Epigenetic mechanisms, including histone acetylation, regulate learning and memory and underlie Alzheimer's disease and related dementia (ADRD). Acetyl-CoA synthetase 2 (ACSS2), an enzyme generating acetyl-CoA, locally regulates histone acetylation and gene expression in neuronal nuclei. This regulatory mechanism may be a promising target for therapeutic intervention in neurodegenerative diseases. Previously, we showed that systemic ACSS2 knockout mice, although largely normal in physiology, exhibit memory deficits. Here, we investigated whether increasing ACSS2 levels could protect neurons against disease and age-associated cognitive decline. Given the role of tau in ADRD, we used primary hippocampal neurons that mimic the sporadic development of tau pathology and the P301S transgenic mouse model for tau-induced memory decline. Our results show that ACSS2 upregulation mitigates tau-induced transcriptional alterations, enhances neuronal resilience against tau pathology, improves long-term potentiation, and ameliorates memory deficits. Additionally, boosting histone acetylation through ACSS2 countered age-related memory decline. These findings indicate that increasing ACSS2 is highly effective in countering age- and tau-induced transcriptome changes, preserving elevated levels of synaptic genes, and safeguarding synaptic integrity. These findings position ACSS2 as a key epigenetic regulator of cognitive aging and ADRD, highlighting its potential for targeted therapeutics to enhance brain resilience and function.

Fibrillar aggregates of the natively disordered protein α-synuclein (αS) are hallmarks of Parkinson's disease and related neurodegenerative disorders termed synucleinopathies. Here, we used micromap (μMap) photoproximity labeling to determine the interactomes of αS monomers and fibrils in mouse brain lysate to better understand both the loss of healthy function and gain of toxic function aspects of synucleinopathies. Several αS variants were synthesized and characterized, showing that the small size (1 kDa) of the Ir catalyst attached through a Cys-maleimide linkage makes it minimally perturbing to αS, with a narrow labeling radius that allows one to identify interactome differences between different regions of αS. Monomer and fibril interactomes were compared to each other and to previous proximity labeling data sets for validation, and several examples of further investigations are demonstrated, including Western blotting, affinity pulldowns, fluorescence and super-resolution microscopy, and μMap in primary neurons.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of upper and lower motor neurons and pathological accumulation of TAR DNA-binding protein 43 (TDP-43). Microglial activation is a prominent feature of ALS pathology, and the receptor tyrosine kinase Axl, a marker of disease-associated microglia (DAM), has been proposed as a potential therapeutic target. Here, we investigated Axl signaling and its pharmacological inhibition in the inducible rNLS8 mouse model of ALS. Disease progression in the brain of these mice was accompanied by robust microgliosis, reflected by increased Iba1 Immunoreactivity in the hippocampus and by elevated Axl protein at later stages. Treatment with Bemcentinib (BGB), a selective Axl inhibitor, resulted in central nervous system penetration by the drug and evidence of target engagement as revealed by increased plasma soluble Axl and modulation of downstream signaling pathways in the brain, including reduced total Stat3 and Akt. Nevertheless, BGB administration failed to attenuate microglial or astrocytic reactivity in the brain and did not improve motor function, clasping behavior, or weight loss. In contrast, suppressed expression of the pathogenic TDP-43 cytoplasmic protein rapidly restored motor performance and body weight, confirming the reversibility of these phenotypes in the rNSL8 model. These data further establish Axl protein upregulation as a component of the microglial response in TDP-43-driven cortical neurodegeneration but suggest that Axl inhibition after BGB treatment was insufficient to counteract neuroinflammation or functional decline in the rNLS8 mouse model of ALS.

