MBP-α-syn tg mice treated with the LV-NR-R80Q-apoB vector showed no change in the transcription of α-syn mRNA indicating that the change in α-syn protein was due to degradation and not transcriptional alterations (Additional file 3: Figure S3). Consistent with this finding, double labeling and confocal microscopy studies with the oligodendroglial marker p25 and α-syn showed that the total number of oligodendrocytes were not significantly decreased in the striatum, however the levels of α-syn per oligodendroglial cell showed a trend to reduction between animals treated with LV-Control and LV- NR-R80Q-apoB, although this reduction was not statistically significant (Additional file 4: Figure S4A, B). Taken together, these results suggest that the intracellular level of α-syn within individual oligodendrocytes is not significantly affected by the treatment, and that the recombinant neurosin is primarily cleaving the α-syn that propagates from cell-to-cell.
Neurosin-R80Q-apoB reduces the accumulation of α-syn in astrocytes and α-syn is cleared via microglia
Neurosin-R80Q-apoB ameliorates neurodegenerative pathology in MBP-α-syn tg mice
Neurosin-R80Q-apoB ameliorated myelination deficits in the MBP-α-syn tg mice
Immunoblot analysis of myelin basic protein (MBP) from dissected brain homogenates revealed a significant reduction in total MBP protein in the LV-Control treated MBP-α-syn tg mice compared to non-tg mice (Fig. 7e, f). Treatment with LV-NR-R80Q-apoB increased levels of MBP protein to levels greater than those in non-tg mice (Fig. 7e, f). The MBP protein levels increased at the post-transcriptional level, as real-time PCR analysis of the LV-NR-R80Q-apoB treated MBP-α-syn tg mice showed no change in transcription levels of MBP mRNA (Additional file 2: Figure S2B). Furthermore, CNP protein expression is associated with increased oligodendrocytes process extension and myelination . Compared to the LV-Control-injected MBP-α-syn tg mice, CNP protein levels were increased in MBP-α-syn tg mice treated with LV-NR-R80Q-apoB indicating that the presence of elevated neurosin improved the oligodendroglial process extension and branching in the MBP mouse model (Fig. 7e).
Neurosin-R80Q-apoB reduces behavioral deficits in MBP-α-syn tg mice
We have developed a novel brain penetrating neurosin protein for systemic delivery to the CNS with the potential for treatment of synucleinopathies. In addition to the stabilizing point mutation (R80Q), our fusion protein contained a C-terminal apoB tag that facilitates trafficking of the protein into the CNS via the LDL-R. We showed that the LV-NR-R80Q-apoB reduced the accumulation of α-syn and the propagation of α-syn from oligodendrocytes to astroglia, ameliorated the neurodegenerative pathology including myelination and reversed the behavioral deficits in the MBP-α-syn tg mouse model of MSA. This is the first application of this stabilized neurosin for a therapeutic purpose and represents a novel approach for treatment of a unique synucleinopathy that affects to a greater extent the oligodendrocytes. We chose to test the NR-R80Q-apoB in an MSA model because this construct distributes in multiple brain regions and in MSA there is more diffuse and widespread disease when compared to other synucleinopathies such as PD.
Although neurosin is predominantly expressed in the CNS, many kallikreins exhibit aberrant expression in peripheral tumors such as those of breast and prostate. Interestingly, in both these tumors there is a reduced expression of the kallikreins PSA, kallikrein 6 and NES1 that is associated with cell outgrowth, suggesting that kallikrein expression may have an anti-tumor function in these tissues. In fact, recent investigation of KLK5 has shown that expression in breast tissue regulates several anti-oncogenic miRNAs [40, 41]. In the CNS, neurosin displays remarkably restricted substrate specificity, lacking chymo-, trypsin-, urokinase-, or elastase- specific substrate specificity. In fact, other than α-syn, the primary CNS substrate appears to be PAR-2 where cleavage occurs at S37 to activate the receptor; however, neurosin fails to activate PAR-1 or PAR-4. Activation of PAR-2 by excess neurosin could be potentially harmful to neurons as it elicits elevations in intracellular Ca+ levels , and elevated intracellular Ca+ has been shown to be neurotoxic in cultured hippocampal neurons . However, in this study examination of neuronal cells immunolabeled with NeuN did not show any significant loss of neurons following systemic NR-R80Q-apoB; on the contrary, we found a rescue effect. Further studies should examine off-target effects such as these.
