Direct detection of alpha synuclein oligomers in vivo

Background: Rat models of Parkinson’s disease are widely used to elucidate the mechanisms underlying disease etiology or to investigate therapeutic approaches. Models were developed using toxins such as MPTP or 6-OHDA to specifically target dopaminergic neurons resulting in acute neuronal loss in the substantia nigra or by using viral vectors to induce the specific and gradual expression of alpha synuclein in the substantia nigra. The detection of alpha- synuclein oligomers, the presumed toxic species, in these models and others has been possible using only indirect biochemical approaches to date. Here we coinjected AAVs encoding alpha-synuclein fused to the N- or C-terminal half of VenusYFP in rat substantia nigra pars compacta and describe for the first time a novel viral vector rodent model with the unique ability to directly detect and track alpha synuclein oligomers ex vivo and in vivo. Results: Viral coinjection resulted in widespread VenusYFP signal within the nigrostriatal pathway, including cell bodies in the substantia nigra and synaptic accumulation in striatal terminals, suggestive of in vivo alpha-synuclein oligomers formation. Transduced rats showed alpha-synuclein induced dopaminergic neuron loss in the substantia nigra, the appearance of dystrophic neurites, and gliosis in the striatum. Moreover, we have applied in vivo imaging techniques in the living mouse to directly image alpha-synuclein oligomers in the cortex. Conclusion: We have developed a unique animal model that provides a tool for the Parkinson’s disease research community with which to directly detect alpha- synuclein oligomers in vivo and screen therapeutic approaches targeting alpha-synuclein oligomers

Safety and immunogenicity of 2-dose heterologous Ad26.ZEBOV, MVA-BN-Filo Ebola vaccination in healthy and HIV-infected adults: A randomised, placebo-controlled Phase II clinical trial in Africa.

BACKGROUND: We investigated safety, tolerability, and immunogenicity of the heterologous 2-dose Ebola vaccination regimen in healthy and HIV-infected adults with different intervals between Ebola vaccinations. METHODS AND FINDINGS: In this randomised, observer-blind, placebo-controlled Phase II trial, 668 healthy 18- to 70-year-olds and 142 HIV-infected 18- to 50-year-olds were enrolled from 1 site in Kenya and 2 sites each in Burkina Faso, Cote d'Ivoire, and Uganda. Participants received intramuscular Ad26.ZEBOV followed by MVA-BN-Filo at 28-, 56-, or 84-day intervals, or saline. Females represented 31.4% of the healthy adult cohort in contrast to 69.7% of the HIV-infected cohort. A subset of healthy adults received booster vaccination with Ad26.ZEBOV or saline at Day 365. Following vaccinations, adverse events (AEs) were collected until 42 days post last vaccination and serious AEs (SAEs) were recorded from signing of the ICF until the end of the study. The primary endpoint was safety, and the secondary endpoint was immunogenicity. Anti-Ebola virus glycoprotein (EBOV GP) binding and neutralising antibodies were measured at baseline and at predefined time points throughout the study. The first participant was enrolled on 9 November 2015, and the date of last participant's last visit was 12 February 2019. No vaccine-related SAEs and mainly mild-to-moderate AEs were observed among the participants. The most frequent solicited AEs were injection-site pain (local), and fatigue, headache, and myalgia (systemic), respectively. Twenty-one days post-MVA-BN-Filo vaccination, geometric mean concentrations (GMCs) with 95% confidence intervals (CIs) of EBOV GP binding antibodies in healthy adults in 28-, 56-, and 84-day interval groups were 3,085 EU/mL (2,648 to 3,594), 7,518 EU/mL (6,468 to 8,740), and 7,300 EU/mL (5,116 to 10,417), respectively. In HIV-infected adults in 28- and 56-day interval groups, GMCs were 4,207 EU/mL (3,233 to 5,474) and 5,283 EU/mL (4,094 to 6,817), respectively. Antibody responses were observed until Day 365. Ad26.ZEBOV booster vaccination after 1 year induced an anamnestic response. Study limitations include that some healthy adult participants either did not receive dose 2 or received dose 2 outside of their protocol-defined interval and that the follow-up period was limited to 365 days for most participants. CONCLUSIONS: Ad26.ZEBOV, MVA-BN-Filo vaccination was well tolerated and immunogenic in healthy and HIV-infected African adults. Increasing the interval between vaccinations from 28 to 56 days improved the magnitude of humoral immune responses. Antibody levels persisted to at least 1 year, and Ad26.ZEBOV booster vaccination demonstrated the presence of vaccination-induced immune memory. These data supported the approval by the European Union for prophylaxis against EBOV disease in adults and children ≥1 year of age. TRIAL REGISTRATION: ClinicalTrials.gov NCT02564523

