Novel oligodendroglial alpha synuclein viral vector models of multiple system atrophy: studies in rodents and nonhuman primates

Transgenic mouse models overexpressing human α-syn under oligodendroglia-specific promoters such as PLP, MBP, and CNP have been developed to study MSA.

“Transgenic (tg) mouse models overexpressing human α-syn under different oligodendroglia-specific promoters, such as proteolipid protein (PLP) [31], myelin basic protein (MBP) [51], and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) [67], have been developed to study MSA.”

• cites [31] Kahle PJ, Neumann M, Ozmen L, Muller V, Jacobsen H, Spooren W et al (2002) Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes. EMBO Rep 3(6):583–588 sourceSupported
• cites [51] Shults CW, Rockenstein E, Crews L, Adame A, Mante M, Larrea G et al (2005) Neurological and neurodegenerative alterations in a transgenic mouse model expressing human alpha-synuclein under oligodendrocyte promoter: implications for multiple system atrophy. J Neurosci 25:10689–10699 sourceSupported
• cites [67] Yazawa I, Giasson BI, Sasaki R, Zhang B, Joyce S, Uryu K et al (2005) Mouse model of multiple system atrophy α-synuclein expression in oligodendrocytes causes glial and neuronal degeneration. Neuron 45:847–859 sourceNot checked — no full text of the source

To date, none of the available transgenic mouse lines have been able to recapitulate the specific striatonigral degeneration or olivopontocerebellar atrophy seen in human MSA.

“To date, none of the available tg mouse lines have been able to recapitulate the specific striatonigral degeneration or olivopontocerebellar atrophy as seen in the human disorder [3].”

• cites [3] Bleasel JM, Halliday GM, Kim WS (2016) Animal modeling an oligodendrogliopathy-multiple system atrophy. Acta Neuropathol Commun 4:12. doi: 10.1186/s40478-016-0279-6 sourceSupported

AAV capsid cell tropisms obtained in rodents are not always predictive of transduction specificity in primates.

“Moreover, the AAV capsid cell tropisms obtained in rodents are not always predictive of the transduction specificity in primates.”

• cites [11] Dodiya HB, Bjorklund T, Stansell J 3rd, Mandel RJ, Kirik D, Kordower JH. Differential transduction following basal ganglia administration of distinct pseudotyped AAV capsid serotypes in nonhuman primates. Mol Ther. 2010 Mar;18(3):579-87. doi: 10.1038/mt.2009.216 . Epub 2009 Sep 22. PubMed PMID: 19773746; PubMed Central PMCID: PMC2839447. sourceCould not verify

  The Discussion (page unavailable) says the primate results confirm earlier rodent findings on the tropism and transduction patterns of the serotypes studied. It does not address whether rodent capsid cell tropisms sometimes fail to predict transduction specificity in primates; agreement in this study does not rule out such failures elsewhere. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

The Olig001 vector was engineered by capsid shuffling and directed evolution to transduce oligodendrocytes in the striatum and corpus callosum of rodents.

“The vector was engineered by capsid shuffling and directed evolution to transduce oligodendrocytes in the striatum and corpus callosum of rodents [44].”

• cites [44] Powell SK, Khan N, Parker CL, Samulski RJ, Matsushima G, Gray SJ, McCown TJ (2016) Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism. Gene Ther 23(11):807–814. doi: 10.1038/gt.2016.62 Epub 2016 Sep 15 sourcePartly supported

  The Introduction and Results and Discussion describe Olig001 as generated by capsid shuffling and directed evolution and show preferential transduction of oligodendrocytes after injection into the rat striatum. The cited paper’s copy does not report transduction in the corpus callosum, so that anatomical part of the claim is unsupported. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Alterations in lipid composition are restricted to affected regions in human MSA samples.

“Additionally, alterations in lipid composition are restricted to affected regions in human MSA samples [12].”