Neurosin protein expression is observed in oligodendrocytes with reduced expression during demyelination [21, 44] and knockout of neurosin reduces MBP protein in the spinal cord of experimental mice indicating that neurosin regulates myelination via MBP . Although the exact role of neurosin activity on myelination or maturation of MBP is not known, we did observe an increase in myelination and MBP following delivery of the recombinant NR-R80Q-apoB to the MBP-α-syn tg mouse. This is consistent with the possibility that neurosin might improve the demyelination phenotype in the mice by modulating MBP. This was observed both by electron microscopy and LFB staining of the corpus callosum. Interestingly, we did not observe increased neurosin protein in the oligodendrocytes; however, we did observe a reduction in the number of α-syn positive oligodendrocytes in the corpus callosum. This is probably due to the lack of LDL-R on these cells preventing the uptake of the neurosin-apoB fusion protein. In addition, we did not observe a change in the expression of the MBP mRNA, suggesting that the change in MBP we observe is at the post-transcriptional level. The increased MBP in the corpus callosum may be due to effects of extracellular neurosin directly on MBP either by stabilizing or by processing the protein. Alternatively, the increased MBP may be due to an overall increase in the overall health of oligodendrocyte cells due to an improvement in the extracellular environment caused by the reduction of extracellular α-syn by neurosin.
Without specific evidence that neurosin is degrading α-synuclein extracellularly or intracellularly, we cannot definitively rule out other hypotheses and it is clear that further experimental data is needed to better understand how neurosin reduces α-syn in astrocytes. However, neurosin taken up by astrocytes via the LDL-R would be targeted to the lysosome through native LDL-R pathway leading to an acidic environment . Neurosin is known to function best in a neutral environment  providing further evidence that little if any α-synuclein degradation would be occurring in astrocyte lysosomes and instead most would be occurring in the extracellular environment.
Taken together these findings suggest that this might be the result of a combination of reduced extracellular α-syn and reduced propagation of α-syn. Interestingly, double labeling studies showed co-localization of α-syn to microglial cells in the MBP-α-syn mice treated with LV-NR-R80Q-apoB, suggesting that in the presence of neurosin, extracellular α-syn was cleared via the microglia. This is consistent with previous studies using anti-α-syn antibodies showing that microglial cells are capable of clearing α-syn [46–48]. A recent study showed that neurosin had reduced degradation of extra-cellular α-syn when in the presence of lipids or fatty acids . The reduction in total α-syn we observed might be due to higher than normal levels of neurosin in the extra-cellular environment leading to proteolysis, increased degradation by microglia or increased intra-cellular proteolysis in astrocytes.
Considering many synucleinopathies involve the accumulation of α-syn in neurons, astrocytes and oligodendroglial cells and involve the propagation of α-syn from cell-to-cell, the use of NR-R80Q-apoB should be considered a potential therapeutic for other synucleinopathies such as PD and DLB. Systemic delivery of the recombinant protein by intra-peritoneal infusion followed by transport across the BBB for treatment of the whole brain would facilitate a non-invasive and broad-based therapeutic option for numerous α-syn related pathologies.
Construction of lentivirus vectors
The mouse Neurosin cDNA (Open Biosystems) was PCR amplified and cloned into the third generation self-inactivating lentivirus vector  with the CMV promoter driving expression producing the vector LV-Neurosin as previously described . The lentivirus vector expressing the human wild-type α-syn has been previously described. The R80Q neurosin mutation was generated as described  using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Addition of the 38 amino acid apoB LDL-R binding domain to the c-terminus of both wild-type neurosin and the R80Q neurosin was performed by site directed mutagenesis addition of a unique AgeI restriction site in frame followed by cloning of the oligonucleotide coding for the apoB peptide [29, 30, 32]. Lentiviruses expressing the neurosin (LV-wt-NR), neurosin-apoB (LV-wt-NR-apoB), neurosin R80Q (LV-NR-R80Q), neurosin R80Q-apoB (LV-NR-R80Q-apoB, α-synuclein (LV-α-syn) or empty vector (LV-Control) were prepared by transient transfection in 293T cells .
