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

Inhibition of the NFAT pathway alleviates amyloid β neurotoxicity in a mouse model of Alzheimer's disease

Amyloid β (Aβ) peptides, the main pathological species associated with Alzheimer’s disease (AD), disturb intracellular calcium homeostasis, which in turn activates the calcium-dependent phosphatase calcineurin (CaN). CaN activation induced by Aβ leads to pathological morphological changes in neurons, and overexpression of constitutively active calcineurin is sufficient to generate a similar phenotype, even without Aβ. Here, we tested the hypothesis that calcineurin mediates neurodegenerative effects via activation of the nuclear transcription factor of activated T-cells (NFAT). We found that both spine loss and dendritic branching simplification induced by Aβ exposure were mimicked by constitutively active NFAT, and abolished when NFAT activation was blocked using the genetically encoded inhibitor VIVIT. When VIVIT was specifically addressed to the nucleus, identical beneficial effects were observed, thus enforcing the role of NFAT transcriptional activity in Aβ-related neurotoxicity. In vivo, when VIVIT or its nuclear counterpart were overexpressed in a transgenic model of Alzheimer’s disease via a gene therapy approach, the spine loss and neuritic abnormalities observed in the vicinity of amyloid plaques were blocked. Overall, these results suggest that NFAT/calcineurin transcriptional cascades contribute to Aβ synaptotoxicity, and may provide a new specific set of pathways for neuroprotective strategies

Amyloid beta induces the morphological neurodegenerative triad of spine loss, dendritic simplification, and neuritic dystrophies through calcineurin activation

Amyloid beta containing plaques are surrounded by dystrophic neurites in the Alzheimer disease (AD) brain, but whether and how plaques induce these neuritic abnormalities remain unknown. We tested the hypothesis that soluble oligomeric assemblies of Aβ, which surround plaques, induce calcium mediated secondary cascades that lead to dystrophic changes in local neurites. We show that soluble Aβ oligomers lead to activation of the calcium-dependent phosphatase CaN (PP2B) which in turn activates the transcriptional factor nuclear factor of activated T cells (NFAT). Activation of these signaling pathways, even in the absence of Aβ, is sufficient to produce a virtual phenocopy of Aβ induced dystrophic neurites, dendritic simplification, and dendritic spine loss in both neurons in culture and in the adult mouse brain. Importantly, the morphological deficits in the vicinity of Aβ deposits in a mouse model of AD are ameliorated by CaN inhibition, supporting the hypothesis that CaN/NFAT are aberrantly activated by Aβ, and that CaN/NFAT activation is responsible for disruption of neuronal structure near plaques. In accord with this, we also detect increased levels of an active form of CaN and NFATc4 in the nuclear fraction from the cortex of patients with AD. Thus, Aβ appears to mediate the neurodegeneration of AD, at least in part, by activation of CaN and subsequent NFAT-mediated downstream cascades

Tau protein liquid–liquid phase separation can initiate tau aggregation

Abstract The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases

Enhance mechanical property and electrical conductivity simultaneously of Sn–Cu–Co solder alloys by directional solidification

Improving mechanical properties of solder alloy can result in a decrease of its conductivity. The decrease in conductivity of the solder increases the generation of Joule heat and reduces the reliability of the joint. This problem can be solved by adjusting the solidification parameters of solders. In this paper, the effect of the growth rate (V) on the microstructure evolution, microhardness and electrical conductivity of directionally solidified (DS) Sn-0.7Cu-xCo (x = 0.5, 1.0, 1.5, 2.0, mass %) alloys was investigated. The Sn-0.7Cu-xCo alloys consists of a Sn-based solid solution (Sn-ss), Cu6Sn5 phase, and CoSn2 phase. The volume fraction of the CoSn2 phase increases with increasing Co content. In the DS specimen, the CoSn2 phases change from block-shaped to a long strip shape with a long axis direction close to the heat flow direction. With increasing V, the growth direction tends to become disordered due to the weakening of the heat flow effect. The microhardness of DS Sn-0.7Cu-xCo alloys increases with increasing V due to an increase of solution strengthening effect. The microhardness of DS Sn-0.7Cu-xCo alloys increases with increasing Co content under the same V. The conductivity of the DS Sn-0.7Cu-xCo alloys decreases with increasing V. The conductivity of the DS Sn-0.7Cu-xCo alloy is even better than that of the as-cast Sn-0.7Cu alloy when V ≤ 20 μm/s. The findings in the research provide a new way to improve the strength of solder without reducing its conductivity

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

Specific detection of tau seeding activity in Alzheimer's disease using rationally designed biosensor cells

Background: The prion-like propagation of tau in neurodegenerative disorders implies that misfolded pathological tau can recruit the normal protein and template its aggregation. Here, we report the methods for the development of sensitive biosensor cell lines for the detection of tau seeding activity. Results: We performed the rational design of novel tau probes based on the current structural knowledge of pathological tau aggregates in Alzheimer's disease. We generated Förster resonance energy transfer (FRET)-based biosensor stable cell lines and characterized their sensitivity, specificity, and overall ability to detect bioactive tau in human samples. As compared to the reference biosensor line, the optimized probe design resulted in an increased efficiency in the detection of tau seeding. The increased sensitivity allowed for the detection of lower amount of tau seeding competency in human brain samples, while preserving specificity for tau seeds found in Alzheimer's disease. Conclusions: This next generation of FRET-based biosensor cells is a novel tool to study tau seeding activity in Alzheimer's disease human samples, especially in samples with low levels of seeding activity, which may help studying early tau-related pathological events.</p

Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9

The capacity of certain adeno-associated virus (AAV) vectors to cross the blood–brain barrier after intravenous delivery offers a unique opportunity for noninvasive brain delivery. However, without a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in nontarget cells or organs. To refine this approach, the present study characterizes the transduction profiles of new self-complementary AAV9 (scAAV9) expressing the green fluorescent protein (GFP) either under an astrocyte (glial fibrillary acidic (GFA) protein) or neuronal (Synapsin (Syn)) promoter, after intravenous injection of adult mice (2 × 1013 vg/kg). ScAAV9-GFA-GFP and scAAV9-Syn-GFP robustly transduce astrocytes (11%) and neurons (17%), respectively, without aberrant expression leakage. Interestingly, while the percentages of GFP-positive astrocytes with scAAV9-GFA-GFP are similar to the performances observed with scAAV9-CBA-GFP (broadly active promoter), significant higher percentages of neurons express GFP with scAAV9-Syn-GFP. GFP-positive excitatory as well as inhibitory neurons are observed, as well as motor neurons in the spinal cord. Additionally, both activated (GFAP-positive) and resting astrocytes (GFAP-negative) express the reporter gene after scAAV9-GFA-GFP injection. These data thoroughly characterize the gene expression specificity of AAVs fitted with neuronal and astrocyte-selective promoters after intravenous delivery, which will prove useful for central nervous system (CNS) gene therapy approaches in which peripheral expression of transgene is a concern