Ageing is accompanied by declining memory function, with extremely heterogeneous manifestation in the human population1. Brain-extrinsic factors influencing cognitive decline, such as gastrointestinal signals, have emerged as attractive targets for peripheral interventions2-6, but the underlying mechanisms remain largely unclear. Here, by charting a high-resolution map of microbiome ageing and its functional consequences throughout the lifespan of mice, we identify a mechanism by which inhibition of gut-brain signalling during ageing results in impaired neuronal activation in the hippocampus and loss of memory encoding. Specifically, accumulation of gut bacteria that produce medium-chain fatty acids, such as Parabacteroides goldsteinii, can drive peripheral myeloid cell inflammation through GPR84 signalling. As a result, the function of vagal afferent neurons is impaired, the interoceptive signal received by the brain is weakened and hippocampal function declines. We leverage this pathway to define interventions that enhance memory in aged mice, such as phage targeting of Parabacteroides, GPR84 inhibition and restoration of vagal activity. These findings indicate a key role for interoceptive dysfunction in brain ageing and suggest that interoceptomimetics that stimulate gut-brain communication may counteract age-associated cognitive decline.

A bidirectional relationship between seizures and neurodegenerative disease has been established with neurodegenerative pathology found in late-onset epilepsy patients, increased risk of seizures in tauopathies, and accelerated Alzheimer's disease progression in patients with epileptiform activity. Tau pathology spreads between interconnected neuronal networks, driving disease progression. We hypothesized that seizures would promote tau propagation throughout the brain in a tauopathy mouse model. To explore the brain-wide relationship between tau pathology and seizure activity, we crossed the T40PL-GFP mouse, which contains a pathogenic MAPT mutation tagged with GFP, with targeted recombination in active population (TRAP; T40PL-TRAP) mice to label all seizure activated neurons with tdTomato. We triggered tau propagation in these mice with intracerebral seeding of human AD brain-derived tau lysate and induced seizures with pentylenetetrazol (PTZ) kindling. With light sheet microscopy, we imaged and mapped tau-GFP and tdT levels throughout whole brain. We found that PTZ induced seizures worsened tau pathology in brain regions with increased tdT levels, including the hippocampus and cortex, and in the fiber tracts in T40PL-TRAP mice. We also found that seizure-activated (tdT+) neurons were more likely to develop somatic tau pathology compared to the surrounding (tdT-) populations. Overall, these data demonstrate that seizures can enhance tau pathology propagation.

Cerebral glucose hypometabolism in Alzheimer’s disease (AD) leads to enhanced metabolism of fatty acids (FAs) and branched-chain amino acids (BCAAs) as a compensatory mechanism. While there have been some 13C labeled studies investigating the metabolism of FAs and BCCAs, their clinical translation is challenging. In this study, we investigated the potential of measuring neurometabolic perturbations through macromolecular signal at 0.9 ppm (MM09) in proton magnetic resonance (1H MR) spectrum. This signal represents a composite macromolecular signal with contributions from lipids and BCAA associated methyl resonances and may be sensitive to metabolic alterations occurring during glucose hypometabolism in AD. MM09 levels were measured from localized 1H MR spectra in the hippocampus and thalamus/hypothalamus of male and female APPNL−F/NL−F (AD) mice. In addition, the levels of glutamate in these regions were also recorded as it is known to be reduced under glucose hypometabolism in AD. We further studied the metabolic association of MM09 with glutamate in Pearson correlation plots. To find the statistical significance of difference two-way ANOVA analysis with post-hoc Tukey HSD tests were used. Male AD mice exhibited significantly reduced MM09 (15.42 ± 1.32 vs. 16.93 ± 1.15 mM; p = 0.008) and glutamate levels (15.27 ± 1.65 vs. 17.24 ± 1.21 mM; p = 0.004) in the hippocampus. Female AD mice did not show any changes in glutamate or MM09 levels. MM09 also showed a strong positive correlation with glutamate (R = 0.74; p < 0.0001). The observed reductions in MM09 and glutamate in male AD mice are consistent with neurometabolic alterations associated with impaired glucose metabolism, whereas the absence of such changes in female AD mice may reflect sex-specific metabolic resilience. The strong association between MM09 and glutamate suggests that MM09 may capture neurochemical changes linked to metabolic adaptations in AD. Because the MM09 resonance occurs in a relatively uncrowded region of the 1H MR spectrum, it may represent a promising spectroscopic marker for investigating metabolic shifts in AD and warrants further evaluation in clinical studies.

