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The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

Figure S3. Synaptic density marker staining and IBA1+ cell numbers in inner retina of EAE mice at PID42. a Quantification of PSD-95 (a) and SYP (b) staining by positive staining area in the inner retina of EAE mouse (n = 15) and CFA control (n = 9). c IBA1+ cell number in inner retina of EAE mice. Quantification is represented as mean ± SEM. Significance was determined by two-tailed, unpaired Student’s t-test with P

Figure S5. SMI31, MBP and SMI32 staining in the optic nerve of EAE and CFA control mice. a SMI31 and MBP staining in different region of optic nerve of in EAE and CFA control mice at PID16 and PID42, respectively. b SMI32 and MBP staining in different region of optic nerve of EAE and CFA control mice at PID16 and PID42, respectively. CFA group was from control of PID 42. Scale bar =50â Îźm. (PDF 4950 kb)

Figure S2. RGC numbers in PID 16 EAE and in CFA control mice vs healthy controls. a Brn3a staining at PID16. b Quantification of RGC number at PID 16. c RGC number of central retina at early stage EAE (PID16). d RGC number of middle retina at early stage EAE (PID16). e RGC number of peripheral retina at early stage EAE (PID16). f RGC density of naïve and CFA control mice. g There were no difference in BS between female and male mice. N.S. = no significant difference. Red scale bar =1 mm. White scale bar =20 μm. (PDF 519 kb)

Figure S6. GFAP and IBA1 staining in the optic nerve of EAE and CFA control mice. a GFAP and IBA1 staining in different region of optic nerve of in EAE and CFA control mice at PID16 and PID42, respectively. b IBA1 and iNOS staining in different region of optic nerve of EAE and CFA control mice at PID16 and PID42, respectively. CFA group was from control of PID 42. Scale bar =50â Îźm. (PDF 5320 kb)

Figure S1. Semi-automated analysis of Brn3a-postive retinal ganglion cell density across whole flat mount retinas. a Whole flat mount retina stained with 3 different antibodies, NeuN, a neuronal specific marker, and RBPMS and Brn3a, RGC specific markers. Scale bar =50 μm. b Comparison of RGC number from automatic and manual count in random selected EAE (n = 5, red labels) and wild-type mice (n = 6, blue labels). c Comparison of RGC density in subregions of whole retina from automatic and manual count in random selected EAE (n = 5) and wild-type mice (n = 6). Automated analysis accurately reflected regional differences. d The RGC density between left and right retinas of the same mouse were comparable. e Comparision of RGC number counted by different persons. N.S. = no significant difference. (PDF 890 kb)

Figure S4. CD4+ T-cell staining in the optic nerve of EAE and CFA control mice. a CD4 staining in different degion of cross sectioned optic nerve of EAE mouse at PID 16 and PID 42, respectively. CFA group was from control of PID 42 since 2 CFA groups had no difference. Scale bar =50â Îźm. (PDF 388 kb)

The schematic diagram depicts the time schedule of intervention and analyses performed. Numerals represent the days experiments were conducted. On 1th day we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in 8 months WT, GBA +/L444P , SNCA−/−, GBA +/L444P SNCA−/− mice. On 6th day, the pole and grip strength were performed. On 7th day, mice were sacrificed for indicated studies. Following are animal numbers used for these studies: behavioral (n = 10), neurochemical (n = 5), immunohistochemistry (n = 5), and biochemical studies (n = 4) per each treatment group. (PDF 110 kb)

GBA overexpression inhibits MPTP-reduced mitochondrial protein level in GBA +/L444P mice. a Immunoblots of SDHA, PDH, VDAC, TH, GBA, and α-synuclein from AAV5-Con injected WT, AAV5-hGBA injected WT, AAV5-Con injected heterozygous, and AAV5-hGBA injected heterozygous mice treated with saline or MPTP. VMB lysates were immunoblotted with anti-SDHA, anti-PDH, anti-VDAC, anti-TH, and anti-GBA antibodies. b SDHA, c PDH, d VDAC, e TH, f GBA, and g α-synuclein expression levels were normalized against β-actin. Error bars represent the mean ± S.E.M. (n = three mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P

MPTP-induced α-synuclein oligomer. a Filter trap soluble-α- synuclein oligomer species assay from ventral midbrain of WT and GBA +/L444P mice with or without MPTP. Error bars represent the mean ± S.E.M. (n = four mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. ***P