• cites [12] Don AS, Hsiao JH, Bleasel JM, Couttas TA, Halliday GM, Kim W (2014) Altered lipid levels provide evidence for myelin dysfunction in multiple system atrophy. Acta Neuropathol Commun 2(1):150. doi: 10.1186/s40478-014-0150-6 sourcePartly supported

  The Results sections on galactosylceramide and sphingomyelin (page not provided) report reduced lipid levels in MSA-affected motor-cortex white matter but not in the unaffected visual-cortex region examined. The paper distinguishes those changes in absolute levels from lipid-species distribution, which did not significantly change; it also did not test every brain region. Thus the regional finding is supported, but a change in lipid composition or restriction to all affected regions is broader than the findings establish. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Currently available animal models of MSA are limited to 3 transgenic mouse lines overexpressing human α-syn under PLP, MBP, and CNP promoters.

“Currently available animal models of MSA are limited to 3 tg mouse lines overexpressing human α-syn under proteolipid protein (PLP) promoter [31], myelin basic protein (MBP) promoter [51], and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter [67].”

• cites [31] Kahle PJ, Neumann M, Ozmen L, Muller V, Jacobsen H, Spooren W et al (2002) Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes. EMBO Rep 3(6):583–588 sourceSupported
• cites [51] Shults CW, Rockenstein E, Crews L, Adame A, Mante M, Larrea G et al (2005) Neurological and neurodegenerative alterations in a transgenic mouse model expressing human alpha-synuclein under oligodendrocyte promoter: implications for multiple system atrophy. J Neurosci 25:10689–10699 sourceSupported
• cites [67] Yazawa I, Giasson BI, Sasaki R, Zhang B, Joyce S, Uryu K et al (2005) Mouse model of multiple system atrophy α-synuclein expression in oligodendrocytes causes glial and neuronal degeneration. Neuron 45:847–859 sourceNot checked — no full text of the source

Decreased neurotrophic support of oligodendroglia, as shown by reductions of glial derived neurotrophic factor expression levels, occurs in MSA transgenic mice and human MSA samples.

“Moreover, decreased neurotrophic support of oligodendroglia, as shown by reductions of glial derived neurotrophic factor expression levels in MSA tg mice and human MSA samples [56], furthers the idea that oligodendrocyte dysfunction is an early pathological event leading to secondary neurodegeneration related to retrograde axonal disease.”

• cites [56] Ubhi K, Rockenstein E, Mante M, Inglis C, Adame A, Patrick C et al (2010) Neurodegeneration in a transgenic mouse model of multiple system atrophy is associated with altered expression of Oligodendroglial-derived Neurotrophic factors. J Neurosci 30(18):6236–6246. doi: 10.1523/JNEUROSCI.0567-10.2010 sourceSupported

Overexpression of α-syn using the MBP promoter showed the most classical distribution of pathology, with both the basal ganglia and cerebellum displaying extensive pathology.

“Overexpression of α-syn using the MBP promoter showed the most classical distribution of pathology, with both the basal ganglia and cerebellum displaying extensive pathology [51].”

• cites [51] Shults CW, Rockenstein E, Crews L, Adame A, Mante M, Larrea G et al (2005) Neurological and neurodegenerative alterations in a transgenic mouse model expressing human alpha-synuclein under oligodendrocyte promoter: implications for multiple system atrophy. J Neurosci 25:10689–10699 sourcePartly supported

  The Discussion reports extensive α-synuclein accumulation and associated neurodegeneration in the basal ganglia and cerebellum of MBP-promoter mice. It also describes more extensive alterations in these regions than in another model, but does not establish that the MBP model had the *most classical* distribution of pathology. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Overexpression of α-syn in cell culture models dramatically impaired the maturation of 2 separate oligodendrocyte precursor cell lines, shown by significant reductions of MBP during maturation.

“In support of this, overexpression of α-syn in cell culture models dramatically impaired the maturation of 2 separate oligodendrocyte precursor cell lines, shown by significant reductions of MBP during maturation [13].”