IAPR Workshop 017 CV- Speaal Hardware and Industrial Applications OCT.12-14. 1988. Tokyo A SEGMENT-BASED MATCHING ALGORITHM IN TRINOCULAR VISION

ABSTRACT been proposed to improve matching accurac

CHIP reduces the amount of human α-syn in the striatum.

<p>Striatal homogenates were biochemically analyzed for α-syn levels. Higher molecular weight α-syn species are visible in native-PAGE (A). Band quantification revealed a 43% reduction in α-syn aggregates measured in the syn+CHIP group (+CHIP) compared to the syn-CHIP group (−CHIP) (B). Denatured samples were also run on SDS-PAGE to evaluate the total amount of α-syn. The higher band at ∼31.3 KD and lower band at ∼22.4 KD detected with anti-α-syn correspond to V1S and SV2 respectively (C). A 26% and a 69% reduction were measured in both V1S and SV2 respectively in animals expressing CHIP (D and E).</p

NeuN quantification in the presence of CHIP.

<p>Blinded stereological analysis of NeuN immunopositive cells in coronal sections across the SNpc was performed. Sections were co-stained with anti TH antibody (A., purple) and NeuN (B., red). A binary image was produced where the SNpc is delineated according to the TH immunostaining (C., blue). Comparison of percent cell loss per animal revealed discrepancies in the TH measured lesion compared to NeuN in the presence of CHIP (E), unlike animals not expressing CHIP (D). We found a 20–30% difference in lesions determined by NeuN to that determined by TH in the presence of CHIP (F). A significant 23% NeuN lesion was measured in the syn–CHIP group whereas no significant lesion was measured in the venus+CHIP group and syn+CHIP (F). Scale bars 200 µm. For the purpose of illustrating the image analysis conducted in this assay only representative images of CHIP animal are presented.</p

TH cell loss in the presence of CHIP.

<p>Unbiased blinded stereological analysis of TH immunopositive cells in coronal sections across the SNpc was performed using DAB (A). Cell loss is determined by the ratio of TH positive cells ipsilateral (right, asterisk) to contralateral (left). A 34% lesion and a 35% lesion were measured in the syn–CHIP group (n = 10, p<0.005) and syn+CHIP group (n = 7, p<0.005) respectively (B). The venus+CHIP group expressing only venusYFP and CHIP had a 44% lesion (n = 4, p<0.05). Representative images are displayed. Scale bar 500 µm.</p

Reduced venusYFP fluorescence in the presence of CHIP.

<p>Coronal sections of the SNpc were immunostained with anti-Myc (alexa-635, purple) to evaluate co-expression of CHIP with α-syn aggregates (venusYFP, green). Images revealed extensive co-expression of CHIP and α-syn aggregates demonstrating efficient viral co-transduction (A). Coronal Sections were imaged for venusYFP fluorescence and α-syn immunostaining (red) to evaluate the level of α-syn aggregates (B). Image analysis demonstrates in syn+CHIP group (+CHIP) a significant reduction in the overall venusYFP fluorescence and no change in α-syn immunostaining compared to syn-CHIP (−CHIP)(C). Detailed image analysis shows a significant 35% reduction in the number of venusYFP positive cells in the syn+CHIP group compared to syn-CHIP (D) as well as a 4-fold reduction in venusYFP fluorescence per cell compared to control (E). Representative images are displayed. Scale bars 200 µm.</p

