Determinants of astrocytic pathology in stem cell models of primary tauopathies.

Astrocytic tau aggregates are seen in several primary and secondary tauopathies, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and chronic traumatic encephalopathy (CTE). In all of these diseases, astrocytic tau consists mostly of the longer (4R) tau isoform, even when adjacent neuronal aggregates consist of a mixture of 3- and 4R tau, as in CTE. Even the rare astrocytic tau aggregates seen in Pick's disease appear to contain both 3R and 4R tau. The reasons for this, and the mechanisms by which astrocytic tau aggregates form, remain unclear. We used a combination of RNA in situ hybridization and immunofluorescence in post-mortem human brain tissue, as well as tau uptake studies in human stem cell-derived astrocytes, to determine the origins of astrocytic tau in 4R tauopathies. We found no differences in tau mRNA expression between diseases or between tau positive and negative astrocytes within PSP. We then found that stem cell-derived astrocytes preferentially take up long isoform (4R) recombinant tau and that this uptake is impaired by induction of reactivity with inflammatory stimuli or nutritional stress. Astrocytes exposed to either 3R or 4R tau also showed downregulation of genes related to astrocyte differentiation. Our findings suggest that astrocytes preferentially take up neuronal 4R tau from the extracellular space, potentially explaining why 4R tau is the predominant isoform in astrocytic tau aggregates.

Determinants of Astrocytic Pathology in Stem Cell Models of Primary Tauopathies.

Astrocytic tau aggregates are seen in several primary and secondary tauopathies, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and chronic traumatic encephalopathy (CTE). In all cases, astrocytic tau consists exclusively of the longer (4R) tau isoform, even when adjacent neuronal aggregates consist of a mixture of 3- and 4R tau, as in CTE. The reasons for this and the mechanisms by which astrocytic tau aggregates form remain unclear. We used a combination of RNA in situ hybridization and immunofluorescence in post-mortem human brain tissue, as well as tau uptake studies in human stem cell-derived astrocytes, to determine the origins of astrocytic tau in 4R tauopathies. We found that astrocytes across tauopathies do not upregulate tau mRNA expression between diseases or between tau-positive and -negative astrocytes within PSP. We then found that stem cell-derived astrocytes preferentially take up long isoform (4R) labeled recombinant tau and that this uptake is impaired by induction of reactivity with inflammatory stimuli or nutritional stress. Astrocytes exposed to either 3R or 4R tau also showed downregulation of genes related to astrocyte differentiation. Our findings suggest that astrocytes preferentially take up neuronal 4R tau from the extracellular space, which potentially explains why astrocytic tau aggregates contain only 4R tau, and that tau uptake is impaired by decreased nutrient availability or neuroinflammation, both of which are common in the aging brain.

Thioflavin S Staining and Amyloid Formation Are Unique to Mixed Tauopathies.

Tau phosphorylation, aggregation, and toxicity are the main drivers of neurodegeneration in multiple tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau. Although aggregation and amyloid formation are often assumed to be synonymous, the ability of tau aggregates in different diseases to form amyloids in vivo has not been systematically studied. We used the amyloid dye Thioflavin S to look at tau aggregates in mixed tauopathies such as AD and primary age-related tauopathy, as well as pure 3R or 4R tauopathies such as Pick's disease, progressive supranuclear palsy, and corticobasal degeneration. We found that aggregates of tau protein only form thioflavin-positive amyloids in mixed (3R/4R), but not pure (3R or 4R), tauopathies. Interestingly, neither astrocytic nor neuronal tau pathology was thioflavin-positive in pure tauopathies. As most current positron emission tomography tracers are based on thioflavin derivatives, this suggests that they may be more useful for differential diagnosis than the identification of a general tauopathy. Our findings also suggest that thioflavin staining may have utility as an alternative to traditional antibody staining for distinguishing between tau aggregates in patients with multiple pathologies and that the mechanisms for tau toxicity may differ between different tauopathies.