• cites [13] Ettle B, Reiprich S, Deusser J, Schlachetzki JC, Xiang W, Prots I et al (2014) Intracellular alpha-synuclein affects early maturation of primary oligodendrocyte progenitor cells. Mol Cell Neurosci 62:68–78. doi: 10.1016/j.mcn.2014.06.012 sourcePartly supported

  The Results (‘Interference of h-aSyn with primary OPC maturation’; page unavailable) report significantly reduced MBP during early differentiation after h-aSyn expression, with levels restored by day 6. But this study used primary rat OPC cultures, not two separate OPC cell lines; the Discussion identifies the CG4 cell-line finding as an earlier study. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Drastic demyelination observed in MSA patients is not accompanied by a severe loss of mature oligodendrocytes.

“The drastic demyelination observed in MSA patients is not accompanied by a severe loss of mature oligodendrocytes [49, 14]”

• cites [49] Salvesen L, Ullerup BH, Sunay FB, Brudek T, Løkkegaard A, Agander TK et al (2015) Changes in total cell numbers of the basal ganglia in patients with multiple system atrophy - a stereological study. Neurobiol Dis 74:104–113. doi: 10.1016/j.nbd.2014.11.008 Epub 2014 Nov 21 sourceNot checked — no full text of the source
• cites [14] Ettle B, Schlachetzki JC, Winkler J (2016) Oligodendroglia and myelin in neurodegenerative diseases: more than just bystanders? Mol Neurobiol 53(5):3046–3062. doi: 10.1007/s12035-015-9205-3 sourceSupported

Three months after injection of Olig001-α-syn in rhesus macaques, robust soluble and aggregated α-syn was seen throughout the caudate, putamen, and corpus callosum of all 3 primates, as shown by LB509 and pSer-129 immunoreactivity.

“Histologically, 3-months following injection of Olig001-α-syn, robust soluble and aggregated α-syn was seen throughout the caudate, putamen, and corpus callosum of all 3 primates, as shown by LB509 (Fig. 3a) and pSer-129 α-syn immunoreactivity (Fig. 3b).”

There was slightly more co-expression of GFP and NeuN in rodent striatum and corpus callosum than in similar brain regions in NHPs (3.7% rodents vs. 0.7% primates, F(1,5)=31.7, p=0.002).

“In contrast to glial co-expression, there was slightly more co-expression of GFP and NeuN in rodent striatum and corpus callosum than in similar brain regions in NHPs [3.7% rodents vs. 0.7% primates (F1,5) = 31.7, p = 0.002]”

In NHPs, 0.23-1.42% of GFP+ cells co-localized with NeuN.

“with only 0.23–1.42% of GFP+ cells co-localized with NeuN”

Intrastriatal injection of Olig001-GFP in rats produced widespread transduction of cells, with intense GFP+ fluorescence in the dense white matter of the corpus callosum and white matter bundles coursing the striatum in all injected animals.

“As expected, histological analysis 4-weeks following injection of Olig001-GFP revealed widespread transduction of cells, as shown by intense GFP+ fluorescence in the dense white matter of the corpus callosum and white matter bundles coursing the striatum in all injected animals (Fig. 1a).”

In Olig001-α-syn injected monkeys, marked demyelination was observed in the corpus callosum.

“The corpus callosum represents a pure white matter region in which marked demyelination was observed in monkeys injected with Olig001-α-syn (Fig. 4a).”

In rats, 94-97% of GFP+ cells in the corpus callosum and striatum co-localized with oligodendrocyte marker Olig2.

“Stereological quantification indicated that 94–97% of GFP+ cells in the corpus callosum and striatum co-localized with oligodendrocyte marker Olig2, indicating that Olig001 transduction was highly specific for oligodendroglia (Fig. 1d top two panels).”

In rats, 2.9-4.7% of GFP+ cells co-localized with neuronal marker NeuN.