Establishment of in vitro cultures
For these experiments we used the rat neuroblastoma cell line B103. This model was selected because overexpression of α-syn in these cells results in mitochondrial alterations, reduced cell viability, defective neurite outgrowth and abnormal accumulation of oligomeric α-syn . For all experiments, cells were infected with LVs at a multiplicity of infection (MOI) of 20.
The B103 cells were grown as described above and plated onto poly L-lysine coated glass coverslips at a density of 5 × 104 cells. Five hours after plating, cells were infected with the LV-Neurosin vectors and incubated for 48 h. Control experiments were performed with cells infected with LV-Control. For co-culture analysis, 5 × 104 B103 cells were plated onto poly L-lysine coated glass coverslips and infected with LV-Neurosin vectors or onto 12 well cell culture inserts containing a 0.4 μm PET membrane (Fisher Scientific) and infected with LV-α-syn vectors. Cultures were incubated separately for 6 h to allow cells to attach and then co-cultured until analysis.
BV2 microglia cells were grown as previously described  and plated on poly L-lysine coated glass coverslips at a density of 5 × 104 cells for 24 h. Cultured supernatent from LV-NR-R80Q-apoB or LV-Control infected B103 cells was collected and passed through a 0.22 μm filter to remove any cells. The supernatent was then incubated with 500nM of previously oligomerized α-syn  for 32 h at 37 °C and then added to BV2 cells for 1 h at 37 °C, 5 % CO2. At the conclusion of all experiments, cells were washed twice with ice-cold PBS followed by fixation with cold 4 % PFA.
Treatment of animals
Mice expressing human α-syn under the control of the Myelin Basic Protein (MBP) promoter (MBP-α-syn tg) were generated as previously described . In this study we used the MBP1 line, as animals express an intermediate level of α-syn compared to the other lines and they are more viable and less aggressive. MBP-α-syn tg mice develop progressive accumulation of α-syn inclusions in oligodendrocytes along the axonal tracts in the brainstem, basal ganglia, cerebellum, corpus callosum, and neocortex, leading to neurodegeneration in the neocortex and to loss of dopaminergic fibers in the basal ganglia. Non-tg and MBP-α-syn tg mice received a single intra-peritoneal injection of LV-Control (1.0 × 109 TDU/mouse) or LV-NR-R80Q-apoB (1.0 × 109 TDU/mouse) (n = 10/group) in a volume of 300 μl. We, and others have previously shown that this method of vector delivery results in lentiviral transduction of the liver (sinusoidal cells and to a lesser extent hepatocytes) and the spleen (splenocytes) where the secreted protein is expressed and transported by the blood to the blood–brain barrier [32, 55]. Treatment lasted 3 months with all mice between 9 and 10 months of age by the end of the study. All experiments were carried out in accordance with the guidelines laid down by the NIH regarding the care and use of animals for experimental procedures and approved by the Institutional Animal Care and Use Committee at the University of California at San Diego (UCSD) under protocol #S02221.
Context-dependent learning in an open field was analyzed using a Kinder SmartFrame Cage Rack Station activity monitor system (Kinder Scientific, Poway, CA), in 3-dimensional space using a 7 × 15 beam configuration. Data collection began when an animal was placed in the test chamber. Animals were evaluated for 10 min for three consecutive days, given a two-day dishabituation period, followed by a fourth trial .
Real-time PCR analysis
Total RNA was extracted from the mouse anterior hemibrain using a Qiagen RNeasy kit and following the instructions of the manufacturer. RNA concentration was determined and 0.5 μg of RNA per sample were used for reverse transcription to cDNA using a High capacity cDNA reverse transcription kit (Applied Biosystems). cDNA solutions were diluted 1:10 in ultrapure water and 4 μl of this dilution were used per reaction. Real time PCR (qPCR) was performed using Fast SYBR Master Mix and primers for human α-syn, mouse MBP, TNFα, IL-1ß, IL-6, IL-10 and ß-actin as internal control [35, 57]. qPCR reactions were run in a StepOnePlus Real-Time PCR system (Applied Biosystems) and ΔΔCt calculations were made using StepOne software (Applied Biosystems).
Hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [54, 58]. For immunoblot analysis, 20 μg of total protein per lane was loaded on 4–12 % Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. Membranes were probed with antibodies against full-length human α-syn (SYN211, Life Technologies), human neurosin (R&D Systems) or the epitope tag V5 (Life Technologies). Incubation with primary antibody was followed by species-appropriate incubation with secondary antibody tagged with horseradish peroxidase (Santa Cruz Biotechnology) and visualization with enhanced chemiluminescence.
Heparinized blood (plasma) (diluted 1:10 in PBS) and CSF (undiluted) samples were loaded on 4-12 % Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes and probed with antibodies against human neurosin (R&D Systems) as described above. Analysis of all immunoblot was performed with a Versadoc XL imaging apparatus (BioRad) using β-actin (Sigma) levels as a loading control.
Immunohistochemistry and electron microscopy
At the end of the experiment, CSF and blood were collected and animals were transcardially perfused with physiological saline and brains were collected. Brains were divided sagittally into right and left hemibrains. The left hemibrain was fixed in 4 % paraformaldehyde in phosphate buffered saline and serially sectioned in the vibratome at 40 μm and stored at −20 °C in cryoprotective medium. The right hemibrain was snap-frozen and stored at −80 °C for subsequent protein extraction. Sections were immunostained with antibodies against α-syn (Chemicon), NeuN (neuronal marker, Millipore), GFAP (astroglial marker, Millipore), Iba-1 (microglia, Wako), Olig-2 (oligodendrocytes, Cell Signaling) and p25 (oligodendrocytes, IBL) and imaged with an Olympus BX54 bright field digital microscope or a laser scanning confocal microscope. Digital images were analyzed with the ImageQuant 1.43 program (NIH) to determine numbers of α-syn aggregates, neurons, astrogliosis and microgliosis [28, 46]. A minimum of 100 cells with 4 fields were counted per animals and expressed as the average number of positive cells per field (230 μm × 184 μm). Stereological analysis of NeuN, Iba-1 and α-syn immunoreactivity was conducted by the dissector method using the Stereo-Investigator System (MBF Bioscience), and the results were averaged and expressed as cell counts per 0.1 mm3. Additional sections were stained with luxol fast blue (LFB, Acros) in order to visualize the myelin layers and imaged on the Olympus BX54 bright field digital microscope . Optical density measurements were obtained using the ImageQuant software, and quantification performed by correcting against background signal levels.
For electron microscopy, vibratome sections were postfixed in 1 % glutaraldehyde, treated with osmium tetraoxide, embedded in epon araldite and sectioned with the ultramicrotome (Leica). Grids were analyzed with a Zeiss OM 10 electron microscope as previously described . Cells were randomly acquired from 3 grids, and electron micrographs were obtained at a magnification of 5,000X and 25,000X.
Double immunolabeling and confocal microscopy
To determine the co-localization between α-syn or neurosin and cellular markers, double-labeling experiments were performed as previously described . Vibratome sections were immunolabeled with antibodies against S100 (astroglia), Iba1 (microglia), Olig-2 (oligodendrocytes), p25 (oligodendrocytes) or NeuN (neurons), and the immunoreactive structures were detected with FITC-tagged secondary antibody (Vector Laboratories, 1:75) and the V5 tagged neurosin with the Tyramide Signal Amplification™-Direct system (NEN Life Sciences), while α-syn was detected with an antibody specific for human α-syn (SYN211, Sigma) and a FITC-tagged secondary antibody (Vector Laboratories, 1:75). Cell nuclei were stained using ProLong® Gold Antifade with DAPI (4',6-diamidino-2-phenylindole) (Molecular Probes). Sections were imaged with a Zeiss 63X objective on an Axiovert 35 microscope (Zeiss) with an attached MRC1024 laser scanning confocal microscope (BioRad) with an optical image of 1 μm thick with fluorescent signals in co-registry .
Values are expressed as average ± standard error of the mean (SEM). To determine the statistical significance we used one-way analysis of variance with post-hoc Dunnett’s test when comparing to the control condition. Additional comparisons were done using Tukey-Kramer or Fisher post hoc tests. The differences were considered to be significant if p values were less than 0.05.
This work was supported by the MSA Coalition and the NIH through grants: AG18440, NS044233, AG5131, AG0222074.
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