Aims

Alzheimer disease (AD), primary age-related tauopathy (PART) and chronic traumatic encephalopathy (CTE) all harbor 3R/4R-tau immunopositive neurofibrillary tangles (NFTs), yet they differ in the spatial development of these NFTs, which may depend on the presence and distribution of β-amyloid. In the hippocampus, PART and CTE display an early predilection for neurofibrillary degeneration in the CA2 subregion, whereas neurofibrillary degeneration in AD typically begins in the entorhinal cortex and CA1 subregion. CTE also displays a unique predilection for the CA4 subregion. This study aims to identify protein expression differences in NFTs, normal neurons and the microenvironments within the hippocampal subregions of these disorders.

Methods

Using Nanostring’s GeoMx™ Digital Spatial Profiling (DSP), differences in the expression of 73 proteins between AD (n=8), PART (n=7), and CTE (n=5) were compared within NFT-bearing neurons, non-NFT-bearing neurons and the microenvironments in the entorhinal cortex and hippocampal subregions (Figure 1).

Results

Analyses identified several proteins displaying differential expression when comparing AD, PART and CTE. These included differences in p-tau epitopes and glial markers in the subregions, as well as proteins involved in proteostasis, the complement pathway and amyloid processing (Figures 2-5).

Conclusions

In conclusion, PART and CTE cases generally display protein expression patterns that are more similar to each other, as opposed to AD. However, there are differences between PART and CTE in p-tau epitope expression in the hippocampal subregions, certain microglial markers and proteins involved in proteostasis which may help explain NFT distribution differences and could point toward disease-specific differences in the mechanism of NFT development in these disorders.

Aims

The propagation of aggregated and hyperphosphorylated tau protein is a characteristic feature of Alzheimer's disease (AD). However, the molecular processes governing its unconventional protein secretion (UPS) remain unclear. A recent study has found that tau levels in the interstitial fluid and cerebrospinal fluid (CSF) are increased during wakefulness compared to sleep. Moreover, we previously evidenced that tau phosphorylation follows a circadian pattern driven by the variations in body temperature (BT) during the sleep-wake cycle. Thus, we hypothesized that circadian BT variations play a significant role in UPS-mediated tau secretion.

Methods

In two neuronal models, we examined the impact of various temperatures (35-38°C), mimicking those experienced during the sleep-wake cycle, on UPS-related tau secretion. In wild-type mice, we evaluated changes in the expression levels of UPS-related proteins during the sleep-wake cycle, and following 6 hours of sleep deprivation (SD). Additionally, we subjected hTau mice (human tau overexpression) to hypo- or hyperthermia in order to influence tau levels in their CSF.

Results

We demonstrated that wakefulness-like temperatures promote tau secretion. This process is primarily driven by the caspase-3-mediated cleavage of tau into TauC3. Furthermore, we identified that phosphatidylinositol 4,5-bisphosphate (PIP₂) and syndecan-3 (SDC3) are upregulated at higher temperatures, and act as key players in tau export. Observations in mice revealed that wakefulness, as well as SD and mild-hyperthermia, correlate with increased expression of TauC3 and UPS-related proteins, ultimately resulting in elevated tau levels in CSF.

Conclusions

This study unveils a novel connection between BT fluctuations during the sleep-wake cycle and tau propagation, shedding light on potential therapeutic avenues for neurodegenerative diseases. These results also emphasize the importance of considering disruptions in both sleep and thermoregulation in the elderly population susceptible to AD.

Aims

Regional distribution of neurofibrillary lesions consisting of abnormally hyperphosphorylated tau correlates strongly with the progression of Alzheimer’s disease (AD). Misfolded proteopathic tau templates the conversion of naive tau into a pathological state in a prion-like fashion, which underlies the spreading of tau pathology in the brain. However, whether hyperphosphorylation triggers tau aggregation or hyperphosphorylation occurs after aggregation is under much debate.

Methods

In the present study, by using oligomeric tau derived from AD brain (AD O-tau) as proteopathic tau seeds, we determined the role of hyperphosphorylation in seeded tau aggregation in cultured cells and in vivo in mice.

Results

We found that tau aggregation seeded by AD O-tau was accompanied by site-specific hyperphosphorylation of tau and by reduction in the level of PP2A catalytic subunit (PP2Ac) that reacts with monoclonal 1D6 and polyclonal R123d antibodies to PP2Ac and in PP2A activity. Hyperphosphorylated tau was found to be enriched site-specifically in RIPA-insoluble aggregates. Phospho-blocking mutations at Ser202 and/or Thr217 of tau inhibited AD O-tau–seeded tau aggregation and the decline of 1D6/R123d-PP2Ac level. Reduction of 1D6/R123d-PP2Ac was correlated with tau aggregation and site-specific hyperphosphorylation. Knockdown and overexpression of PP2Ac enhanced and suppressed tau aggregation, respectively, in cultured cells and in vivo.