“Conversely, there was very little off target transduction of neurons, with only 2.9–4.7% of GFP+ cells co-localizing with neuronal marker NeuN (Fig. 1d third panel)”

Olig001 displayed oligodendrocyte-specific tropism in all NHP animals injected.

“Olig001 displayed oligodendrocyte specific tropism in all animals injected.”

GFP+ cells occupied a volume of 3.873 ± 0.304 mm3 in the rat striatum.

“We also quantified the spread of the injected vector, and found that GFP+ cells occupied a volume of 3.873 ± 0.304 mm3 in the striatum (Fig. 1c).”

Following proteinase K treatment, abundant pSer-129 immunoreactive α-syn inclusions remained throughout the striatum of Olig001-α-syn injected monkeys, indicating formation of insoluble GCIs.

“Following PK treatment, abundant pSer-129 immunoreactive α-syn inclusions remained apparent throughout the striatum of Olig001-α-syn injected monkeys (Fig. 3e), indicating the formation of insoluble GCIs, as seen in MSA patients.”

There was no difference between the phenotypic transduction of oligodendrocytes in rodents and primates (F(1,5)=3.4, p>0.12).

“Moreover, there was no difference between the phenotypic transduction of oligodendrocytes in rodents and primates [F(1,5) = 3.4, p > 0.12].”

In rats, Olig001-GFP showed little to no expression in neurons or astrocytes.

“Histological analysis showed widespread expression of GFP throughout the striatum and corpus callosum with >95% of GFP+ cells co-localizing with oligodendroglia and little to no expression in neurons or astrocytes.”

Stereological cell counts revealed an estimated 477,448.33 ± 157,773.29 GFP+ cells in NHPs.

“Stereological cell counts revealed an estimated 477,448.33 ± 157,773.29 GFP+ cells (Fig. 2b)”

In nonhuman primates, 4 weeks after intracerebral injection of Olig001-GFP, there was a large number of GFP+ cells in the caudate, putamen, and corpus callosum.

“In concurrence with the results from our rat transduction experiment, 4-weeks following intracerebral injection of Olig001-GFP, histological analysis displayed a large number of GFP+ cells in the primate caudate, putamen, and corpus callosum (Fig. 2a).”

In Olig001-α-syn injected NHPs, striatal pSer-129 α-syn immunoreactivity correlated with regions of profound demyelination of striasomes and internal capsule fibers.

“Furthermore, striatal pSer-129 α-syn immunoreactivity in Olig001-α-syn injected NHPs correlated with regions of profound demyelination of striasomes and internal capsule fibers, shown by reduction of Luxol Fast Blue staining in level matched sections (Fig. 4b).”

Regions of demyelination corresponded to pSer-129 immunoreactivity in level matched sections.

“Regions of demyelination corresponded to pSer-129 immunoreactivity in level matched sections (Fig. 4a).”

In rats, 0.18-0.49% of GFP+ cells co-localized with astrocyte marker GFAP.

“and virtually no transduction of astrocytes, with only 0.18–0.49% of GFP+ cells co-localizing with astrocyte marker GFAP (Fig. 1d bottom panel).”

pSer-129+ aggregates covered 124.8 ± 2.18 mm3 of the striatum.

“which covered 124.8 ± 2.18 mm3 of the striatum (Fig. 3d).”

Confocal microscopy confirms the cytoplasmic localization of α-syn in oligodendrocytes.

“Confocal microscopy confirms the cytoplasmic localization of α-syn in oligodendrocytes, shown by LB509 immunoreactivity (red channel) wrapped around nuclear Olig2 signal (green channel) (scale bar 300 μm)”

In NHPs, 0.06-0.12% of GFP+ cells co-localized with GFAP.

“and only 0.06–0.12% co-localized with GFAP (Fig. 2d, middle and bottom panel, respectively).”

Stereological quantification estimated 126,574 ± 11,303 GFP+ cells in the striatum and corpus callosum of rats.