Amyloidoses are predominantly associated with the accumulation of persistent aggregates of a particular protein. For example, the protein α-synuclein characteristically aggregates in Parkinson's disease (PD), while amyloid beta and tau deposits are associated with Alzheimer's disease (AD). However, α-synuclein-positive inclusions have been reportedly found in some tauopathies, and vice versa; tau-positive inclusions can be found in synucleinopathies. This suggests that there may be coexistence or crosstalk between these proteinopathies. This coexistence suggests that the simultaneous presence of these misfolded proteins may amplify pathogenic mechanisms. However, the crosstalk between these two types of proteopathies remains poorly understood. We now determine the structure of α-synuclein fibrils that directly promote tau aggregation by cryogenic electron microscopy. Helical reconstruction at 2.6 Å resolution reveals a new α-synuclein fibril polymorph we term "strain B"; its core is unique, incorporating both the N- and C-termini of α-synuclein. The design of peptides meant to inhibit the formation of this structure demonstrates that the C-terminal domain fragment (D105-E115) of α-synuclein is critical for the formation of "strain B" fibrils and may play a key role in its interaction with tau. We hypothesize that the unique structure of pathological α-synuclein significantly contributes to tau co-aggregation and plays a role in the intricate interactions among Alzheimer's, Parkinson's, and other neurodegenerative diseases. These findings open new avenues for drug targeting, discovery, and improve our understanding of neurodegenerative pathology.

Neuronal hyperexcitability is a hallmark of amyotrophic lateral sclerosis (ALS), but its relationship with the TDP-43 aggregates that comprise the predominant pathology in over 90% of ALS cases remains unclear. Emerging evidence indicates that TDP-43 pathology induces neuronal hyperexcitability, which may contribute to excitotoxic neuronal death. To characterize TDP-43 mediated network excitability changes in a disease-relevant model, we performed in vivo continuous electroencephalography monitoring and ex vivo acute hippocampal slice electrophysiology in rNLS8 mice (males and females), which express human TDP-43 with a defective nuclear localization signal (hTDP-43ΔNLS). Surprisingly, we identified the presence of seizures in ∼64% of rNLS8 mice beginning ∼2.5 weeks after transgene induction (off-DOX). More broadly, we observed longitudinal changes in cortical EEG patterns and circuit hyperexcitability preceding neurodegeneration of vulnerable hippocampal subfields. Consistent with previous reports, we have observed broad dysregulation of AMPA subunit expression in mice expressing hTDP-43ΔNLS. These changes were most pronounced in the hippocampus, where we hypothesized they promote hyperexcitability and ultimately, excitotoxic cell death. Interestingly, hippocampal injection of AAV encoding inhibitory DREADDs (hM4Di) and daily activation with CNO ligand rescued anxiety deficits on the elevated zero maze but did not reduce neurodegeneration. Moreover, therapeutic doses of the antiseizure medications, valproic acid and levetiracetam, did not improve behavior or prevent neurodegeneration. These results highlight the complex relationship between TDP-43-mediated neuronal hyperexcitability and neurodegeneration. Although targeting hyperexcitability may ameliorate some behavioral deficits, our study suggests it may not be sufficient to halt or slow neurodegeneration in TDP-43-related proteinopathies.

Structurally diverse tau filaments form proteinaceous aggregates in a heterogeneous group of neurodegenerative diseases called tauopathies 1 . The factors extrinsic to the highly ordered core structure that influence tau filament stability are not well understood. Here, we found that polyubiquitinated tau filaments from Alzheimer’s disease and vacuolar tauopathy human brain tissue exhibit distinct seeding patterns in mice, in association with differences in tau filament ultrastructure determined by cryo-electron microscopy. Interestingly, chemical modulation of the polarity of polyubiquitin adjacent to the tau core with the small molecule ubistatin B resulted in the repositioning of poorly structured densities towards positively charged residues on the highly structured core filament, leading to shifting of the protofilament-protofilament interface of certain vacuolar tauopathy tau filaments. These results suggest that the structure of tau filaments that are associated with different seeding activities in vivo can be influenced by post-translational modifications.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the presence of phosphorylated tau neurofibrillary tangles and extracellular deposits of amyloid beta plaques (Aβ) in the brain. Microglia cells have been proposed to be involved in amyloid plaque formation since activated microglia produce inflammatory cytokines that contribute to a hostile neuronal environment, exacerbating AD pathogenesis. We aim to evaluate if the pharmacological inhibition of the myeloid/microglial receptor tyrosine kinase AXL, with bemcentinib (BGB) could be used as a novel therapeutic approach for AD. The objective is to evaluate the efficacy of the BGB inhibiting Axl activity in the brain of the 5xFAD mouse model, which recapitulates pathological hallmarks of AD, including Aβ plaques formation and overactive microglia. We evaluated target engagement and performed an efficacy study in 5xFAD and WT mice orally dosed with BGB and Vehicle (Veh) as control. Mice were dosed twice a day for 1 week. Mice were anesthetized to collect CSF and plasma and perfused to obtain their brains. In the present work, we have used the pharmacological Axl inhibitor BGB, to evaluate the therapeutic potential of targeting Axl signaling in 5xFAD mice. We have evaluated the effect of Axl inhibition measuring changes in total Axl levels and its soluble fragment (sAxl) across the hippocampus, plasma, and CSF of 5xFAD mice and WT mice. As expected, we found a significant upregulation of Aβ plaques and microglia in 5xFAD mice, and interestingly, a concomitant upregulation of Axl levels in the hippocampus of 5xFAD mice. We observed, although not statistically significant, a trend of increasing levels of sAxl in CSF and plasma of mice receiving BGB. Unfortunately, no significant effect of BGB drug was detected in 5xFAD mice when Aβ-plaque burden and Iba1 immunoreactivity was measured after the treatment. Overall, the preliminary findings do not show a significant effect of Axl inhibitor BGB in the brain. The reduced BBB penetrance of the drug could be a potential explanation for lack of significant changes in Axl levels and microglia activation. Further studies of increasing dosing time and cohort size could potentially improve the outcome.

Seizures are highly comorbid with Alzheimer’s disease (AD). We and others have demonstrated worsened pathological and cognitive outcomes in AD patients with seizure history and after seizure induction in AD mouse models. Central to AD progression is the spread of tau along neuronal connections, which can be modelled by intracerebral injection of human AD brain derived tau lysate (AD-tau), but whether seizures impact the spread of tau is unknown. We hypothesized that seizures would worsen tau spread and that neurons activated during seizures would have increased susceptibility to develop and possibly transmit tau pathology. To investigate seizure-tau interactions, we crossed the 5XFAD mouse model with targeted recombination in active populations mice (TRAP; 5X-/WT-TRAP) to permanently label seizure-activated neurons. We injected AD-tau unilaterally into the hippocampus and overlying cortex (1 µg/site) at three months of age in 5X-TRAP and WT-TRAP littermates. Seizures were then induced with pentylenetetrazol (PTZ) kindling 2-3 weeks following surgery and seizure-activated neurons were induced to express tdTomato on the final day of kindling via 4-hydroxytamoxifen administration. Three months following AD-tau seeding, 5X-/WT-TRAP brains were serially sectioned and underwent immunofluorescent slide scanning. Scanned brain images were then registered to the Allen Brain Atlas and mapped for seizure-activated neurons (tdTomato+) and tau pathology (phospho-tau Ser202/Ser205; AT8). We found that 5X-TRAP mice had increased tau pathology compared to WT-TRAP in hippocampal subregions ipsilateral to AD-tau injection, including the dentate gyrus (p<0.05, n = 5-10/group) and subiculum (p<0.01, n = 5-9/group), regardless of seizure kindling, without changes in the contralateral hippocampus. Seizure induction resulted in increased tau spread in remote interconnected brain regions, including the ipsilateral and contralateral thalamo-cortical regions (p<0.05, n = 5-9/group). In addition, we found that, compared to neurons from a saline-treated 5XFAD mouse, thalamic seizure-activated (tdTomato+) neurons showed significantly increased somatic AT8 levels (p<0.05, n = 8-11), while surrounding (tdTomato-) neurons from 5XFAD mice did not, suggesting that these thalamic, seizure-activated neurons preferentially facilitate tau spread. Together, our data demonstrate that seizures increase tau spread in mouse models and identify seizures and seizure-activated neurons as therapeutic targets to slow pathological AD progression.

