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The molecular etiology of tau-derived neurodegeneration remains poorly understood, reflected in the low success rate of clinical trials. Hence, aquiring a better understanding the molecular basis of tauopathies is a critical need. To develop a versatile and reproducible system to study tau aggregation with high spatiotemporal control through optogenetics that will aid in investigating the differences in tau aggregation kinetics, the burden the burden of tau isoforms, and mutations and that will be suitable for high-throughput analysis of tauopathy-related mechanisms. We engineered an optogenetic cellular model using the CRY2 protein that when light-activated enables spatiotemporal control over tau aggregation. A panel of CRY2-tau constructs expressing all six neuronal isoforms and known mutations was created to assess aggregation kinetics and cellular burden due to imbalances in tau proteoforms. Tauopathy-associated factors will be assayed with biochemistry, and challenge optogenetic tau aggregation with their overexpression and knockdown. Experiments in patient-derived human induced pluripotent stem cell models will be performed supporting the project's translational nature. HEK293 and SH-SY5Y cells expressing our constructs showed mCherry stable tau inclusions after light stimulation, which are not present when the backbone alone is transfected and are less frequent without light stimulus. These inclusions where positive for phosphorylation markers consistent with Alzheimer disease when assessed by immunocytochemistry. Western blotting analysis confirmed the presence of high molecular weight tau molecules consistent with dimmers and higher aggregates and live-imaging assessments of different tau proteoforms suggest different CRY2-tau inclusion formation kinetics. These data support the feasibility of generating tau aggregates with pathological features in our cellular system. This approach has the potential to enhance the screening process of possible tau aggregation modulators allowing analysis in real time of tau aggregation in a precise manner with high spatiotemporal resolution while. This model may r be amenable to drug screening.

Background The molecular etiology of tau‐derived neurodegeneration remains poorly understood, reflected in the low success rate of clinical trials. Hence, aquiring a better understanding the molecular basis of tauopathies is a critical need. Objective To develop a versatile and reproducible system to study tau aggregation with high spatiotemporal control through optogenetics that will aid in investigating the differences in tau aggregation kinetics, the burden the burden of tau isoforms, and mutations and that will be suitable for high‐throughput analysis of tauopathy‐related mechanisms. Method We engineered an optogenetic cellular model using the CRY2 protein that when light‐activated enables spatiotemporal control over tau aggregation. A panel of CRY2‐tau constructs expressing all six neuronal isoforms and known mutations was created to assess aggregation kinetics and cellular burden due to imbalances in tau proteoforms. Tauopathy‐associated factors will be assayed with biochemistry, and challenge optogenetic tau aggregation with their overexpression and knockdown. Experiments in patient‐derived human induced pluripotent stem cell models will be performed supporting the project’s translational nature. Result HEK293 and SH‐SY5Y cells expressing our constructs showed mCherry stable tau inclusions after light stimulation, which are not present when the backbone alone is transfected and are less frequent without light stimulus. These inclusions where positive for phosphorylation markers consistent with Alzheimer disease when assessed by immunocytochemistry. Western blotting analysis confirmed the presence of high molecular weight tau molecules consistent with dimmers and higher aggregates and live‐imaging assessments of different tau proteoforms suggest different CRY2‐tau inclusion formation kinetics. Conclusion These data support the feasibility of generating tau aggregates with pathological features in our cellular system. This approach has the potential to enhance the screening process of possible tau aggregation modulators allowing analysis in real time of tau aggregation in a precise manner with high spatiotemporal resolution while. This model may r be amenable to drug screening.

The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.

