Neurofibrillary tangle-bearing neurons have reduced risk of cell death in mice with Alzheimer's pathology.

A prevailing hypothesis is that neurofibrillary tangles play a causal role in driving cognitive decline in Alzheimer's disease (AD) because tangles correlate anatomically with areas that undergo neuronal loss. We used two-photon longitudinal imaging to directly test this hypothesis and observed the fate of individual neurons in two mouse models. At any time point, neurons without tangles died at >3 times the rate as neurons with tangles. Additionally, prior to dying, they became >20% more distant from neighboring neurons across imaging sessions. Similar microstructural changes were evident in a population of non-tangle-bearing neurons in Alzheimer's donor tissues. Together, these data suggest that nonfibrillar tau puts neurons at high risk of death, and surprisingly, the presence of a tangle reduces this risk. Moreover, cortical microstructure changes appear to be a better predictor of imminent cell death than tangle status is and a promising tool for identifying dying neurons in Alzheimer's.

Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Reconstitution of the Alzheimer's Disease Tau Core Structure from Recombinant Tau297-391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM.

The protein tau misfolds into disease-specific fibrillar structures in more than 20 neurodegenerative diseases collectively referred to as tauopathies. To understand and prevent disease-specific mechanisms of filament formation, in vitro models for aggregation that robustly yield these different end point structures will be necessary. Here, we used cryo-electron microscopy (cryo-EM) to reconstruct fibril polymorphs taken on by residues 297-391 of tau under conditions previously shown to give rise to the core structure found in Alzheimer's disease (AD). While we were able to reconstitute the AD tau core fold, the proportion of these paired helical filaments (PHFs) was highly variable, and a majority of filaments were composed of PHFs with an additional identical C-shaped protofilament attached near the PHF interface, termed triple helical filaments (THFs). Since the impact of filament layer quaternary structure on the biological properties of tau and other amyloid filaments is not known, the applications for samples of this morphology are presently uncertain. We further demonstrate the variation in the proportion of PHFs and PHF-like fibrils compared to other morphologies as a function of shaking time and AD polymorph-favoring cofactor concentration. This variation in polymorph abundance, even under identical experimental conditions, highlights the variation that can arise both within a lab and in different laboratory settings when reconstituting specific fibril polymorphs in vitro.

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.

Conformational fingerprinting of tau variants and strains by Raman spectroscopy.

Tauopathies are a group of disorders in which the deposition of abnormally folded tau protein accompanies neurodegeneration. The development of methods for detection and classification of pathological changes in protein conformation are desirable for understanding the factors that influence the structural polymorphism of aggregates in tauopathies. We have previously demonstrated the utility of Raman spectroscopy for the characterization and discrimination of different protein aggregates, including tau, based on their unique conformational signatures. Building on this, in the present study, we assess the utility of Raman spectroscopy for characterizing and distinguishing different conformers of the same protein which in the case of tau are unique tau strains generated in vitro. We now investigate the impact of aggregation environment, cofactors, post-translational modification and primary sequence on the Raman fingerprint of tau fibrils. Using quantitative conformational fingerprinting and multivariate statistical analysis, we found that the aggregation of tau in different buffer conditions resulted in the formation of distinct fibril strains. Unique spectral markers were identified for tau fibrils generated using heparin or RNA cofactors, as well as for phosphorylated tau. We also determined that the primary sequence of the tau monomer influenced the conformational signature of the resulting tau fibril, including 2N4R, 0N3R, K18 and P301S tau variants. These results highlight the conformational polymorphism of tau fibrils, which is reflected in the wide range of associated neurological disorders. Furthermore, the analyses presented in this study provide a benchmark for the Raman spectroscopic characterization of tau strains, which may shed light on how the aggregation environment, cofactors and post-translational modifications influence tau conformation in vivo in future studies.

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein.

Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.

Isoform-selective decrease of glycogen synthase kinase-3-beta (GSK-3ß) reduces synaptic tau phosphorylation, transcellular spreading, and aggregation.

It has been suggested that aberrant activation of glycogen synthase kinase-3-beta (GSK-3ß) can trigger abnormal tau hyperphosphorylation and aggregation, which ultimately leads to neuronal/synaptic damage and impaired cognition in Alzheimer disease (AD). We examined if isoform-selective partial reduction of GSK-3ß can decrease pathological tau changes, including hyperphosphorylation, aggregation, and spreading, in mice with localized human wild-type tau (hTau) expression in the brain. We used adeno-associated viruses (AAVs) to express hTau locally in the entorhinal cortex of wild-type and GSK-3ß hemi-knockout (GSK-3ß-HK) mice. GSK-3ß-HK mice had significantly less accumulation of hyperphosphorylated tau in synapses and showed a significant decrease of tau protein spread between neurons. In primary neuronal cultures from GSK-3ß-HK mice, the aggregation of exogenous FTD-mutant tau was also significantly reduced. These results show that a partial decrease of GSK-3ß significantly represses tau-initiated neurodegenerative changes in the brain, and therefore is a promising therapeutic target for AD and other tauopathies.

