Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade in vitro and in vivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.

X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

Posttranslational Modifications Mediate the Structural Diversity of Tauopathy Strains.

Tau aggregation into insoluble filaments is the defining pathological hallmark of tauopathies. However, it is not known what controls the formation and templated seeding of strain-specific structures associated with individual tauopathies. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of tau filaments from corticobasal degeneration (CBD) human brain tissue. Cryo-EM and mass spectrometry of tau filaments from CBD reveal that this conformer is heavily decorated with posttranslational modifications (PTMs), enabling us to map PTMs directly onto the structures. By comparing the structures and PTMs of tau filaments from CBD and Alzheimer's disease, it is found that ubiquitination of tau can mediate inter-protofilament interfaces. We propose a structure-based model in which cross-talk between PTMs influences tau filament structure, contributing to the structural diversity of tauopathy strains. Our approach establishes a framework for further elucidating the relationship between the structures of polymorphic fibrils, including their PTMs, and neurodegenerative disease.

Microglia-mediated T cell infiltration drives neurodegeneration in tauopathy.

Extracellular deposition of amyloid-ß as neuritic plaques and intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles are two of the characteristic hallmarks of Alzheimer's disease1,2. The regional progression of brain atrophy in Alzheimer's disease highly correlates with tau accumulation but not amyloid deposition3-5, and the mechanisms of tau-mediated neurodegeneration remain elusive. Innate immune responses represent a common pathway for the initiation and progression of some neurodegenerative diseases. So far, little is known about the extent or role of the adaptive immune response and its interaction with the innate immune response in the presence of amyloid-ß or tau pathology6. Here we systematically compared the immunological milieux in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not those with amyloid deposition developed a unique innate and adaptive immune response and that depletion of microglia or T cells blocked tau-mediated neurodegeneration. Numbers of T cells, especially those of cytotoxic T cells, were markedly increased in areas with tau pathology in mice with tauopathy and in the Alzheimer's disease brain. T cell numbers correlated with the extent of neuronal loss, and the cells dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of interferon-γ and PDCD1 signalling both significantly ameliorated brain atrophy. Our results thus reveal a tauopathy- and neurodegeneration-related immune hub involving activated microglia and T cell responses, which could serve as therapeutic targets for preventing neurodegeneration in Alzheimer's disease and primary tauopathies.

Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.

Missense mutations in the gene encoding the microtubule-associated protein TAU (current and approved symbol is MAPT) cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human TAU in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas-mediated gene editing to model frontotemporal dementia caused by the TAU P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for Tau P251L display age-dependent neurodegeneration, display metabolic defects, and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies, we performed single-cell RNA sequencing on approximately 130,000 cells from brains of Tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified. Gene expression was also altered in glial cells, suggestive of non-cell-autonomous regulation. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell type-specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy that faithfully recapitulates the genetic context and phenotypic features of the human disease, and use the results of comprehensive single-cell sequencing analysis to outline pathways of neurotoxicity and highlight the potential role of non-cell-autonomous changes in glia.

Structure-based classification of tauopathies.

The ordered assembly of tau protein into filaments characterizes several neurodegenerative diseases, which are called tauopathies. It was previously reported that, by cryo-electron microscopy, the structures of tau filaments from Alzheimer's disease1,2, Pick's disease3, chronic traumatic encephalopathy4 and corticobasal degeneration5 are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a new three-layered fold. Moreover, the structures of tau filaments from globular glial tauopathy are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs, instead resembling the four-layered fold of corticobasal degeneration. The AGD fold is also observed in ageing-related tau astrogliopathy. Tau protofilament structures from inherited cases of mutations at positions +3 or +16 in intron 10 of MAPT (the microtubule-associated protein tau gene) are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, the structures of tau filaments from cases of familial British dementia and familial Danish dementia are the same as those from cases of Alzheimer's disease and primary age-related tauopathy. These findings suggest a hierarchical classification of tauopathies on the basis of their filament folds, which complements clinical diagnosis and neuropathology and also allows the identification of new entities-as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of globular glial tauopathy and PSP.

Chaperoning shape-shifting tau in disease.