Alzheimer's disease (AD) is characterized by neocortical dissemination of neurofibrillary tangles (NFTs) while primary age-related tauopathy (PART) has NFTs largely confined to the hippocampus and adjacent structures. Thus, PART and AD represent two extremes of a spectrum of NFT spread. We investigated epigenetic mechanisms of interindividual variation in NFT spread. We evaluated DNA methylation (DNAm) in frontal cortex by Infinium EPIC BeadChip array in the multi-center PART Working Group cohort (PWG, N = 398), controlling for age, sex, and batch effects. Using SeSAMe and a false discovery rate of p<0.05, we identified differentially methylated positions (DMPs) associated with PART pathology quantitated from immunohistochemically-stained sections. To assess amyloid effects, we compared this set with DMPs associated with neuritic amyloid plaque using the CERAD score. We identified novel DMPs not previously implicated in AD. We validated our findings in the Religious Orders Study and Memory and Aging Project cohort (ROSMAP, N = 707). We then related DMPs to differential expression of linked genes and performed enrichment analyses using KnowYourCG. We identified DMPs associated with NFTs [PWG: 8 novel / 8 total; ROSMAP: 31 novel / 68 total]. In the PWG cohort, of the 550 total [539 novel] DMPs that associated with CERAD score, there was no overlap with NFT-associated DMPs. In the ROSMAP cohort, 57 total [3 novel] DMPs are associated with both NFTs and diagnosis of PART vs. AD and are linked to genes related to T-cells and axonal transport. In contrast, the 11 total [10 novel] DMPs associated with NFTs alone are linked to genes related to synaptic signaling, heparin sulfate proteoglycan biosynthesis, and microtubule architecture. DNA methylation distinguishes PART and AD brain. We identify tau-specific DMPs that account for variance in NFT burden among aging individuals, both in the absence and presence of amyloid plaques in PART and AD, respectively. In PART, tau-DMPs are fully orthogonal to the set of amyloid-DMPs. In contrast, in AD, the tau-DMPs have both amyloid-associated and amyloid-independent subsets with separate gene enrichment profiles. Therefore, distinct epigenetic events may drive pathology within the limbic system compared to those that drive tau spread to neocortex.

Background Alzheimer’s disease (AD) is characterized by neocortical dissemination of neurofibrillary tangles (NFTs) while primary age‐related tauopathy (PART) has NFTs largely confined to the hippocampus and adjacent structures. Thus, PART and AD represent two extremes of a spectrum of NFT spread. We investigated epigenetic mechanisms of interindividual variation in NFT spread. Method We evaluated DNA methylation (DNAm) in frontal cortex by Infinium EPIC BeadChip array in the multi‐center PART Working Group cohort (PWG, N = 398), controlling for age, sex, and batch effects. Using SeSAMe and a false discovery rate of p<0.05, we identified differentially methylated positions (DMPs) associated with PART pathology quantitated from immunohistochemically‐stained sections. To assess amyloid effects, we compared this set with DMPs associated with neuritic amyloid plaque using the CERAD score. We identified novel DMPs not previously implicated in AD. We validated our findings in the Religious Orders Study and Memory and Aging Project cohort (ROSMAP, N = 707). We then related DMPs to differential expression of linked genes and performed enrichment analyses using KnowYourCG. Result We identified DMPs associated with NFTs [PWG: 8 novel / 8 total; ROSMAP: 31 novel / 68 total]. In the PWG cohort, of the 550 total [539 novel] DMPs that associated with CERAD score, there was no overlap with NFT‐associated DMPs. In the ROSMAP cohort, 57 total [3 novel] DMPs are associated with both NFTs and diagnosis of PART vs. AD and are linked to genes related to T‐cells and axonal transport. In contrast, the 11 total [10 novel] DMPs associated with NFTs alone are linked to genes related to synaptic signaling, heparin sulfate proteoglycan biosynthesis, and microtubule architecture. Conclusion DNA methylation distinguishes PART and AD brain. We identify tau‐specific DMPs that account for variance in NFT burden among aging individuals, both in the absence and presence of amyloid plaques in PART and AD, respectively. In PART, tau‐DMPs are fully orthogonal to the set of amyloid‐DMPs. In contrast, in AD, the tau‐DMPs have both amyloid‐associated and amyloid‐independent subsets with separate gene enrichment profiles. Therefore, distinct epigenetic events may drive pathology within the limbic system compared to those that drive tau spread to neocortex.

