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

Cellular anatomy of the mouse primary motor cortex.

An essential step toward understanding brain function is to establish a structural framework with cellular resolution on which multi-scale datasets spanning molecules, cells, circuits and systems can be integrated and interpreted1. Here, as part of the collaborative Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based anatomical description of one exemplar brain structure, the mouse primary motor cortex, upper limb area (MOp-ul). Using genetic and viral labelling, barcoded anatomy resolved by sequencing, single-neuron reconstruction, whole-brain imaging and cloud-based neuroinformatics tools, we delineated the MOp-ul in 3D and refined its sublaminar organization. We defined around two dozen projection neuron types in the MOp-ul and derived an input-output wiring diagram, which will facilitate future analyses of motor control circuitry across molecular, cellular and system levels. This work provides a roadmap towards a comprehensive cellular-resolution description of mammalian brain architecture

Household, community, sub-national and country-level predictors of primary cooking fuel switching in nine countries from the PURE study

Introduction. Switchingfrom polluting (e.g. wood, crop waste, coal)to clean (e.g. gas, electricity) cooking fuels can reduce household air pollution exposures and climate-forcing emissions.While studies have evaluated specific interventions and assessed fuel-switching in repeated cross-sectional surveys, the role of different multilevel factors in household fuel switching, outside of interventions and across diverse community settings, is not well understood. Methods.We examined longitudinal survey data from 24 172 households in 177 rural communities across nine countries within the Prospective Urban and Rural Epidemiology study.We assessed household-level primary cooking fuel switching during a median of 10 years offollow up (∼2005–2015).We used hierarchical logistic regression models to examine the relative importance of household, community, sub-national and national-level factors contributing to primary fuel switching. Results. One-half of study households(12 369)reported changing their primary cookingfuels between baseline andfollow up surveys. Of these, 61% (7582) switchedfrom polluting (wood, dung, agricultural waste, charcoal, coal, kerosene)to clean (gas, electricity)fuels, 26% (3109)switched between different polluting fuels, 10% (1164)switched from clean to polluting fuels and 3% (522)switched between different clean fuels

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

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

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

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

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

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