Lysosomal calcium concentration. SH-SY5Y cells were transfected with indicated constructs for 48 h. The cells were then labeled with CellLight® Lysosome-RFP (LAMP1; red) and loaded with 0.1 mg/ml of lysosomal calcium indicator Oregon Green BAPTA-1 dextran (BAPTA-1; green) for 12 h. The Oregon Green BAPTA-1 signals that co-localized to lysosome (LAMP-1; red) were used for measuring lysosomal calcium concentration. [Ca2+]lys was measured using ratiometric methods via confocal microscopy. Two-way ANOVA was used to test for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. ***P

Figure S2. Expression of AAV-Rab35 WT and phosphomutants after intracranial injection of AAV. At one week after intracranial injection of AAV-eGFP (control), AAV-Rab35 WT, AAV-Rab35 T75A or AAV-Rab35 T75D into the substantia nigra, mouse brains were prepared for immunoblot (a) and immunohistochemistry (b). (a) Ventral midbrain was dissected from the AAV-injected hemisphere and the tissue was homogenized. Immunoblots were performed with anti-Rab35, anti-TH, and anti-actin antibodies. Exogenous and endogenous Rab35 are indicated by red and blue arrowhead, respectively. (b) Cryosections of AAV-injected brains including the substantia nigra region were immunostained with anti-GFP (green) and anti-TH (red) antibodies. Scale bar, 200 μm. (PPTX 3143 kb)

Figure S4. Expression of Rab35 in adult mouse brain. Analysis of Rab35 protein expression by immunoblot in various brain regions of adult mouse. (PPTX 1820Â kb)

Neuron specific AAV5-hGBA overexpression in SNpc region. a Vector design of AAV5 hGBA. b Representative immunofluorescent images of GFP (green, injection marker), GFAP (red, astrocyte, non-neuronal marker), Tuj1 (Violet, neuronal marker), and DAPI (Blue). (PDF 4671 kb)

Activity of 20S proteasome was measured in WT and GBA +/L444P primary cultured neurons (n = three per each group). Student’s t-test was used to for statistical analysis. *P

Effect of α-synuclein deficiency and GBA overexpression on susceptibility of GBA +/L444P mice to MPTP-induced gliosis. a, c Representative images of immunohistochemistry data for GFAP with low (scale bar, 500 μm) and high magnification (scale bar, 50 μm). b, d Intensities of GFAP positive signals in the SNpc of mice treated with saline or MPTP were quantified and shown as a graph. Error bars represent the mean ± S.E.M (n = four mice per group). Two-way ANOVA was used to test for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P

Figure S1. Immunostaining of cortical neurons transfected with Rab35 mutants. Representative images of embryonic day 15.5 cortical neurons expressing mutants (T72A, T72D or Q67Q) of GFP-conjugated Rab35. Neurons were fixed at one day after transfection and immunostained with anti-GFP, anti-smi312 (axonal marker), and anti-GFAP (astrocyte marker) antibodies. Scale bar, 100 μm. (PPTX 9498 kb)

L444P GBA heterozygous mutation leads to autophagy abnormality. a, b Effect of rapamycin on GBA +/L444P expression. Primary cortical neurons were cultured from WT and GBA +/L444P mice. After 10 DIV, primary neurons were treated with 20 nM of rapamycin (mTOR inhibitor for inducing autophagy) for 24 h. a Representative Immunoblots of GBA. b GBA expression levels were normalized against β-actin and the error bars represent the mean ± S.E.M (n = four per group). c Immunoblots of Autophagy marker proteins SQSTM1/p62, and LC3A/B-I/II. VMB lysates were immunoblotted with anti-SQSTM1/p62, and anti-LC3A/B-I/II antibodies. d SQSTM1/p62, and e LC3A/B-II expression levels were normalized against β-actin. Error bars represent the mean ± S.E.M (n = four mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P

Figure S3. Localization of Rab35 WT and phosphomutants. (a) HEK-293 cells were transfected with V5-tagged Rab35 WT or phosphomutants (T72A or T72D). At 48 h after transfection, cells were harvested and membrane fractionation was performed as described in Methods. Prepared membrane fractions were subjected to SDS-PAGE and immunoblotting with anti-caveolin-1 (plasma membrane marker), anti-EEA1 (early endosomal marker), anti-GOPC (golgi marker), and anti-Hsp90 (cytosol marker) antibodies. 2.5% of total lysates and 30% of membrane fractions were loaded for each immunoblot. (b) Quantification of Rab35 protein levels normalized against input level of Rab35. Data are mean ± SD (n = 3). * p

FoldX measurement for mutational energy effect on stability of binding pocket. Using the FoldX algorithm, which compares the WT structure with the mutant, calculations on WT and mutant at t = 0 and after 100 ns were performed. While WT is nearly zero over that time interval, the N368 mutant increases energy (∆∆G) by 1.5–2 kcal/mol*Å2. This modest increase supports that the mutation distorts the local region via an increase of Gibb’s free energy. (DOCX 16 kb)