Background The accumulation of abnormal tau protein in neurons and glia in the human brain is the defining feature of neurodegenerative diseases known as tauopathies. Progressive supranuclear palsy (PSP), the most common primary tauopathy, is typified by selective vulnerability of dopaminergic neurons and glia in the midbrain leading to an atypical parkinsonian movement disorder. To investigate candidate disease mechanisms underlying PSP, there is a critical need for model systems that more accurately recapitulate the cellular and molecular environment in the human brain. Human induced pluripotent stem cell (hiPSC)‐derived organoid models have emerged as a powerful tool to address this gap. Method Skin biopsies were collected from living clinically diagnosed PSP patients or during autopsy. Fibroblasts were cultured and reprogrammed into hiPSCs using Sendai virus. HiPSCs were maintained with StemCultures FGF2 Discs to improve pluripotency and FACS was performed to confirm pluripotency marker expression. To generate midbrain organoids, hiPSCs were seeded into suspension spinner flasks, patterned using pharmacological directed differentiation, and grown for four months. Reliable patterning was confirmed with qRT‐PCR, immunohistochemistry and immunoblot using a panel of cell‐type specific markers. Astrocytes were extracted from mature organoids, cultured, and screened for astrocyte‐specific markers. Result Fibroblasts have been banked from twenty‐two PSP patients and seven reprogrammed into hiPSCs. Sporadic case status was determined by Sanger sequencing confirming the absence of a MAPT mutation. We found that hiPSCs grown with controlled‐release FGF2 discs were 90‐100% positive for pluripotency markers and negative for off‐target genes. During patterning, organoids displayed morphological and cytoarchitectural patterns consistent with developing neuroectoderm and midbrain. Midbrain neural progenitor and dopaminergic markers such as FOXA2, LMX1A, and TH were positive in a time‐dependent manner, with mature dopaminergic neurons expressing NURR1 and GIRK2 detected by day 30. GFAP‐positive astrocytes appeared around day 100. Astrocytes extracted from mature organoids were positive for multiple astrocyte markers. Conclusion Sporadic PSP patient hiPSCs reliably differentiate into midbrain dopaminergic organoids and astrocytes, resulting in a sporadic tauopathy model containing key cell types affected in PSP. This cell collection is a valuable resource to investigate candidate mechanisms underlying tauopathy and could provide insight into cell‐type specific disease drivers.

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene ( MAPT ) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP ( n  = 84) and controls ( n  = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder.

Background Tauopathies are a group of neurodegenerative diseases where there is pathologic accumulation of hyperphosphorylated tau protein (ptau). The most common tauopathy is Alzheimer’s disease (AD), but chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and argyrophilic grain disease (AGD) are significant health risks as well. Currently, it is unclear what specific molecular factors might drive each distinct disease and represent therapeutic targets. Additionally, there is a lack of biomarkers that can differentiate each disease in life. Recent work has suggested that neuroinflammatory changes might be specific among distinct diseases and offers a novel resource for mechanistic targets and biomarker candidates. Methods To better examine each tauopathy, a 71 immune-related protein multiplex ELISA panel was utilized to analyze anterior cingulate grey matter from 127 individuals neuropathologically diagnosed with AD, CTE, PSP, CBD, and AGD. A partial least square regression analysis was carried out to perform unbiased clustering and identify proteins that are distinctly correlated with each tauopathy correcting for age and gender. Receiver operator characteristic and binary logistic regression analyses were then used to examine the ability of each candidate protein to distinguish diseases. Validation in postmortem cerebrospinal fluid (CSF) from 15 AD and 14 CTE cases was performed to determine if candidate proteins could act as possible novel biomarkers. Results Five clusters of immune proteins were identified and compared to each tauopathy to determine if clusters were specific to distinct disease. Each cluster was found to correlate with either CTE, AD, PSP, CBD, or AGD. When examining which proteins were the strongest driver of each cluster, it was observed the most distinctive protein for CTE was CCL21, AD was FLT3L, and PSP was IL13. Individual proteins that were specific to CBD and AGD were not observed. CCL21 was observed to be elevated in CTE CSF compared to AD cases ( p  = 0.02), further validating the use as possible biomarkers. Sub-analyses for male only cases confirmed the results were not skewed by gender differences. Conclusions Overall, these results highlight that different neuroinflammatory responses might underlie unique mechanisms in related neurodegenerative pathologies. Additionally, the use of distinct neuroinflammatory signatures could help differentiate between tauopathies and act as novel biomarker candidate to increase specificity for in-life diagnoses.