“Quantification of GFP+ cells in serial sections throughout the rostrocaudal striatum using unbiased stereological cell counting methods estimated that 126,574 ± 11,303 cells in the striatum and corpus callosum were GFP+ (Fig. 1b).”

In Olig001-GFP injected monkeys, GFP expression did not result in demyelination of the corpus callosum.

“Conversely, in Olig001-GFP injected monkeys, expression of GFP did not result in demyelination of the corpus callosum (Fig. 4a), indicating that demyelination is specifically related to α-syn, and not a byproduct of the vector or GFP expression.”

Stereological counts of pSer-129 inclusions estimated 2,606,200 ± 322,083 pSer-129+ aggregates in the striatum.

“Quantification using unbiased stereological counts of pSer-129 inclusions estimates 2,606,200 ± 322,083 pSer-129+ aggregates (Fig. 3c)”

Olig001-α-syn GCIs in the NHP model were resistant to proteinase K digestion, as in MSA.

“As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation.”

• Also stated in: § Intrastriatal injection of Olig001-α-syn produces widespread and specific transduction of oligodendroglia in nonhuman primates · hedged

  “pS129 staining with and without proteinase K digestion indicates the formation of insoluble aggregates throughout the striatum (scale bar 30 μm).”

• AssumptionRests on an assumption: In human MSA, GCIs are resistant to proteinase K digestion.

Confocal microscopy confirmed cytoplasmic localization of α-syn inside oligodendroglia in the model.

“Confocal microscopy confirmed the cytoplasmic localization of α-syn inside oligodendroglia (Fig. 3f), indicating that our model develops GCIs, the pathological hallmark of MSA.”

The transduction volume in NHPs was 52.89 ± 19.17 mm3.

“which had a transduction volume of 52.89 mm3 ± 19.17 mm3 (Fig. 2c).”

In NHPs, 90-94% of GFP+ cells co-localized with oligodendrocyte marker Olig2.

“As with the rats, stereological quantification indicated that Olig001 demonstrated a marked oligodendroglial-specific tropism in the NHP brain, with 90–94% of GFP+ cells co-localized with oligodendrocyte marker Olig2 (Fig. 2d, top panel).”

The percentage of co-expression of GFP and GFAP was not different between rats and NHPs (F(1,5)=5.3, p>0.06).

“The percentage of co-expression of GFP and GFAP to characterize astrocytic transgene expression was also not different between rats and NHPs [F(1,5) = 5.3, p > 0.06].”

The morphology of the GCIs in the model mimics the typical conical half-moon shape inclusions seen in human MSA.

“Moreover, the morphology of the GCIs (Fig. 3f, red channel) mimics the typical conical half-moon shape inclusions seen in the human disease [43].”

• cites [43] Papp MI, Lantos PL (1994) The distribution of oligodendroglial inclusions in multiple system atrophy and its relevance to clinical symptomatology. Brain 117(Pt 2):235–243 sourceNot checked — no full text of the source

Olig001-α-syn caused microglial activation.

“As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation.”

HLA-DR+ microglia in α-syn injected animals had an activated morphology, characterized by bushy or amoeboid morphology with shrunken processes.

“The morphology of HLA-DR+ microglia clearly indicates an activated state, characterized by a bushy or amoeboid morphology with shrunken processes (Fig. 5a high magnification).”

Activated microglia were seen throughout the substantia nigra of Olig001-α-syn injected monkeys.

“Moreover, activated microglia were seen throughout the substantia nigra of Olig001-α-syn injected monkeys (Fig. 5b).”

Microglial activation did not cover the entire area of GFP expression, unlike what was observed with α-syn transduction.

“Microglial activation did not cover the entire area of GFP expression, unlike what was observed with α-syn transduction (Fig. 5a).”

In the substantia nigra of Olig001-GFP injected monkeys, microglia were observed to be ramified or "resting" microglia, shown by long-ramified processes with smaller cell bodies.