Increased mitochondrial calcium levels associated with neuronal death in a mouse model of Alzheimer's disease.

Mitochondria contribute to shape intraneuronal Ca2+ signals. Excessive Ca2+ taken up by mitochondria could lead to cell death. Amyloid beta (Aß) causes cytosolic Ca2+ overload, but the effects of Aß on mitochondrial Ca2+ levels in Alzheimer's disease (AD) remain unclear. Using a ratiometric Ca2+ indicator targeted to neuronal mitochondria and intravital multiphoton microscopy, we find increased mitochondrial Ca2+ levels associated with plaque deposition and neuronal death in a transgenic mouse model of cerebral ß-amyloidosis. Naturally secreted soluble Aß applied onto the healthy brain increases Ca2+ concentration in mitochondria, which is prevented by blockage of the mitochondrial calcium uniporter. RNA-sequencing from post-mortem AD human brains shows downregulation in the expression of mitochondrial influx Ca2+ transporter genes, but upregulation in the genes related to mitochondrial Ca2+ efflux pathways, suggesting a counteracting effect to avoid Ca2+ overload. We propose lowering neuronal mitochondrial Ca2+ by inhibiting the mitochondrial Ca2+ uniporter as a novel potential therapeutic target against AD.

Experimental evidence for the age dependence of tau protein spread in the brain.

The incidence of Alzheimer's disease (AD), which is characterized by progressive cognitive decline that correlates with the spread of tau protein aggregation in the cortical mantle, is strongly age-related. It could be that age predisposes the brain for tau misfolding and supports the propagation of tau pathology. We tested this hypothesis using an experimental setup that allowed for exploration of age-related factors of tau spread and regional vulnerability. We virally expressed human tau locally in entorhinal cortex (EC) neurons of young or old mice and monitored the cell-to-cell tau protein spread by immunolabeling. Old animals showed more tau spreading in the hippocampus and adjacent cortical areas and accumulated more misfolded tau in EC neurons. No misfolding, at any age, was observed in the striatum, a brain region mostly unaffected by tangles. Age and brain region dependent tau spreading and misfolding likely contribute to the profound age-related risk for sporadic AD.

A flow cytometry-based in vitro assay reveals that formation of apolipoprotein E (ApoE)-amyloid beta complexes depends on ApoE isoform and cell type.

Apolipoprotein E (ApoE) is a secreted apolipoprotein with three isoforms, E2, E3, and E4, that binds to lipids and facilitates their transport in the extracellular environment of the brain and the periphery. The E4 allele is a major genetic risk factor for the sporadic form of Alzheimer's disease (AD), and studies of human brain and mouse models have revealed that E4 significantly exacerbates the deposition of amyloid beta (Aß). It has been suggested that this deposition could be attributed to the formation of soluble ApoE isoform-specific ApoE-Aß complexes. However, previous studies have reported conflicting results regarding the directionality and strength of those interactions. In this study, using a series of flow cytometry assays that maintain the physiological integrity of ApoE-Aß complexes, we systematically assessed the association of Aß with ApoE2, E3, or E4. We used ApoE secreted from HEK cells or astrocytes overexpressing ApoE fused with a GFP tag. As a source of soluble Aß peptide, we used synthetic Aß40 or Aß42 or physiological Aß secreted from CHO cell lines overexpressing WT or V717F variant amyloid precursor protein (APP). We observed significant interactions between the different ApoE isoforms and Aß, with E4 interacting with Aß more strongly than the E2 and E3 isoforms. We also found subtle differences depending on the Aß type and the ApoE-producing cell type. In conclusion, these results indicate that the strength of the ApoE-Aß association depends on the source of Aß or ApoE.

Tau protein liquid-liquid phase separation can initiate tau aggregation.

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.

Isoform- and cell type-specific structure of apolipoprotein E lipoparticles as revealed by a novel Forster resonance energy transfer assay.

Apolipoprotein E (apoE) has an important role in the pathogenesis of Alzheimer's disease with its three isoforms having distinct effects on disease risk. Here, we assessed the conformational differences between those isoforms using a novel flow cytometry-Forster resonance energy transfer (FRET) assay. We showed that the conformation of intracellular apoE within HEK cells and astrocytes adopts a directional pattern; in other words, E4 adopts the most closed conformation, E2 adopts the most open conformation, and E3 adopts an intermediate conformation. However, this pattern was not maintained upon secretion of apoE from astrocytes. Intermolecular interactions between apoE molecules were isoform-specific, indicating a great diversity in the structure of apoE lipoparticles. Finally, we showed that secreted E4 is the most lipidated isoform in astrocytes, suggesting that increased lipidation acts as a folding chaperone enabling E4 to adopt a closed conformation. In conclusion, this study gives insights into apoE biology and establishes a robust screening system to monitor apoE conformation.

Characterization of TauC3 antibody and demonstration of its potential to block tau propagation.

The spread of neurofibrillary tangle (NFT) pathology through the human brain is a hallmark of Alzheimer's disease (AD), which is thought to be caused by the propagation of "seeding" competent soluble misfolded tau. "TauC3", a C-terminally truncated form of tau that is generated by caspase-3 cleavage at D421, has previously been observed in NFTs and has been implicated in tau toxicity. Here we show that TauC3 is found in the seeding competent high molecular weight (HMW) protein fraction of human AD brain. Using a specific TauC3 antibody, we were able to substantially block the HMW tau seeding activity of human AD brain extracts in an in vitro tau seeding FRET assay. We propose that TauC3 could contribute to the templated tau misfolding that leads to NFT spread in AD brains.

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.

Formation, release, and internalization of stable tau oligomers in cells.

Tau is a neuronal microtubule-binding protein that, in Alzheimer's disease and other neurodegenerative diseases, can form oligomeric and large fibrillar aggregates, which deposit in neurofibrillary tangles. Tau's physiological state of multimerization appears to vary across conditions, and a stable dimeric form of soluble tau has been suggested from experiments using recombinant tau in vitro. We tested if tau dimerization or oligomerization, also occurs in cells, and if soluble tau oligomers are relevant for the release and internalization of tau. We developed a sensitive tau split-luciferase assay to show the rapid intracellular formation of stable tau dimers that are released and taken up by cells. Our data further suggest that tau dimerization can be accelerated slightly by aggregation catalysts. We conclude that tau oligomers are a stable physiological form of tau, and that tau oligomerization does not necessarily lead to tau aggregation.

Seed-competent high-molecular-weight tau species accumulates in the cerebrospinal fluid of Alzheimer's disease mouse model and human patients.

OBJECTIVE: Cerebrospinal fluid (CSF) tau is an excellent surrogate marker for assessing neuropathological changes that occur in Alzheimer's disease (AD) patients. However, whether the elevated tau in AD CSF is just a marker of neurodegeneration or, in fact, a part of the disease process is uncertain. Moreover, it is unknown how CSF tau relates to the recently described soluble high-molecular-weight (HMW) species that is found in the postmortem AD brain and can be taken up by neurons and seed aggregates. METHODS: We have examined seeding and uptake properties of brain extracellular tau from various sources, including interstitial fluid (ISF) and CSF from an AD transgenic mouse model and postmortem ventricular and antemortem lumbar CSF from AD patients. RESULTS: We found that brain ISF and CSF tau from the AD mouse model can be taken up by cells and induce intracellular aggregates. Ventricular CSF from AD patients contained a rare HMW tau species that exerted a higher seeding activity. Notably, the HMW tau species was also detected in lumbar CSF from AD patients, and its levels were significantly elevated compared to control subjects. HMW tau derived from CSF of AD patients was seed competent in vitro. INTERPRETATION: These findings suggest that CSF from an AD brain contains potentially bioactive HMW tau species, giving new insights into the role of CSF tau and biomarker development for AD. Ann Neurol 2016;80:355-367.

Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity.

In Alzheimer's disease and tauopathies, tau protein aggregates into neurofibrillary tangles that progressively spread to synaptically connected brain regions. A prion-like mechanism has been suggested: misfolded tau propagating through the brain seeds neurotoxic aggregation of soluble tau in recipient neurons. We use transgenic mice and viral tau expression to test the hypotheses that trans-synaptic tau propagation, aggregation, and toxicity rely on the presence of endogenous soluble tau. Surprisingly, mice expressing human P301Ltau in the entorhinal cortex showed equivalent tau propagation and accumulation in recipient neurons even in the absence of endogenous tau. We then tested whether the lack of endogenous tau protects against misfolded tau aggregation and toxicity, a second prion model paradigm for tau, using P301Ltau-overexpressing mice with severe tangle pathology and neurodegeneration. Crossed onto tau-null background, these mice had similar tangle numbers but were protected against neurotoxicity. Therefore, misfolded tau can propagate across neural systems without requisite templated misfolding, but the absence of endogenous tau markedly blunts toxicity. These results show that tau does not strictly classify as a prion protein.

Direct visualization of CHIP-mediated degradation of alpha-synuclein in vivo: implications for PD therapeutics.

Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies, a pathological hallmark comprised mostly of aggregated alpha synuclein. Accumulating evidence demonstrates the association of smaller oligomeric aggregates to disease etiology and many therapeutic approaches are aimed at inhibiting and reducing the aggregation process. Molecular chaperones and co-chaperones play a key role in protein homeostasis and have potential as therapeutics to inhibit alpha synuclein associated toxicity. Here we use a gene therapy approach to evaluate the applicability of the Hsp70 co-chaperone CHIP (C-terminal Hsp70 interacting protein) as a therapeutic candidate and examine its direct effect on alpha synuclein aggregates in vivo. Utilizing a novel viral vector mediated rat model to directly detect alpha synuclein aggregates, we show that CHIP can mediate the degradation of alpha synuclein aggregates in vivo. However, our studies also reveal that CHIP may potentially degrade tyrosine hydroxylase which would compromise the applicability of CHIP as a therapeutic approach for Parkinson's disease.

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.

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.