Aging-related cognitive decline is associated with brain structural changes and synaptic loss. However, the molecular mechanisms of cognitive decline during normal aging remain elusive. Using the GTEx transcriptomic data from 13 brain regions, we identified aging-associated molecular alterations and cell-type compositions in males and females. We further constructed gene co-expression networks and identified aging-associated modules and key regulators shared by both sexes or specific to males or females. A few brain regions such as the hippocampus and the hypothalamus show specific vulnerability in males, while the cerebellar hemisphere and the anterior cingulate cortex regions manifest greater vulnerability in females than in males. Immune response genes are positively correlated with age, whereas those involved in neurogenesis are negatively correlated with age. Aging-associated genes identified in the hippocampus and the frontal cortex are significantly enriched for gene signatures implicated in Alzheimer's disease (AD) pathogenesis. In the hippocampus, a male-specific co-expression module is driven by key synaptic signaling regulators including , , and ; while in the cortex, a female-specific module is associated with neuron projection morphogenesis, which is driven by key regulators including , and . In the cerebellar hemisphere, a myelination-associated module shared by males and females is driven by key regulators such as , , , , and , which have been implicated in the development of AD and other neurodegenerative diseases. This integrative network biology study systematically identifies molecular signatures and networks underlying brain regional vulnerability to aging in males and females. The findings pave the way for understanding the molecular mechanisms of gender differences in developing neurodegenerative diseases such as AD.

Background Progressive supranuclear palsy (PSP) is the most common primary tauopathy, with a constellation of pathological features including 4R‐tau positive neurofibrillary tangles and tufted astrocytes. Most PSP cases are sporadic and associated with common structural variation in the 17q21.31 MAPT locus as well as other loci, including EIF2AK3 which is critical for the integrated stress response (ISR). Despite these known genetic risk associations, mechanisms underlying disease pathogenesis are unclear. To investigate candidate mechanisms, there is a critical need for model systems that better recapitulate the cellular complexity of the human brain. Induced pluripotent stem cell (iPSC) patient‐derived organoid models are a powerful tool to study molecular and cellular changes in a disease‐relevant genomic context. Method Single‐nucleus RNA sequencing (snRNA‐seq) was performed in the subthalamic nucleus region from autopsy PSP and control brains. Transcriptional differences were validated by immunohistochemistry (IHC) using antibodies for ISR activation markers and phosphorylated tau (p‐tau). Fibroblasts grown from sporadic PSP patient skin were reprogrammed into iPSCs, and midbrain organoids were generated in spinner flasks through pharmacological directed differentiation. Total tau, tau isoform, p‐tau, and ISR activation markers were assessed in PSP and control organoids. Result Differential gene expression and pathway analysis in PSP brain snRNA‐seq data identified dysregulated EIF2 signaling, a target of the ISR, in vulnerable cell types. Histological validation in autopsy brain tissue showed PSP‐vulnerable brain regions had the highest frequency of ISR activation, while no activation was detected in protected brain regions. ISR activation positively correlated with tau burden and was localized to p‐tau+ neurons and astrocytes. PSP organoids contained increased high molecular weight p‐tau and 4R‐tau, a higher ratio of p‐tau:total tau, and different ISR activation levels compared to controls. Conclusion SnRNA‐seq and neurohistological data reveals ISR dysregulation in disease‐affected cell types in PSP brain tissue. ISR activation positively associates with tau burden in vulnerable brain regions in PSP, providing a potential mechanistic link with EIF2AK3 genetic risk. PSP patient‐derived organoids recapitulate key disease‐relevant features, including elevation of toxic tau proteoforms and ISR dysregulation. This sporadic PSP organoid model will provide insight into cell‐type specific drivers of neurodegeneration that underlie sporadic tauopathy.