Conclusions

These findings strongly suggest that site-specific hyperphosphorylation of tau is essential for its seeded aggregation. Tau pathogenesis may involve a vicious cycle of tau site–specific hyperphosphorylation and aggregation and the reduction of PP2Ac. Thus, interrupting this vicious cycle may curb tau pathogenesis and the spreading of tau pathology in AD and related tauopathies.

Aims

Accumulation of tau in neurofibrillary tangles (NFTs) is a pathological hallmark of Alzheimer’s Disease (AD) brains. Kinases heavily contribute to the pathological phosphorylation and aggregation of tau in AD. Previously, we showed that the neuronally expressed G protein-coupled receptor (GPCR) kinase 2 (GRK2) positively correlates with soluble tau levels and is associated with NFTs in the human AD brain. However, whether GRK2 is causally involved in tau pathogenesis remains unknown.

Methods

We investigated the impact of GRK2 in tau phosphorylation and aggregation using CRISPR/Cas9 HEK293 modified cell lines, control and AD patient-derived induced neurons (iNs), optogenetic tools, and mass spectrometry analysis.

Results

Manipulating GRK2 levels induces global changes in the tau phosphoproteome, specifically increasing levels of several tau phosphorylation (pTau) sites upon GRK2 overexpression. Using a novel optogenetic system to induce tau aggregation (optoTAU) we show that tau aggregation, and specifically soluble tau, is reduced in Grk2-deficient cells. We also show that GRK2 directly interacts with unmodified, phosphorylated, and misfolded tau species. Moreover, we find that GRK2 modulates pTau levels in a kinase activity-independent manner. Specifically, ERK-mediated phosphorylation of GRK2 at residue S670 is protective against pTau formation. Intriguingly, levels of S670 are downregulated in AD patient brains. Pharmacological inhibition of GRK2 in human iNs increases both ERK activation and GRK2-s670 levels, while decreasing pTau and reactive-oxygen stress in AD-derived iNs. Lastly, Grk2 deficiency enhances tau ubiquitination and interaction with HSP90, which further increases tau degradation, protecting the cells from aggresome formation upon proteasome inhibition.

Conclusions

These studies causally implicate GRK2 as a multifactorial modulator of tau pathobiology through changes in phosphorylation, aggregation, and degradation of tau and support further investigation of GRK-mediated therapeutic intervention strategies for AD.

Aims

Abnormal intracellular accumulation of Tau aggregates is a hallmark of Alzheimer's disease (AD) and other Tauopathies, such as Frontotemporal dementia (FTD).

FTD in particular is characterized by Tau deposits in neurons and astrocytes, suggesting that cells other than neurons might be affected and contribute distinctively to disease progression. Pathological Tau can undergo a range of post-translational modifications (PTMs) that might trigger or modulate disease pathology. Recent studies indicate that modification of Tau by Small ubiquitin-like modifier SUMO affects Tau hyperphosphorylation and aggregation in neuronal cells. However, the consequences of this modification have not been investigated in astrocytes or microglial cells

The goal of our study was to evaluate whether SUMO conjugation modifies the neurodegenerative disease pathology associated with the aggregation-prone mutant Tau P301L, in neurons as well as glial cells.

Methods

We used viral approaches to express mutant Tau P301L and SUMO2 in the hippocampus of wild type mice. We assessed Tau distribution and phosphorylation by immunostaining and western blot. We assessed neuronal toxicity and performed cell count and shape descriptor analyses on astrocytes and microglial cells.

Results

We found that mutant Tau is toxic not only to neurons but also to glial cells, and that SUMO2 counteracts this cytotoxicity in all cell types. In particular we found that SUMO2 reduces both phosphorylation and aggregation of mutant forms of Tau. In addition, we showed that tau induced a significant reduction in the number, average size and perimeter of both astrocytes and microglial cells and that SUMO2 prevents these pathological changes induced by tau.

Conclusions

Our results uncover an endogenous neuroprotective mechanism in our FTD model, whereby SUMO2 conjugation reduces Tau neuropathology, and protects against toxic effects of Tau in astroglial cells as well.

Aims

Neuronal nuclei are normally smoothly surfaced spheroids. In Alzheimer’s disease (AD) and other tauopathies, though, they often develop visually conspicuous invaginations. We investigated mechanisms and functional consequences of neuronal nuclear invagination in tauopathies.

Methods

Nuclear invagination was assayed by immunofluorescence in brain, and in cultured mouse cortical neurons before and after exposure to extracellular tau oligomers (xcTauOs) made from recombinant tau or isolated from hTau mouse brain. Nucleocytoplasmic transport was assayed in cultured neurons. Gene expression was investigated using nanoString nCounter technology and qRT-PCR.