Pathological tau spreads via neuronal connections in Alzheimer's disease (AD). Given the high incidence and deleterious consequences of epileptiform activity in AD, we hypothesized that neuronal hyperactivity and seizures exacerbate tau spread. To examine the impacts of brain-wide network and population hyperactivity on tau spread, we created a novel mouse model involving the cross of targeted recombination in active populations (TRAP) and the 5 times familial AD mice (5X-TRAP) that allows for the permanent labelling of seizure-activated neurons. To explore the effects of seizures on tau spread, we injected these mice with human AD brain-derived tau to induce pathological tau spread, and induced seizures with pentylenetetrazol (PTZ) kindling. Brain mapping revealed that seizures increased tau spread in 5X-TRAP mice, which correlated extensively with memory deficits in PTZ kindled 5X-TRAP mice. Using computational models, we found data supportive of increased anterograde tau spread in 5X-TRAP mice and that regional neuronal activity levels were predictive of tau pathology. On a cellular level, we found that hyperactive neurons drive elevated tau propagation in 5X-TRAP mice. We also found corroborating evidence of increased tau spread in AD patients with a seizure history compared to those without. Our study identifies neuronal hyperactivity and seizures as key, targetable factors underlying AD progression.

Measuring splicing and chromatin accessibility simultaneously in frozen tissues remains challenging. Here we combined single-cell isoform RNA sequencing and assay for transposase accessible chromatin (ScISOr–ATAC) to interrogate the correlation between these modalities in single cells in human and rhesus macaque frozen cortical tissue samples. Applying a previous definition of four ‘cell states’ in which the transcriptome and chromatin accessibility are coupled or decoupled for each gene, we demonstrate that splicing patterns in one cell state can differ from those of another state within the same cell type. We also use ScISOr–ATAC to measure the correlation of chromatin and splicing across brain cell types, cortical regions and species (macaque and human) and in Alzheimer’s disease. In macaques, some excitatory neuron subtypes show brain-region-specific splicing and chromatin accessibility. In human and macaque prefrontal cortex, strong evolutionary divergence in one molecular modality does not necessarily imply strong divergence in another modality. Finally, in Alzheimer’s disease, oligodendrocytes show high dysregulation in both chromatin and splicing. Joint profiling of chromatin and splicing in the brain uncovers shared and distinct patterns.