Postmortem Cerebellar Volume Is Not Reduced in Essential Tremor: A Comparison with Multiple System Atrophy and Controls.

BACKGROUND: Essential tremor (ET) is a common movement disorder in which cerebellar microscopic and volume alterations have been repeatedly reported although with disagreement between studies. However, pronounced heterogeneity was found with regard to cerebellar volume alterations. OBJECTIVE: This study aimed to assess postmortem cerebellar volume in subjects with or without ET, as compared with subjects with multiple system atrophy (MSA), a well-established cerebellar neurodegeneration. METHODS: Cases with ET (n = 29), MSA (n = 7), and non-demented control cases without any movement disorder (n = 22) were selected from the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), a longitudinal clinicopathological study with annual research-dedicated clinical assessments by neuropsychologists, subspecialist movement disorders, and cognitive/behavioral neurologists, with comprehensive neuropathological examinations after death. Group comparisons were controlled for common age-related neurodegenerative and cerebrovascular pathologies. Cerebellar volumes were calculated using digital images of slices taken at the time of autopsy, immediately after brain removal and before fixation. RESULTS: Cerebellar volume was not reduced in ET subjects compared to controls. The two groups did not differ in terms of incidental cerebrovascular and Alzheimer's disease neuropathology. In contrast, cerebellar volume was significantly reduced in subjects with MSA when compared to ET and control subjects. CONCLUSION: In a well-characterized cohort, postmortem cerebellar volume measurements suggest that there are no volume alterations in ET when compared to controls, in contrast to significant cerebellar atrophy in subjects with MSA.

Characterization of the Tau Interactome in Human Brain Reveals Isoform-Dependent Interaction with 14-3-3 Family Proteins.

Despite exhibiting tau phosphorylation similar to Alzheimer's disease (AD), the human fetal brain is remarkably resilient to tau aggregation and toxicity. To identify potential mechanisms for this resilience, we used co-immunoprecipitation (co-IP) with mass spectrometry to characterize the tau interactome in human fetal, adult, and Alzheimer's disease brains. We found significant differences between the tau interactome in fetal and AD brain tissue, with little difference between adult and AD, although these findings are limited by the low throughput and small sample size of these experiments. Differentially interacting proteins were enriched for 14-3-3 domains, and we found that the 14-3-3-ß, η, and γ isoforms interacted with phosphorylated tau in Alzheimer's disease but not the fetal brain. Since long isoform (4R) tau is only seen in the adult brain and this is one of the major differences between fetal and AD tau, we tested the ability of our strongest hit (14-3-3-ß) to interact with 3R and 4R tau using co-immunoprecipitation, mass photometry, and nuclear magnetic resonance (NMR). We found that 14-3-3-ß interacts preferentially with phosphorylated 4R tau, forming a complex consisting of two 14-3-3-ß molecules to one tau. By NMR, we mapped 14-3-3 binding regions on tau that span the second microtubule binding repeat, which is unique to 4R tau. Our findings suggest that there are isoform-driven differences between the phospho-tau interactome in fetal and Alzheimer's disease brain, including differences in interaction with the critical 14-3-3 family of protein chaperones, which may explain, in part, the resilience of fetal brain to tau toxicity.

Fyn depletion ameliorates tauP301L-induced neuropathology.

The Src family non-receptor tyrosine kinase Fyn has been implicated in neurodegeneration of Alzheimer's disease through interaction with amyloid ß (Aß). However, the role of Fyn in the pathogenesis of primary tauopathies such as FTDP-17, where Aß plaques are absent, is poorly understood. In the current study, we used AAV2/8 vectors to deliver tauP301L to the brains of WT and Fyn KO mice, generating somatic transgenic tauopathy models with the presence or absence of Fyn. Although both genotypes developed tau pathology, Fyn KO developed fewer neurofibrillary tangles on Bielschowsky and Thioflavin S stained sections and showed lower levels of phosphorylated tau. In addition, tauP301L-induced behavior abnormalities and depletion of synaptic proteins were not observed in the Fyn KO model. Our work provides evidence for Fyn being a critical protein in the disease pathogenesis of FTDP-17.