Direct detection of alpha synuclein oligomers in vivo

Abstract Background Rat models of Parkinson’s disease are widely used to elucidate the mechanisms underlying disease etiology or to investigate therapeutic approaches. Models were developed using toxins such as MPTP or 6-OHDA to specifically target dopaminergic neurons resulting in acute neuronal loss in the substantia nigra or by using viral vectors to induce the specific and gradual expression of alpha synuclein in the substantia nigra. The detection of alpha- synuclein oligomers, the presumed toxic species, in these models and others has been possible using only indirect biochemical approaches to date. Here we coinjected AAVs encoding alpha-synuclein fused to the N- or C-terminal half of VenusYFP in rat substantia nigra pars compacta and describe for the first time a novel viral vector rodent model with the unique ability to directly detect and track alpha synuclein oligomers ex vivo and in vivo. Results Viral coinjection resulted in widespread VenusYFP signal within the nigrostriatal pathway, including cell bodies in the substantia nigra and synaptic accumulation in striatal terminals, suggestive of in vivo alpha-synuclein oligomers formation. Transduced rats showed alpha-synuclein induced dopaminergic neuron loss in the substantia nigra, the appearance of dystrophic neurites, and gliosis in the striatum. Moreover, we have applied in vivo imaging techniques in the living mouse to directly image alpha-synuclein oligomers in the cortex. Conclusion We have developed a unique animal model that provides a tool for the Parkinson’s disease research community with which to directly detect alpha- synuclein oligomers in vivo and screen therapeutic approaches targeting alpha-synuclein oligomers

Chronic treatment with novel small molecule Hsp90 inhibitors rescues striatal dopamine levels but not α-synuclein-induced neuronal cell loss.

Hsp90 inhibitors such as geldanamycin potently induce Hsp70 and reduce cytotoxicity due to α-synuclein expression, although their use has been limited due to toxicity, brain permeability, and drug design. We recently described the effects of a novel class of potent, small molecule Hsp90 inhibitors in cells overexpressing α-synuclein. Screening yielded several candidate compounds that significantly reduced α-synuclein oligomer formation and cytotoxicity associated with Hsp70 induction. In this study we examined whether chronic treatment with candidate Hsp90 inhibitors could protect against α-synuclein toxicity in a rat model of parkinsonism. Rats were injected unilaterally in the substantia nigra with AAV8 expressing human α-synuclein and then treated with drug for approximately 8 weeks by oral gavage. Chronic treatment with SNX-0723 or the more potent, SNX-9114 failed to reduce dopaminergic toxicity in the substantia nigra compared to vehicle. However, SNX-9114 significantly increased striatal dopamine content suggesting a positive neuromodulatory effect on striatal terminals. Treatment was generally well tolerated, but higher dose SNX-0723 (6-10 mg/kg) resulted in systemic toxicity, weight loss, and early death. Although still limited by potential toxicity, Hsp90 inhibitors tested herein demonstrate oral efficacy and possible beneficial effects on dopamine production in a vertebrate model of parkinsonism that warrant further study

Comparison of higher dose SNX-0723 and SNX-9114 effects on nigrostriatal toxicity. A–D

<p>) Photos show low power images of injected SN (right) and contralateral uninjected side (left) for each drug treatment group. Black (medial) and white (lateral) squares indicate regions of interest for higher magnification photos shown. There is modest cell loss on the side of the lesion for all treatments. <b>E</b>) Graph of stereological counts (mean ±SEM) of TH-positive cells in the SN ipsilateral and contralateral to AAV-α-synuclein lesion. Numbers at base of bars indicate N for each group. Analysis of variance revealed no significant differences among treatment groups.</p