Cytoplasmic inclusions of TDP-43 are the primary pathology in the majority of ALS and FTLD cases. Recent reports in cell and animal models suggest TDP-43 pathology may enhance neuronal excitability, which could contribute to neurodegeneration via excitotoxicity. Dox-regulatable rNLS8 mice express human TDP-43 with mutations in the nuclear localization signal (hTDP-43NLSm) to promote cytoplasmic accumulation. Preliminary electrophysiological data indicate these mice develop robust hyperexcitability in brain circuits along with generalized seizures. To test whether reducing hyperexcitability may be therapeutic, we administered the anti-epilepsy drug, levetiracetam, to rNLS8 mice and assessed its effects on behavior and neurodegeneration. hTDP-43NLSm expression was induced by replacing doxycycline-chow with normal mouse feed (off-DOX) in a cohort of 16 adult rNLS8 mice, approximately equal numbers male and female. Starting 1 week off-DOX, 100mg/kg levetiracetam (LEV) or 0.5% w/w hydroxypropyl methylcellulose with 1% Tween-80 vehicle (VEH) was administered by daily gavage. At 4 weeks off-DOX, mice were subjected to a behavior battery including cognitive (Y maze, elevated zero maze, 3-chamber social interaction test) and motor (accl. rotarod, open field) tests. Brains were collected for histology at 6 weeks off-DOX. No improvement in behavioral deficits was observed in LEV treated mice. Preliminary analysis indicated a potential mild reduction in gross neurodegeneration. Mean hippocampus mass at 6 weeks off-DOX was similar for both groups (21.7mg LEV, 22.mg VEH; Student’s t-test, p = 0.84) but the LEV treated group showed a trend towards reduced cortical atrophy (mean motor cortex thickness on H&E stained sections: 943µm LEV, 870µm VEH; Student’s t-test, p = 0.12). Daily levetiracetam administration was not effective in significantly reducing disease severity. Future studies will examine the effect of orthogonal approaches, such as AAV encoded chemogenetics, and characterize downstream mediators of TDP-43 -induced hyperexcitability, which may serve as more potent and specific therapeutic targets.

Granulovacuolar degeneration (GVD) is a common feature in Alzheimer’s disease (AD) and other tauopathies such as corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Pick’s disease (PiD). Despite its prevalence, much remains unknown about the role of GVD in the pathobiology of these diseases. Morphologically, GVD is characterized by intraneuronal membrane-bound vacuoles, also known as GVD bodies (GVBs). Previously, it has been observed that in vitro and in vivo mouse models of tauopathy can also induce GVB formation. Nevertheless, the in vivo models in particular remain poorly characterized. Herein, we provide a more systematic characterization of GVD in the in vivo mouse models to better enable elucidation of GVD and its relation to tau pathology and neurodegeneration. Brain sections were retrieved from various tauopathy mouse models. GVB distribution was characterized via immunohistochemistry using CK1δ as the primary GVB marker. Co-staining with a phospho-tau antibody (AT8) confirmed the presence of tau pathology in all of our models. CK1δ+ GVBs were detected in multiple tauopathy mouse models, including PS19 as well as AD-tau-injected wild type, 5xFAD, and 6hTau mice. The level of GVBs differ between mouse models and correlate with the level of tau pathology that was developed in these mice. In particular for the 6hTau mice, where the amount of GVBs was the most robust, temporal characterization of GVB formation shows the most significant increase in the number of GVB+ neurons from 1 to 3 months post-AD-tau injection. At the single cell level, the number of CK1δ+ puncta also increased over time. By 3 mpi, GVBs were detected beyond the hippocampus (site of injection) and in the cortical regions where tau pathology was also present. Finally, 6hTau mice injected with CBD-, PSP-, and PiD-tau all developed GVBs, albeit at variable levels that parallel the extent of tau pathology. GVB development tracks with tau pathology development in our in vivo mouse models. The increase in GVBs within a cell over time suggests that GVBs continue to form as tau pathology matures. This induction of GVBs by tau pathology is irrespective of the different tau strains from different tauopathies.