Results

Invaginated neuronal brain nuclei were twice as abundant in human AD as in cognitively normal adults, and were similarly increased in transgenic mouse AD and pure tauopathy models. In cultured mouse neurons, nuclear invagination was induced by xcTauOs within 1 hour of exposure by an intracellular tau-dependent mechanism - in tau knockout neurons, xcTauOs were endocytosed effectively, but increased nuclear invagination was not observed unless the cells were transduced with a lentivirus that drives human tau expression. xcTauOs impaired nucleo­cytoplasmic transport, increased histone H3 tri-methylation at lysine 9 and altered gene expression, especially by increasing the level of tau mRNA.

Conclusions

By a mechanism that requires intracellular tau, xcTauOs are probably a primary cause of nuclear invagination in vivo, and by extension, impaired nucleocytoplasmic transport, altered chromatin organization and pathogenic gene expression changes. The increase in tau mRNA induced by xcTauOs emphasizes the possible existence of a positive feedback loop whereby xcTauO uptake drives production of more intracellular tau, which in turn fuels production of more xcTauOs. The rapidity with which these effects occur in cultured neurons following initial xcTauO exposure implicates xcTauOs as seminal factors in the pathogenesis of AD and other tauopathies.

Aims

Objectives: Tauopathies are a class of neurodegenerative diseases characterized by tau aggregation in the brain. Tauopathies are also marked by a cascade of events, such as shifts in transcriptional patterns, changes in RNA binding proteins, disruptions in synaptic functioning, neurotransmitter imbalances, and impaired cellular clearance mechanisms. We sought to determine whether there are upstream drivers of these pleiotropic phenotypes.

Methods

Methods: Transcriptomic data was generated from iPSC-derived neurons from MAPT IVS10+16 and isogenic controls; microRNA-induced neurons (miNs) directly reprogrammed from patient fibroblasts carrying the MAPT IVS10+16 mutation and non-carrier controls; and frontotemporal dementia brains (FTD; MAPT mutation carriers) and neuropathology-free controls. Integrative analyses were performed to identify differentially expressed genes as well as the transcription factors (TF) that regulate these genes.

Results

Results: We found that 215 genes were differentially expressed in iPSC-derived MAPT mutant neurons and in FTD brains. These genes are enriched in pathways associated with neurofilament assembly and synaptic regulation and are regulated by the TFs PAX6, POU3F2, ZIC1, and ZIC2. In miNs, we identified 291 genes that were differentially expressed in MAPT mutant neurons and in FTD brains. These genes are enriched in pathways associated with calcium homeostasis, endolysosomal regulation, and synapse function, and are regulated by the TFs PPARA, STAT3, and ZFHX3. The 56 genes dysregulated in all three systems were enriched in apoptosis, autophagy, and mitochondrial function, and are regulated by the TFs EBF1, HES1, and LEF1. All these TFs were also dysregulated in mouse models of tauopathy.

Conclusions

Conclusions: Together, these findings suggest that MAPT mutations disrupt key regulatory TFs that drive broad differential gene expression in pathways involved in neuronal health, synaptic function, and endolysosomal function and contribute to the pathophysiology of disease.

Aims

Tauopathies including Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) feature the progressive accumulation of aggregated tau protein in “selectively vulnerable” neurons. AD and FTLD feature unique tau aggregate conformations or “strains,” which are hypothesized to underlie the different disease phenotypes observed across tauopathies. However, the mechanisms that underlie tau strains and their impact on tau aggregation and neurotoxicity are not known. I aim to use CRISPRi-based screening techniques in induced pluripotent stem cell (iPSC)-derived excitatory neurons (iNeurons) that stably propagate tau aggregates to identify regulators of tau aggregation and toxicity across distinct tau strains.

Methods

Typical iNeuron tauopathy models do not readily accumulate higher-order tau aggregates. To more readily study mechanisms of tau aggregation, I created a novel iPSC model that stably expresses full-length tau fused to FRET-compatible fluorophores (FL-tau). Tau aggregation was induced in FL-tau iNeurons by seeding with tau fibrils derived from different human tauopathies. This model stably propagates FRET-positive tau aggregates, which are maintained after differentiation into iNeurons.

Results

In a pilot CRISPRi screen performed in FL-tau aggregate containing iNeurons, modulation of several kinases, chaperones, and heat shock proteins modulated tau aggregation. In addition, inhibition of the lesser-known ubiquitin-like UFMylation pathway reduced tau aggregation. Previous work has shown UFMylation regulates p62 activity and autophagy in the setting of ER stress.

Conclusions

Together, these results suggest several distinct pathways modulate tau aggregation. Future work will examine the mechanisms by which specific chaperons and the UFMylation pathway regulate tau aggregation across distinct tau strains, and whether changes to these pathways correlate with tau aggregation in human AD and FTLD subject histopathologic samples.