Many neurodegenerative diseases, including Alzheimer's, originate from the conversion of proteins into pathogenic conformations. The microtubule-associated protein tau converts into ß-sheet-rich amyloid conformations, which underlie pathology in over 25 related tauopathies. Structural studies of tau amyloid fibrils isolated from human tauopathy tissues have revealed that tau adopts diverse structural polymorphs, each linked to a different disease. Molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. New data supports the model that chaperones selectively recognize different conformations of tau to limit the accumulation of proteotoxic species. The challenge now is to understand how chaperones influence disease processes across different tauopathies, which will help guide the development of novel conformation-specific diagnostic and therapeutic strategies.

Novel tau filament fold in corticobasal degeneration.

Corticobasal degeneration (CBD) is a neurodegenerative tauopathy-a class of disorders in which the tau protein forms insoluble inclusions in the brain-that is characterized by motor and cognitive disturbances1-3. The H1 haplotype of MAPT (the tau gene) is present in cases of CBD at a higher frequency than in controls4,5, and genome-wide association studies have identified additional risk factors6. By histology, astrocytic plaques are diagnostic of CBD7,8; by SDS-PAGE, so too are detergent-insoluble, 37 kDa fragments of tau9. Like progressive supranuclear palsy, globular glial tauopathy and argyrophilic grain disease10, CBD is characterized by abundant filamentous tau inclusions that are made of isoforms with four microtubule-binding repeats11-15. This distinguishes such '4R' tauopathies from Pick's disease (the filaments of which are made of three-repeat (3R) tau isoforms) and from Alzheimer's disease and chronic traumatic encephalopathy (CTE) (in which both 3R and 4R isoforms are found in the filaments)16. Here we use cryo-electron microscopy to analyse the structures of tau filaments extracted from the brains of three individuals with CBD. These filaments were identical between cases, but distinct from those seen in Alzheimer's disease, Pick's disease and CTE17-19. The core of a CBD filament comprises residues lysine 274 to glutamate 380 of tau, spanning the last residue of the R1 repeat, the whole of the R2, R3 and R4 repeats, and 12 amino acids after R4. The core adopts a previously unseen four-layered fold, which encloses a large nonproteinaceous density. This density is surrounded by the side chains of lysine residues 290 and 294 from R2 and lysine 370 from the sequence after R4.

Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease.

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc-EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc-EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington's disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.

Propagation of Tau Aggregates and Neurodegeneration.

A pathway from the natively unfolded microtubule-associated protein Tau to a highly structured amyloid fibril underlies human Tauopathies. This ordered assembly causes disease and represents the gain of toxic function. In recent years, evidence has accumulated to suggest that Tau inclusions form first in a small number of brain cells, from where they propagate to other regions, resulting in neurodegeneration and disease. Propagation of pathology is often called prion-like, which refers to the capacity of an assembled protein to induce the same abnormal conformation in a protein of the same kind, initiating a self-amplifying cascade. In addition, prion-like encompasses the release of protein aggregates from brain cells and their uptake by neighboring cells. In mice, the intracerebral injection of Tau inclusions induces the ordered assembly of monomeric Tau, followed by its spreading to distant brain regions. Conformational differences between Tau aggregates from transgenic mouse brain and in vitro assembled recombinant protein account for the greater seeding potency of brain aggregates. Short fibrils constitute the major species of seed-competent Tau in the brains of transgenic mice. The existence of multiple human Tauopathies with distinct fibril morphologies has led to the suggestion that different molecular conformers (or strains) of aggregated Tau exist.

Distinct involvement of the cranial and spinal nerves in progressive supranuclear palsy.