“Conversely, in the substantia nigra of Olig001-GFP injected monkeys, microglia were observed to be ramified or “resting” microglia, shown by long-ramified processes with smaller cell bodies (Fig. 5b).”

The demyelination observed in the model is not associated with any obvious reductions in oligodendroglia.

“In line with post-mortem human MSA cases, the demyelination observed in our model is not associated with any obvious reductions in oligodendroglia, further indicating that the loss of myelin is a result of oligodendrocyte dysfunction and not an overt loss of oligodendroglia.”

Intrastriatal injection of Olig001-α-syn produced widespread expression of α-syn throughout white matter regions of the striatum.

“Intrastriatal injection of Olig001-α-syn produced widespread expression of α-syn throughout white matter regions of the striatum.”

In Olig001-GFP injected animals, a minimal inflammatory reaction was displayed, limited to the needle tract and specific site of injection.

“In contrast, Olig001-GFP injected animals displayed a minimal inflammatory reaction, limited to the needle tract and specific site of injection (Fig. 5a, injection site marked by arrows).”

Striatal expression of GFP showed no disruption of striatal white matter.

“Striatal expression of GFP showed no disruption of striatal white matter (Fig. 4b), consistent with the notion that oligodendroglial accumulation of α-syn, but not GFP, causes disruption of oligodendrocyte function.”

There was slightly less co-localization of GFP and NeuN in primates than in rodents [3.7% rodents vs. 0.7% primates (F1,5) = 31.7, p = 0.002].

“Furthermore, there was slightly less co-localization of GFP and NeuN in primates than in rodents [3.7% rodents vs. 0.7% primates (F1,5) = 31.7, p = 0.002], indicating that Olig001 produced less off target transduction in nonhuman primates.”

Marked demyelination was observed throughout striatal white matter, demonstrated by reduced Luxol Fast Blue staining in the corpus callosum and striatal white matter bundles.

“Most notably, marked demyelination was observed throughout striatal white matter, demonstrated by reduced Luxol Fast Blue staining in the corpous callosum and striatal white matter bundles.”

Intrastriatal injection of Olig001-GFP in rhesus macaques demonstrates the same preference for oligodendrocytes as in rodents [F(1,5) = 3.4, p > 0.12].

“Intrastriatal injection of Olig001-GFP in rhesus macaques demonstrates the same preference for oligodendrocytes as in rodents [F(1,5) = 3.4, p > 0.12].”

In Olig001-α-syn injected monkeys, a large inflammatory response (HLA-DR+ activated microglia) was observed throughout the area where the α-syn transgene is expressed, clearly seen in white matter patches of the striatum.

“In α-syn injected animals, a large inflammatory response was observed throughout the area where the α-syn transgene is expressed, clearly seen in white matter patches of the striatum (Fig. 5a).”

In the current study, the affinity of Olig001 to transduce oligodendroglia was conserved between species.

“In the current study, we observed that the affinity of Olig001 to transduce oligodendroglia was conserved between species.”

Inclusions of α-syn were found to localize inside oligodendroglia, shown by co-localization of LB509 and oligodendroglia marker Olig2.

“Inclusions of α-syn were found to localize inside oligodendroglia, shown by co-localization of LB509 and oligodendroglia marker Olig2.”

Luxol Fast Blue and pSer-129 staining of rhesus macaques 3-months after receiving intrastriatal injections of AAV2-neurturin or stem cells, as well as untreated animals, displayed no demyelination or aggregated α-syn.

“Luxol Fast Blue and pSer-129 staining of rhesus macaques 3-months after receiving intrastriatal injections of AAV2-neurturin [25] or stem cells, as well as untreated animals, displayed no demyelination or aggregated α-syn which supports the specificity of effects observed with Olig001-GFP and Olig001-α-syn.”