OBJECTIVE:Aggregation of misfolded α-synuclein (aSyn) within the brain is the pathologic hallmark of Lewy body diseases (LBDs), including Parkinson's disease (PD), and dementia with Lewy bodies (DLB) disease. Although evidence exists for aSyn "strains," conformations with distinct biological properties, biomarkers for PD versus DLB are lacking. Here, we used monoclonal antibodies selective for two different in vitro aSyn species - termed strain A and B - to evaluate human brain tissue, cerebrospinal fluid (CSF), and plasma. METHODS:Using these antibodies, we characterized specific aSyn species in human specimens from neurologically normal individuals and individuals with LBD using enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. We also characterized aSyn species immunoprecipitated from brain lysate or plasma with these antibodies using seed amplification assays (SAAs) and a cellular model. RESULTS:Surprisingly, levels of strain A and B aSyn species were higher in plasma from individuals with PD compared to DLB in 2 independent cohorts. Lower levels of plasma aSyn strain A species predicted a faster rate of cognitive decline in individuals with PD. Furthermore, strain A and strain B aSyn species were undetectable in CSF, and their levels in brain versus plasma did not correlate. Moreover, plasma aSyn species isolated by aSyn strain antibodies could template aSyn fibrillization, and they could seed formation of aSyn inclusions in cells. INTERPRETATION:Our findings suggest that circulating plasma aSyn strains may impact LBD clinical presentation, particularly cognition. The enrichment of these aSyn species in plasma but not CSF also suggests a potential source outside the brain. ANN NEUROL 2025;98:682-698.

Alzheimer’s disease (AD) is pathologically defined by the presence of extracellular Aβ plaque and intracellular tau inclusions. Emerging evidence shows that tau aggregates contain pathogenic bioactivities of templating monomeric tau into filamentous fibrils and propagating through cells. Based on these findings, assays have been developed to detect minute amounts of pathogenic tau in human samples. With great potential, it remains unclear about the sensitivity and fidelity of tau fibrillization due to the presence of pathogenic tau species. To that end, our study focuses on optimizing the Real-Time Quaking-Induced Conversion (RT-QuiC) assay for its potential of detecting and amplifying insoluble tau seeds derived from AD patient. To this end, we utilized recombinant tau monomers t306 and k12, 2R and 3R tau peptide respectively known for their rapid fibrillization and high amplification signals in previous studies. More specifically, we optimized the RT-QuiC assay using both crude and purified brain lysates from AD and control cases. The specificity of the RT-QuiC assay were successfully reproduced by multiple cases of AD and control brain lysates. Moreover, we tested the RT-QuiC assay with purified AD tau seeds and assessed the sensitivity of the assays based on the amount of insoluble tau. To further interrogate the system, we also evaluated the impact of reaction buffer on the ThT-based measurement of tau fibrillization. Our results show that the original RT-QuiC assay can sensitively detect insoluble AD tau seeds at the nM level in our hands. The optimized RT-QuiC assay could differentiate better between AD-seeded reactions and controls with an improved sensitivity up to fM level, with a better separation between AD and control and faster reaction rate. The assay showed a clear dose-dependent response till pM range. Upon optimization, the RT-QuiC assay is both ultrasensitive and specific to AD tau fibrillization. Our findings offer insights into the fibrillization mechanism of tau aggregates and expand our understanding of pathogenic tau seeds in AD. By optimizing the assay to more accessible patient samples in the future, namely CSF and plasma, the RT-QuiC can serve as a potent diagnostic tool for AD tau progressions.