The most frequent neurodegenerative proteinopathies include diseases with deposition of misfolded tau or α-synuclein in the brain. Pathological protein aggregates in the PNS are well-recognized in α-synucleinopathies and have recently attracted attention as a diagnostic biomarker. However, there is a paucity of observations in tauopathies. To characterize the involvement of the PNS in tauopathies, we investigated tau pathology in cranial and spinal nerves (PNS-tau) in 54 tauopathy cases [progressive supranuclear palsy (PSP), n = 15; Alzheimer's disease (AD), n = 18; chronic traumatic encephalopathy (CTE), n = 5; and corticobasal degeneration (CBD), n = 6; Pick's disease, n = 9; limbic-predominant neuronal inclusion body 4-repeat tauopathy (LNT), n = 1] using immunohistochemistry, Gallyas silver staining, biochemistry, and seeding assays. Most PSP cases revealed phosphorylated and 4-repeat tau immunoreactive tau deposits in the PNS as follows: (number of tau-positive cases/available cases) cranial nerves III: 7/8 (88%); IX/X: 10/11 (91%); and XII: 6/6 (100%); anterior spinal roots: 10/10 (100%). The tau-positive inclusions in PSP often showed structures with fibrillary (neurofibrillary tangle-like) morphology in the axon that were also recognized with Gallyas silver staining. CBD cases rarely showed fine granular non-argyrophilic tau deposits. In contrast, tau pathology in the PNS was not evident in AD, CTE and Pick's disease cases. The single LNT case also showed tau pathology in the PNS. In PSP, the severity of PNS-tau involvement correlated with that of the corresponding nuclei, although, occasionally, p-tau deposits were present in the cranial nerves but not in the related brainstem nuclei. Not surprisingly, most of the PSP cases presented with eye movement disorder and bulbar symptoms, and some cases also showed lower-motor neuron signs. Using tau biosensor cells, for the first time we demonstrated seeding capacity of tau in the PNS. In conclusion, prominent PNS-tau distinguishes PSP from other tauopathies. The morphological differences of PNS-tau between PSP and CBD suggest that the tau pathology in PNS could reflect that in the central nervous system. The high frequency and early presence of tau lesions in PSP suggest that PNS-tau may have clinical and biomarker relevance.

Glial reactivity and T cell infiltration in frontotemporal lobar degeneration with tau pathology.

Frontotemporal lobar degeneration with tau (FTLD-tau) is a group of tauopathies that underlie ∼50% of FTLD cases. Identification of genetic risk variants related to innate/adaptive immunity have highlighted a role for neuroinflammation and neuroimmune interactions in FTLD. Studies have shown microglial and astrocyte activation together with T cell infiltration in the brain of THY-Tau22 tauopathy mice. However, this remains to be confirmed in FTLD-tau patients. We conducted a detailed post-mortem study of FTLD-tau cases including 45 progressive supranuclear palsy with clinical frontotemporal dementia, 33 Pick's disease, 12 FTLD-MAPT and 52 control brains to characterize the link between phosphorylated tau (pTau) epitopes and the innate and adaptive immunity. Tau pathology was assessed in the cerebral cortex using antibodies directed against: Tau-2 (phosphorylated and unphosphorylated tau), AT8 (pSer202/pThr205), AT100 (pThr212/pSer214), CP13 (pSer202), PHF1 (pSer396/pSer404), pThr181 and pSer356. The immunophenotypes of microglia and astrocytes were assessed with phenotypic markers (Iba1, CD68, HLA-DR, CD64, CD32a, CD16 for microglia and GFAP, EAAT2, glutamine synthetase and ALDH1L1 for astrocytes). The adaptive immune response was explored via CD4+ and CD8+ T cell quantification and the neuroinflammatory environment was investigated via the expression of 30 inflammatory-related proteins using V-Plex Meso Scale Discovery. As expected, all pTau markers were increased in FTLD-tau cases compared to controls. pSer356 expression was greatest in FTLD-MAPT cases versus controls (P < 0.0001), whereas the expression of other markers was highest in Pick's disease. Progressive supranuclear palsy with frontotemporal dementia consistently had a lower pTau protein load compared to Pick's disease across tau epitopes. The only microglial marker increased in FTLD-tau was CD16 (P = 0.0292) and specifically in FTLD-MAPT cases (P = 0.0150). However, several associations were detected between pTau epitopes and microglia, supporting an interplay between them. GFAP expression was increased in FTLD-tau (P = 0.0345) with the highest expression in Pick's disease (P = 0.0019), while ALDH1L1 was unchanged. Markers of astrocyte glutamate cycling function were reduced in FTLD-tau (P = 0.0075; Pick's disease: P < 0.0400) implying astrocyte reactivity associated with a decreased glutamate cycling activity, which was further associated with pTau expression. Of the inflammatory proteins assessed in the brain, five chemokines were upregulated in Pick's disease cases (P < 0.0400), consistent with the recruitment of CD4+ (P = 0.0109) and CD8+ (P = 0.0014) T cells. Of note, the CD8+ T cell infiltration was associated with pTau epitopes and microglial and astrocytic markers. Our results highlight that FTLD-tau is associated with astrocyte reactivity, remarkably little activation of microglia, but involvement of adaptive immunity in the form of chemokine-driven recruitment of T lymphocytes.

Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly.

Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.

Neurodegeneration risk factor, EIF2AK3 (PERK), influences tau protein aggregation.

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms-unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

Validity and value of metabolic connectivity in mouse models of ß-amyloid and tauopathy.

Among functional imaging methods, metabolic connectivity (MC) is increasingly used for investigation of regional network changes to examine the pathophysiology of neurodegenerative diseases such as Alzheimer's disease (AD) or movement disorders. Hitherto, MC was mostly used in clinical studies, but only a few studies demonstrated the usefulness of MC in the rodent brain. The goal of the current work was to analyze and validate metabolic regional network alterations in three different mouse models of neurodegenerative diseases (ß-amyloid and tau) by use of 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) imaging. We compared the results of FDG-µPET MC with conventional VOI-based analysis and behavioral assessment in the Morris water maze (MWM). The impact of awake versus anesthesia conditions on MC read-outs was studied and the robustness of MC data deriving from different scanners was tested. MC proved to be an accurate and robust indicator of functional connectivity loss when sample sizes ≥12 were considered. MC readouts were robust across scanners and in awake/ anesthesia conditions. MC loss was observed throughout all brain regions in tauopathy mice, whereas ß-amyloid indicated MC loss mainly in spatial learning areas and subcortical networks. This study established a methodological basis for the utilization of MC in different ß-amyloid and tau mouse models. MC has the potential to serve as a read-out of pathological changes within neuronal networks in these models.

The innate immune response in tauopathies.

Tauopathies, which include frontotemporal dementia, Alzheimer's disease, and chronic traumatic encephalopathy, are a class of neurological disorders resulting from pathogenic tau aggregates. These aggregates disrupt neuronal health and function leading to the cognitive and physical decline of tauopathy patients. Genome-wide association studies and clinical evidence have brought to light the large role of the immune system in inducing and driving tau-mediated pathology. More specifically, innate immune genes are found to harbor tauopathy risk alleles, and innate immune pathways are upregulated throughout the course of disease. Experimental evidence has expanded on these findings by describing pivotal roles for the innate immune system in the regulation of tau kinases and tau aggregates. In this review, we summarize the literature implicating innate immune pathways as drivers of tauopathy.

Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies.

BACKGROUND: Accumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer's disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood. METHODS: We used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation. RESULTS: We showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism. CONCLUSION: Our results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Overexpression of pathogenic tau in astrocytes causes a reduction in AQP4 and GLT1, an immunosuppressed phenotype and unique transcriptional responses to repetitive mild TBI without appreciable changes in tauopathy.

Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAPP301L mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAPP301L model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.

Biochemical approaches to assess the impact of post-translational modifications on pathogenic tau conformations using recombinant protein.

Tau protein is associated with many neurodegenerative disorders known as tauopathies. Aggregates of tau are thought of as a main contributor to neurodegeneration in these diseases. Increasingly, evidence points to earlier, soluble conformations of abnormally modified monomers and multimeric tau as toxic forms of tau. The biological processes driving tau from physiological species to pathogenic conformations remain poorly understood, but certain avenues are currently under investigation including the functional consequences of various pathological tau changes (e.g. mutations, post-translational modifications (PTMs), and protein-protein interactions). PTMs can regulate several aspects of tau biology such as proteasomal and autophagic clearance, solubility, and aggregation. Moreover, PTMs can contribute to the transition of tau from normal to pathogenic conformations. However, our understating of how PTMs specifically regulate the transition of tau into pathogenic conformations is partly impeded by the relative lack of structured frameworks to assess and quantify these conformations. In this review, we describe a set of approaches that includes several in vitro assays to determine the contribution of PTMs to tau's transition into known pathogenic conformations. The approaches begin with different methods to create recombinant tau proteins carrying specific PTMs followed by validation of the PTMs status. Then, we describe a set of biochemical and biophysical assays that assess the contribution of a given PTM to different tau conformations, including aggregation, oligomerization, exposure of the phosphatase-activating domain, and seeding. Together, these approaches can facilitate the advancement of our understanding of the relationships between PTMs and tau conformations.