Croft, Cara L.

AbstractBackgroundAlzheimer’s disease (AD) is characterized by the accumulation of neurofibrillary tangles (NFTs), comprised primarily of microtubule‐associated protein tau. NFTs are defining hallmarks of AD and its accumulation into pathological oligomers and fibrils has been associated with cognitive decline in AD and other tauopathies. Despite its pivotal role in the disease process, we still have a poor understanding of how their formation, toxicity, and spread across brain regions is regulated via tau‐associated proteins that regulate the oligomerization, pathological accumulation, and seeding of tau in affected cell types and vulnerable brain regions.MethodWe have established two proximity‐dependent biotin‐identification (BioID) models as novel approaches to identify the composition and proximal molecular environment of tau aggregates. Using an in vitro and ex vivo model approach coupled with mass spectrometry, we have identified proteins that were primarily associated with mutant tau aggregates vs. soluble wildtype tau and stratified them based on significance, molecular pathway and druggability. Immunoblotting and immunofluorescence were performed to assess the interaction of endogenous and overexpressed top candidates with tau, along with visualization with NFTs in the context of human AD pathology to assess their impact on tau‐aggregate formation and toxicity.ResultTo determine the physiological and aggregate specific interacting partners of tau, we are comparing wild type tau (TauWT) with mutant tau (Tau3xMUT) carrying three tauopathy‐associated mutations (A152T/P301L/S320F). The proteomic analysis from our combined proteomic datasets revealed that significantly changed proteins included candidates involved in RNA processing, as well as protein ubiquitination and proteasome degradation. We have begun validating proteins that are enriched in mutant tau in human tissue of AD, as well as primary tauopathies, that may be druggable targets for tauopathies.ConclusionThis study provides novel insights into cellular pathways and molecular mechanisms of neurodegeneration, by identifying in the context of living neurons and brain tissue different functional classes of tau‐associated proteins with relevance for AD pathophysiology. These are expected to include proteins that may mediate the toxic effect of NFTs, facilitate the formation or degradation of pathological tau aggregates, and catalyze posttranslational modifications of tau oligomers and associated proteins.

AbstractBackgroundAlzheimer's disease (AD) and related neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates. A defining hallmark of AD is the formation of neurofibrillary tangles (NFTs) by aggregation of the microtubule‐associated protein tau into pathological oligomers and fibrils. Although NFTs are believed to play a pivotal role in the disease process, we have a poor understanding of how their formation, toxicity, and spread across brain regions is regulated. Recent data suggest that the seeding behavior of tau is governed by patterns of posttranslational modifications and varying filament structures. Understanding how tau‐associated proteins regulate the oligomerization, pathological accumulation, and seeding of tau in affected neurons and glia is of critical importance for therapy development.MethodTo profile the interactome of tau aggregates, we have established proximity‐dependent biotin‐identification (BioID) as a novel method to identify the composition and proximal molecular environment of insoluble protein aggregates in the context of living brain cells and tissue. Using an in vitro and ex vivo model approach coupled with mass spectrometry, we are generating datasets containing top identified proteins, stratified based on significance and molecular pathway. Once we validate their co‐localization with NFTs in the context of human AD pathology, functional characterization of putative modifiers will be performed to assess their impact on tau‐aggregate and toxicity.ResultTo determine the physiological and aggregate specific interacting partners of tau, we are comparing wild type tau (TauWT) with mutant tau (Tau3xMUT) carrying three tauopathy‐associated mutations (A152T/P301L/S320F) that form hyper‐phosphorylated and thioflavin S‐positive aggregates even in the absence of seeding. The proteomic analysis from our pilot study revealed that significantly changed proteins included candidates involved in RNA processing, as well as protein ubiquitination and proteasome degradation.ConclusionThis study provides novel insights into cellular pathways and molecular mechanisms of neurodegeneration, by identifying in the context of living neurons and brain tissue different functional classes of tau‐associated proteins with relevance for AD pathophysiology. These are expected to include proteins that may mediate the toxic effect of NFTs, facilitate the formation or degradation of pathological tau aggregates, and catalyze posttranslational modifications of tau oligomers and associated proteins.

AbstractBackgroundPathological aging (PA) patients are individuals that are cognitively normal prior to death but have abundant and widespread amyloid‐β (Aβ) deposits. These individuals typically have few cored amyloid deposits with variable levels of diffuse parenchymal deposits and/or vascular deposits. There is little or no inflammatory reaction, neuritic pathology or neurofibrillary tangles in the cortex. Previously, we have shown that total Aβ levels in PA brain lysates were similar to the levels in Alzheimer’s disease (AD) brain lysates, with overlap in the length of the Aβ peptides that were deposited and no major differences in SDS‐stable Aβ oligomeric assemblies. Despite these similarities, it is unclear whether PA represents a prodromal phase of AD or if the amyloid pathology of PA patients represents a less pathogenic variant. To explore this idea, we examined the seeding capacity of AD and PA brain homogenates to induce Aβ deposition and to secondarily induce tau pathology.MethodWe determined the seeding capacity of two AD, four PA and two control brain homogenates in transgenic mice expressing APPswe/ind with and without Tau‐P301L by intracerebral injection at neonatal day 0. At either 6, 9, 12 or 18 months, we assessed Aβ deposition and neurofibrillary pathology in all 4 genotypes by immunohistochemical and biochemical methods. Furthermore we examined the seeding activity of human AD brain lysates in brain slice cultures from APP/Tau transgenic mice.ResultIn our newborn mouse injections, we observed widespread Aβ seeding as evidenced by accelerated onset of diffuse parenchymal Aβ deposition that was largely proportional to the burden of parenchyma deposits in patient brains. In all cases, high levels of pial deposition were observed and Aβ42 was the predominant peptide deposited. Tau pathology was not induced in any animal, and with the absence of neuritic plaques there was no induction of tau positive neurites.ConclusionThis study provides evidence that the Aβ conformers associated with diffuse Aβ deposits in the brains of cognitively normal individuals possess seeding activities similar to those found in the brains of AD patients. The findings are consistent with the idea that pathological aging is on a continuum with AD.

It has been challenging to produce ex vivo models of the inclusion pathologies that are hallmark pathologies of many neurodegenerative diseases. Using three-dimensional mouse brain slice cultures (BSCs), we have developed a paradigm that rapidly and robustly recapitulates mature neurofibrillary inclusion and Lewy body formation found in Alzheimer’s and Parkinson’s disease, respectively. This was achieved by transducing the BSCs with recombinant adeno-associated viruses (rAAVs) that express α-synuclein or variants of tau. Notably, the tauopathy BSC model enables screening of small molecule therapeutics and tracking of neurodegeneration. More generally, the rAAV BSC “toolkit” enables efficient transduction and transgene expression from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction.