Aims

Modulating p75^NTR signaling with the small molecule LM11A-31 (C31) was previously shown to prevent synaptic dysfunction, as assessed with long-term potentiation (LTP), in a mouse model of tauopathy (PS19). We ask whether tauopathy-related alterations in gene co-expression, in response to stimulation, can be normalized by C31.

Methods

Wildtype (WT) and PS19 mice were treated with vehicle or C31 for 3 months starting at 6 months of age. Theta burst stimulation (TBS) was used to induce late-phase LTP. Bulk RNA sequencing with cell-type enrichment analysis was performed on unstimulated and stimulated slices to generate activity-dependent profiles of gene co-expression networks using weighted analysis with a soft-threshold power of 18 to achieve scale-free topology R²>0.8 and module size 25.

Results

In PS19 versus WT mice, 16 activity-dependent modules were significantly down-regulated with cell-type enrichment for neurons and 8 were up-regulated with enrichment for glia. Notably, all of the transcriptional co-expression modules altered in PS19 mice were normalized to WT level with C31. Down-regulation of gene transcription modules in PS19 mice, known to be significantly enriched for genes in neurons associated to post-synapse and LTP, were up-regulated upon C31 treatment; while in microglial-, oligodendrogial- and astroglial-associated modules, up-regulated genes in PS19 mice were downregulated with C31. Comparison of mouse and human modules demonstrated that those affected in treated mice have similar up- and down-regulated pattern of expression in human AD.

Conclusions

We identified neuronal and glial mechanisms by which the p75^NTR-modulator C31 might prevent tauopathy-associated synaptic dysfunction and



underlying alterations of human AD-relevant gene co-expression networks.

Aims

Amyloid-beta Positron Emission Tomography (PET) efficiently detects Alzheimer’s disease (AD) pathology in cognitive impaired patients, but lacks specificity regarding the prediction of future cognitive status. The increasing validation of tau PET tracers raises questions about whether this biomarker could predict clinically relevant cognitive decline in the AD continuum.

Methods

We selected 293 subjects [cognitive normal (CN; n=196), mild cognitive impairment (MCI; n=80), dementia (n=17)] with baseline amyloid-beta and tau PET scans that have completed a follow-up of at least two years, from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) dataset. Based on Alzheimer’s Disease Assessment Scale–Cognitive Subscale (ADAS-Cog) and linear mixed-effects models, all individuals were clustered as either fast or slow decliners. Group comparisons and accuracy of discrimination were tested.

Results

The individuals with amyloid-beta positivity and fast cognitive decline showed the highest tau PET binding values independently of baseline cognitive status, compared to the other groups (amyloid-beta negative with fast or slow decline and amyloid-beta positive with slow decline). Baseline tau PET binding could determine fast decliners with a amyloid-beta positive scan with an accuracy of 87% (composite temporal region of interest). Tau PET showed a similarly high accuracy to determine patients with MCI that would be both amyloid-beta positive and show a progress in their diagnosis at follow-up.

Conclusions

High baseline tau load is coupled with both amyloid-beta pathology and an accelerated profile of cognitive decline. Tau PET usage could offer substantial advantages over amyloid-beta PET since it gives information about the underlying pathophysiology and prognosis of the disease.

Aims

We recently discovered a novel tau binding partner, SERPINA5, to be upregulated in Alzheimer’s disease (AD) and co-localize with corticolimbic tau distribution. As tau pathology is also observed in primary tauopathies, we hypothesized that SERPINA5 burden would mirror tau burden in these diseases. Thus, we sought to define the relationship between SERPINA5 and tau in primary tauopathies and compare this relationship to AD.

Methods

Immunohistochemistry was performed on the hippocampus or cingulate in 4 cohorts (n=6 per disease group): 1) AD, 2) 3R+4R primary age-related tauopathy, 3) 4R tauopathies (progressive supranuclear palsy, corticobasal degeneration, argyrophilic grains disease), and 4) 3R tauopathy (Pick’s disease). Burden analyses for SERPINA5 and a phospho-tau marker (AT8) were performed on digitized whole slide images.

Results

Visual interpretation of SERPINA5 immunostaining identified both neuronal and glial inclusions that are commonly AT8-positive in primary tauopathies. There were exceptions to this pattern such as SERPINA5-positive Pick bodies being rarely observed. However, some pathologic structures like ballooned neurons were preferentially stained by SERPINA5. Quantitative analyses confirmed this observation with AT8 and SERPINA5 burden correlating in both the cingulate (R²=0.834, p<0.0001, Figure 1a) and hippocampal CA1 (R²=0.926, p<0.0001, Figure 1d). Group comparisons revealed greater AT8 and SERPINA5 burden in AD compared to primary tauopathies in both the cingulate (Figure 1b-c) and hippocampus (Figure 1e-f) (p<0.0001).

Conclusions

SERPINA5 immunostaining patterns in primary tauopathies reflect tau immunostaining patterns. Our data suggests the SERPINA5-tau interaction may not be specific for AD, which may implicate the role of SERPINA5 more broadly across tauopathies.

Aims

A fundamental gap in the field is the incomplete identification of the mechanisms by which tau promotes cellular dysfunction. Pathological tau shifts the translatome yielding a maladaptive response. However, the molecular mechanisms driving this effect remain unknown. We aimed to establish these mechanisms by defining the impact of pathological tau on translation, the components of the tau-interacting translation machinery, and whether improving translation rescues brain function in models of tauopathy.

Methods

We used surface-sensing of translation (SUnSET) and SUnSET proteomics to identify newly-synthesized proteins in various tauopathy models. We treated rTg4510 mice with GSK2606414, a kinase inhibitor that restores ER stress-mediated translational repression, and measured changes in functional and pathological outcomes using manganese-enhanced MRI, behavior assays, biochemistry and immunohistochemistry. Finally, we performed enhanced-CLIP and ribo-profiling of human Alzheimer’s brains.

Results

We found overall suppression of translation in tauopathy models; numerous proteins involved in translation were significantly decreased in rTg4510 brains, and suppression of tau expression rescued these effects. Restoring translation with GSK2606414 rescued brain function in tau transgenics. eCLIP and ribo-profiling identified RNAs that associate differentially with pathological tau.

Conclusions

These results suggest that proteins that manufacture the translational machinery are not adequately synthesized, leading to a maladaptive response to tau pathology. Overcoming deficits in their translation offers functional benefits. In addition, tau exerts unanticipated and potentially devastating effects on availability of RNAs for translation. Therefore, tau-mediated maladaptive translation may be a pathogenic event in tauopathies, and efforts to rescue these effects are currently underway.

Aims

The templated aggregation of tau is considered a major pathological event driving the progression of Alzheimer’s Disease and tauopathy. Whilst uptake of tau is a well-documented process, the mechanistic details governing the access of tau assemblies to the cytosol are unknown. We propose the entry of tau to the cytosol occurs upstream and is required for seeded aggregation. To investigate we have developed novel cell-based assays to quantify the entry of tau to the cytosol with picomolar sensitivity.

Methods

We established a sensitive luciferase-based assay to quantify the cytosolic entry of tau assemblies to various cell types including iPSC-derived cortical neurons. By manipulating the endogenous cellular machinery, we can dissect the underlying mechanisms of entry whilst probing the relationship to seeded aggregation.

Results

Cytosolic entry of tau in HEK293 occurs via a clathrin- and dynamin-dependent route with a role for vesicular sorting machinery in maintaining tau inside compartments. Conversely, entry to both primary and iPSC-derived cortical neurons occurs via an atypical clathrin- and dynamin-independent route with an insensitivity to endosomal disruption. Cholesterol extraction or intracellular accumulation starkly modifies neuronal entry with concomitant changes to seeded aggregation in neurons and slice cultures. Additionally, knockdown of Niemann-Pick type C1 protein significantly modified entry, informing the mechanism of mutation in a human tauopathy.

Conclusions

Taken together, we have demonstrated that entry to the cytosol is upstream and rate-limiting in seeded aggregation. We have found cell-type dependent entry mechanisms with a distinct role for cholesterol in tau spread and disease, further informing the fundamental biology of neurodegeneration.

Aims

Tau is involved in maintaining neuronal structure. In tauopathies, tau can aggregate to form oligomers (oTau). Although the toxicity of oTau is well established, the mechanistic basis of its actions on neuronal function remains poorly understood. Previously, full-length recombinant oTau was found to disrupt neuronal function, synaptic transmission and plasticity (Hill et al, 2019). In this study, we look to understand how oTau mediates these changes.

Methods

We truncated the tau molecule into two parts: the first 123 amino acids and the remaining 124-441 amino acids. We have used these clinically relevant truncations to elucidate the mechanisms underlying the changes in neuronal properties. We introduced the truncated versions of tau in aggregated form into single hippocampal pyramidal cells in acute mouse brain slices and measured the resultant changes in neuronal properties.

Results

These truncated tau molecules had specific effects on neuronal function, allowing us to assign the actions of full-length tau to different regions of the molecule. We identified one key target for the effects of tau, the voltage-gated sodium channel, which could account for the effects of tau on action potential waveform.

Conclusions

This simple, yet highly effective technique of introducing structurally defined aggregated proteins into single neurons allows unparalleled levels of detail and provides a unique opportunity to understand the underlying pathology for tauopathies. By truncating the tau molecule, we have probed the mechanisms that underlie tau dysfunction, and this increased understanding of tau’s pathological actions will build towards developing future tau-targeting therapies.

Hill et al (2019). eNeuro, 6(5), pp.eNEURO.0166-19.2019.

Aims

Aqueously soluble oligomers of the Aβ protein, rather than monomers or insoluble fibrils, are considered the most bioactive form. Although extensive characterization of synthetic oligomers has been performed, little is known about the structure of oligomers that occur in human brain; instead, they are operationally defined as that non-monomeric material present in the supernatant after ultracentrifugation of an aqueously soluble extract.

Methods

Soluble extracts were prepared in TBS using homogenization or soaking, followed by ultracentrifugation in the SW41Ti rotor at 200,000 g. Re-pelleting was performed in a tabletop centrifuge at 20,000 g. Immunogold labeling used D54D2 (rabbit anti-Aβ N-terminus) or various rabbit anti-phosphotau monoclonal antibodies (Cell Signaling Technologies). ELISA utilized m266 and 21F12 after denaturing samples in 5M guanidine hydrochloride.

Results

We found that short filamentous structures could be re-pelleted from soluble extracts of AD but not control cortex in a concentration-dependent manner, and visualized by negative-stain electron microscopy. Immunogold labeling confirmed the presence of Aβ. Re-pelleting of Aβ filaments from soluble post-200,000 g extracts was enhanced by freezing at least overnight and by adding Triton X-100, Tween-20, and digitonin, but not CHAPSO, but was prevented by high ionic strength. Linear time-dependence in re-pelleting suggested the filaments were not reconstituted during re-pelleting and may be present in the initial post-200,000 g aqueous extract. We also observed paired helical filaments re-pelleting from aqueous extracts labeled by anti-phosphotau immunogold.

Conclusions

These results suggest that at least some high molecular weight oligomers of Aβ and tau in putatively soluble fractions are, and/or become, filamentous.

Aims

The abnormal aggregation of transactive response DNA-binding protein of 43 kDa (TDP-43) in neurons and glia is the defining pathological hallmark of amyotrophic lateral sclerosis (ALS) and multiple forms of frontotemporal lobar degeneration (FTLD). It is also common in other diseases, including Alzheimer's and Parkinson's. However, the structures of pathological aggregated TDP-43 are unknown.

Methods

We used electron cryo-microscopy (cryo-EM) to determine the structures of pathological aggregated TDP-43 extracted from the frontal and motor cortices of individuals that succumbed to ALS with FTLD.

Results

We found a conserved amyloid-like filament structure comprising a single protofilament. The ordered filament core is formed by residues 282 to 360 in the TDP-43 low-complexity domain, which adopt a novel double-spiral-shaped fold. The fold shows no similarity to those of TDP-43 filaments formed in vitro. Abundant glycine and neutral polar residues facilitate numerous turns that restrict β-strand length, resulting in the absence of β-sheet stacking associated with cross-β amyloid structure. An uneven distribution of these residues gives rise to structurally and chemically distinct filament surfaces. External densities adjacent to these surfaces suggest possible ligand binding sites.

Conclusions

This work enhances our understanding of the molecular pathogenesis of ALS and FTLD, revealing the formation of filaments that are structurally distinct from amyloid filaments in other neurodegenerative diseases. The structure of pathological TDP-43 filaments in ALS-FTLD informs the development of accurate disease models, as well as diagnostic and therapeutic agents.