• cites [25] Herzog CD, Dass B, Gasmi M, Bakay R, Stansell JE, Tuszynski M et al (2008) Transgene expression, bioactivity, and safety of CERE-120 (AAV2-neurturin) following delivery to the monkey striatum. Mol Ther 16(10):1737–1744. doi: 10.1038/mt.2008.170 Epub 2008 Aug 26 sourceNot checked — no full text of the source

MSA is characterized neuropathologically by α-syn-positive glial cytoplasmic inclusions (GCIs) in oligodendroglia.

“MSA is a synucleinopathy, however, unlike PD and dementia with Lewy bodies, in which α-syn accumulates and aggregates in neurons, MSA is characterized neuropathologically by α-syn-positive glial cytoplasmic inclusions (GCIs) in oligodendroglia [1, 42, 54, 58, 59].”

• cites [1] Arima K, Uéda K, Sunohara N, Arakawa K, Hirai S, Nakamura M, Tonozuka-Uehara H, Kawai M (1998) NACP/alpha-synuclein immunoreactivity in fibrillary components of neuronal and oligodendroglial cytoplasmic inclusions in the pontine nuclei in multiple system atrophy. Acta Neuropathol 96:439–444 sourceNot checked — no full text of the source
• cites [42] Papp MI, Kahn JE, Lantos PL (1989) Glial cytoplasmic inclusions in the CNS of patients with multiple system atrophy (striatonigral degeneration, olivopontocerebellar atrophy and shy-Drager syndrome). J Neurol Sci 94(1–3):79–100 sourceNot checked — no full text of the source
• cites [54] Tu PH, Galvin JE, Baba M, Giasson B, Tomita T, Leight S et al (1998) Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble alpha-synuclein. Ann Neurol 44:415–422 sourceNot checked — no full text of the source
• cites [58] Wakabayashi K, Hayashi S, Kakita A, Yamada M, Toyoshima Y, Yoshimoto M et al (1998b) Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy. Acta Neuropathol 96:445–452 sourceNot checked — no full text of the source
• cites [59] Wakabayashi K, Yoshimoto M, Tsuji S, Takahashi H (1998a) Alpha-synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy. Neurosci Lett 249:180–182 sourceNot checked — no full text of the source

Historical control groups served to control for the effects of anesthesia, surgery, needle penetration to the striatum, AAV delivery, and other bioactive materials.

“Groups 3 and 4 were historical controls in one of our labs (JHK) and served to control for the effects of 1) anesthesia, 2) surgery, 3) needle penetration to the striatum, and delivery of 4) AAV and 5) other bioactive bioactive materials and all were sacrificed at the same post-operative time as Olig001-α-syn treated animals.”

Olig001 produced less off-target transduction in nonhuman primates than in rodents.

“indicating that Olig001 produced less off target transduction in nonhuman primates”

These data support the concept that this vector can provide novel rodent and nonhuman primate models of MSA.

“These data support the concept that this vector can provide novel rodent and nonhuman primate models of MSA.”

Together, these data indicate the development of the first pathologically-based nonhuman primate model of MSA-P.

“Together, these data indicate the development of the first pathologically-based nonhuman primate model of MSA-P.”

• AssumptionRests on an assumption: No prior pathologically-based nonhuman primate model of MSA-P exists.

The model develops GCIs, the pathological hallmark of MSA.

“Confocal microscopy confirmed the cytoplasmic localization of α-syn inside oligodendroglia (Fig. 3f), indicating that our model develops GCIs, the pathological hallmark of MSA.”

The observed demyelination was not a result of the AAV, as no reductions in myelin were observed in the Olig001-GFP transduced animals.

“Moreover, this was not a result of the AAV, as no reductions in myelin were observed in the Olig001-GFP transduced animals.”

Olig001 produced less off-target transduction in nonhuman primates.

“Furthermore, there was slightly less co-localization of GFP and NeuN in primates than in rodents [3.7% rodents vs. 0.7% primates (F1,5) = 31.7, p = 0.002], indicating that Olig001 produced less off target transduction in nonhuman primates.”