Glaucoma is characterized by progressive optic nerve degeneration that results in irreversible blindness, and it can be considered a neurodegenerative disorder of both the eye and the brain. Increasing evidence suggest that glaucoma shares some common neurodegenerative pathways with Frontotemporal Lobar Degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), and Alzheimer’s Disease (AD) among others. Interestingly, a recent study revealed the presence of abnormal TAR DNA-binding protein 43 (TDP-43) inclusions and aggregates in retinal ganglion cells and other retinal cell types in FTLD-TDP patients; however, the significance of this pathology and its impact on retinal function and optical nerve integrity is unknown. In these patients, optic nerve degeneration has been linked to the spread of aggregated TDP-43 beyond the brain. Thus, this study aims to explore the role TDP-43 mislocalization and aggregation contributes to glaucoma. The expression of the mutant cytoplasmic human TDP-43 transgene (hTDP-43NLSm) in a doxycycline-regulatable TDP-43 transgenic mouse line (rNLS8), was induced for 2, 4 and 6 weeks (i.e., Off-Dox), thus causing a time-dependent cytoplasmic translocation of TDP-43 in neurons. rNLS8 mice were euthanized, perfused, and intact eyeballs collected for histological examination. The expression of hTDP-43 and its phosphorylated form (p409/410) together with cleaved caspase-3 and p62 was examined by immunohistochemistry in 5 µm paraffin -embedded retinal sections. rNLS8 mice kept Off-Dox expressed hTDP-43NLSm in all neuronal layers in the retina, in all the time-points analyzed. Interestingly, in rNLS8 mice at 6 weeks Off-Dox, revealed mild phosphorylation of TDP-43 together with an increase of cleaved caspase-3 and p62 protein immunoreactivity. Thus far, this data indicates the activation of apoptotic and autophagy signaling pathways concomitant to the presence of mislocated and phosphorylated TDP-43. Altogether, this indicates that the rNLS8 transgenic mouse line serves as a good model to evaluate the pathophysiological consequences of TDP-43 mislocalization in retina cells and its role in optic nerve degeneration.

Tau aggregation plays a crucial role in the development of Alzheimer's disease (AD). Developing specific techniques that can isolate pathogenic tau from brain tissue is important for understanding tauopathies and advancing targeted therapies. Here, we develop photoaffinity small molecular probes and a novel method for in situ tissue labeling and investigate their activity in interacting with tau in cells and AD patient brains. Based on the reported chemical structures of tau PET tracers, we designed and synthesized two tau-specific probes, namely, Tau-2 and Tau-4. After validation in cell, mouse model, and patient brain samples, our photolabeling results suggested that Tau-2 effectively labels soluble tau in cell and mouse models, while Tau-4 selectively binds high-molecular-weight tau aggregates in late-stage AD patient brain tissues. Proteomic analysis verified the specific isolation of pathogenic tau from AD brain samples. Collectively, these findings underscore the potential of our photoaffinity probes as powerful tools for investigating tau proteins and neurofibrillary tangles in neurodegenerative diseases.

Oxidative stress, defined as the excessive production of reactive oxygen species (ROS), is a crucial factor in the pathogenesis of various neurodegenerative diseases, including the 4-repeat (4R) tauopathies. Collectively, the 4R tauopathies are characterized by the progressive aggregation of tau protein isoforms with four microtubule-binding domains in and around brain cells. The cyclical relationship between oxidative stress and 4R tau aggregation suggests that a means of imaging ROS noninvasively could be a valuable tool for the study and treatment of 4R tauopathy in both humans and animal models. To demonstrate the potential of the ROS-sensitive positron emission tomography (PET) radiotracer [18F]ROStrace as a means of filling this methodological gap, we performed [18F]ROStrace PET imaging on PS19 mice, which exhibit 4R tau aggregation similar to that seen in human 4R tauopathy. Significant increases in [18F]ROStrace signal became detectable in the hippocampus of 6–11-month-old (mo) PS19 animals and spread to the brainstem, midbrain, and thalamus of 11+ mo animals. Additionally, older PS19 mice displayed higher whole-brain average [18F]ROStrace signal compared to age-matched controls (p = 0.042), and tau pathology consistently colocalized with multiple fluorescent indicators of oxidative stress in PS19 brain samples. These results provide novel evidence that 4R tau aggregation is associated with increased oxidative stress in PS19 mouse brain and advance [18F]ROStrace as a noninvasive technology for the detection of oxidative stress in neurodegenerative diseases involving tau pathology.