FoldX measurement for mutational energy effect on stability of binding pocket. Using the FoldX algorithm, which compares the WT structure with the mutant, calculations on WT and mutant at t = 0 and after 100 ns were performed. While WT is nearly zero over that time interval, the N368 mutant increases energy (∆∆G) by 1.5–2 kcal/mol*Å2. This modest increase supports that the mutation distorts the local region via an increase of Gibb’s free energy. (DOCX 16 kb)

Abnormal processing of self-proteins can produce epitopes presented by major histocompatibility (MHC) proteins to be recognized by specific T cells that escaped central tolerance during thymic selection. Such actions by the acquired immune system are widely held to produce autoimmune disorders such as Type-1 diabetes. While not considered to possess autoimmune features, neurodegenerative diseases are characterized by the altered processing of specific proteins. One of the major pathological features of Parkinson’s disease are the presence of intraneuronal aggregrates known as Lewy bodies and neurites composed of α-synuclein. Genetic studies associate Parkinson’s disease with DRB5*01 and DRB1*15:01 MHC alleles. To address the hypothesis that Parkinson’s disease is associated with T cell recognition of epitopes derived from α-synuclein and presented by specific MHC alleles, we recruited 67 Parkinson’s disease patients and 36 age-matched non-Parkinson’s healthy controls. We found that a defined set of peptides derived from α-synuclein, act as antigenic epitopes displayed by these alleles and drive helper and cytotoxic T cell responses in Parkinson’s disease patients. We identified two antigenic regions in α-synuclein, the first near the N terminus (aa31-46), which was bound by DRB1*15:01 and DRB5*01:01, and the second near the C terminus, which required phosphorylation of an amino acid residue (S129). These α-synuclein epitopes were shown to arise from natural processing of both extracellular native α-synuclein and the fibrilized form associated with Parkinson’s disease pathogenesis. These responses may explain the association of Parkinson’s disease with alleles of the acquired immune system.

The schematic diagram depicts the time AAV5 injection schedule of intervention and analyses performed. Numerals represent the days’ experiments were conducted. For stereotaxic injection of AAV5-GFP and AAV5-hGBA, 8-month-old mice of indicated genotypes were anesthetized with pentobarbital (60 mg/kg). An injection cannula (26.5 gauge) was stereotaxically applied to the substantia nigra pars compacta (SNpc). After AAV5 stereotaxic injection for 1 month, we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in WT, and GBA +/L444P mice. On 6th day the pole and grip strength test were performed. On 7th day, mice were sacrificed for indicated studies. Following are animal numbers used for these studies: behavioral (n = 6-8), neurochemical (n = 4), immunohistochemistry (n = 4), and biochemical studies (n = 4) per each treatment group. (PDF 126 kb)

The schematic diagram depicts the time AAV5 injection schedule of intervention and analyses performed. Numerals represent the days’ experiments were conducted. For stereotaxic injection of AAV5-GFP and AAV5-hGBA, 8-month-old mice of indicated genotypes were anesthetized with pentobarbital (60 mg/kg). An injection cannula (26.5 gauge) was stereotaxically applied to the substantia nigra pars compacta (SNpc). After AAV5 stereotaxic injection for 1 month, we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in WT, and GBA +/L444P mice. On 6th day the pole and grip strength test were performed. On 7th day, mice were sacrificed for indicated studies. Following are animal numbers used for these studies: behavioral (n = 6-8), neurochemical (n = 4), immunohistochemistry (n = 4), and biochemical studies (n = 4) per each treatment group. (PDF 126 kb)

The schematic diagram depicts the time schedule of intervention and analyses performed. Numerals represent the days experiments were conducted. On 1th day we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in 8 months WT, GBA +/L444P , SNCA−/−, GBA +/L444P SNCA−/− mice. On 6th day, the pole and grip strength were performed. On 7th day, mice were sacrificed for indicated studies. Following are animal numbers used for these studies: behavioral (n = 10), neurochemical (n = 5), immunohistochemistry (n = 5), and biochemical studies (n = 4) per each treatment group. (PDF 110 kb)

Clinical features of PD patients homozygous for the PINK1 p.I368N mutation are summarized. Abbreviations: RBD: REM sleep behavior disorder; MMSE: Mini Mental State Examination; DBS: deep brain stimulation. (DOCX 16Â kb)

Clinical features of PD patients homozygous for the PINK1 p.I368N mutation are summarized. Abbreviations: RBD: REM sleep behavior disorder; MMSE: Mini Mental State Examination; DBS: deep brain stimulation. (DOCX 16Â kb)