GCIs are composed mainly of α-syn.

“These inclusions were later described to be composed mainly of α-syn [43].”

• cites [43] Papp MI, Lantos PL (1994) The distribution of oligodendroglial inclusions in multiple system atrophy and its relevance to clinical symptomatology. Brain 117(Pt 2):235–243 sourceNot checked — no full text of the source

Previous studies by other groups targeting oligodendroglia using wild-type rAAVs with expression driven by MBP promoters had low overall transduction of oligodendrocytes.

“Previous studies by other groups target oligodendroglia using wild-type rAAVs with expression driven by MBP promoters, however the overall transduction of oligodendrocytes was still low [8, 34].”

• cites [8] Chen H, McCarty DM, Bruce AT, Suzuki K, Suzuki K (1998) Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus. Gene Ther 5:50–58 sourceNot checked — no full text of the source
• cites [34] Lawlor PA, Bland RJ, Mouravlev A, Young D, During MJ (2009) Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates. Mol Ther 17:1692–1702 sourceNot checked — no full text of the source

Early pathological changes seen in MSA were demonstrated in the monkeys 3-months following transduction.

“Early pathological changes seen in MSA were demonstrated in the monkeys 3-months following transduction.”

The differing post-injection time course of the Olig001-GFP and Olig001-α-syn injected monkeys does not allow for a perfect comparison.

“While the differing post-injection time course of the Olig001-GFP and Olig001-α-syn injected monkeys does not allow for a perfect comparison, there is an enormous amount of data from numerous NHP studies following striatal injection of different transgenes that never reported demyelination or α-syn aggregation.”

Accumulation of α-syn leads to dysfunction, rather than overt loss, of oligodendroglia.

“The drastic demyelination observed in MSA patients is not accompanied by a severe loss of mature oligodendrocytes [49, 14], indicating that accumulation of α-syn leads to dysfunction, rather than overt loss of oligodendroglia.”

Constitutive expression of α-syn under oligodendroglia-specific promoters may incorporate developmentally expressed α-syn in the pathology observed in these models.

“Moreover, the constitutive expression of α-syn under oligodendroglia-specific promoters may also incorporate developmentally expressed α-syn in the pathology observed in these models.”

Oligodendrocyte dysfunction is an early pathological event leading to secondary neurodegeneration related to retrograde axonal disease.

“Moreover, decreased neurotrophic support of oligodendroglia, as shown by reductions of glial derived neurotrophic factor expression levels in MSA tg mice and human MSA samples [56], furthers the idea that oligodendrocyte dysfunction is an early pathological event leading to secondary neurodegeneration related to retrograde axonal disease.”

Experimental modeling of MSA critically relies on the overexpression of α-syn in oligodendroglia.

“As such, experimental modeling of MSA critically relies on the overexpression of α-syn in oligodendroglia.”

Accumulation and aggregation of α-syn in oligodendroglia is specific to the disease process.

“In contrast, oligodendrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from MSA patients expressed α-syn, whereas iPSCs from PD and healthy controls do not [10] suggesting that the accumulation and aggregation of α-syn in oligodendroglia is specific to the disease process.”

The model recapitulated other pathological events observed in MSA, such as immune and inflammatory activation.

“Our model recapitulated other pathological events observed in MSA, such as immune and inflammatory activation.”

In previous work, a capsid and/or transgene related inflammatory response peaks at 2-weeks post-injection and then retracts to the needle track.

“In our previous work, we have seen that a capsid and/or transgene related inflammatory response peaks at 2-weeks post-injection and then retracts to the needle track [46], similarly to what is seen with the Olig001-GFP injected monkeys.”

• cites [46] Reimsnider S, Manfredsson FP, Muzyczka N, Mandel RJ (2007) Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat. Mol Ther 15(8):1504–1511 sourceNot checked — no full text of the source