Increased Tau Expression Correlates with Neuronal Maturation in the Developing Human Cerebral Cortex.

Although best known for its role in Alzheimer's disease (AD), tau is expressed throughout brain development, although it remains unclear when and which cell types this expression occurs and how it affects disease states in both fetal and neonatal periods. We thus sought to map tau mRNA and protein expression in the developing human brain at the cellular level using a combination of existing single-cell RNA sequencing (sc-RNAseq) data, RNA in situ hybridization (RNAscope), and immunohistochemistry (IHC). Using sc-RNAseq, we found that tau mRNA expression begins in radial glia but increases dramatically as migrating neuronal precursors mature. Specifically, TBR1+ maturing neurons and SYN+ mature neurons showed significantly higher mRNA expression than GFAP+/NES+ radial glia or TBR2+ intermediate progenitors. By RNAscope, we found low levels of tau mRNA in subventricular zone (SVZ) radial glia and deep white matter intermediate progenitors, with an increase in more superficially located maturing and mature neurons. By total-tau IHC, the germinal matrix and SVZ showed little protein expression, although both RNAscope and sc-RNAseq showed mRNA, and Western blotting revealed significantly less protein in those areas compared with more mature regions. Induced pluripotent stem cell (iPSC)-derived cortical organoids showed a similar tau expression pattern by sc-RNAseq and RNAscope. Our results indicate that tau increases with neuronal maturation in both the developing fetal brain and iPSC-derived organoids and forms a basis for future research on regulatory mechanisms triggering the onset of tau gene transcription and translation, which may represent potential therapeutic targets for neurodegenerative tauopathies and neurodevelopmental disorders.

ß-amyloid and tau pathology in the aging feline brain.

Domestic cats (Felis catus) are known to develop cognitive impairment, and several small series have demonstrated both ß-amyloid and tau aggregation, including neurofibrillary tangles, with age, making them a promising physiologic model of Alzheimer disease (AD). We therefore report the largest feline autopsy cohort to date of 32 cats ranging from 1.5 to 22.1 years of age, with systematic neuropathologic assessment according to NIA-Alzheimer's Association Criteria. Formalin-fixed paraffin-embedded tissue sections of brain were obtained retrospectively from cats autopsied at the Iowa State College of Veterinary Medicine. We found ß-amyloid staining, predominantly in Cortical Layers IV and VI in 27 of the 32 cats used in the study, with four of these animals showing tau-positive tangles and neuropil threads. In 75% of these cases (3/4), tau deposition was limited to entorhinal cortex, while one case showed diffuse positive staining throughout the hippocampal formation and neocortex. This last case showed positive staining for all phospho-tau-specific antibodies tested, similar to the pattern seen in human AD. Interestingly, we saw a higher ratio of pretangles to tangles than that in human AD, and none of the cases showed neuritic plaques on any of the stains used. Our findings indicate that aging domestic cats spontaneously develop both ß-amyloid and tau pathology similar, but not identical to that seen in human AD. This suggests that the domestic cat may serve as a potential model for mechanistic and therapeutic AD studies, but that additional research is needed to identify differences between the neuropathology of aging in humans and felines.

Tau Phosphorylation and Aggregation in the Developing Human Brain.

Tau hyperphosphorylation, mostly at serine (Ser) or threonine (Thr) residues, plays a key role in the pathogenesis of Alzheimer disease (AD) and other tauopathies. Rodent studies show similar hyperphosphorylation in the developing brain, which may be involved in regulating axonal growth and plasticity, but detailed human studies are lacking. Here, we examine tau phosphorylation by immunohistochemistry and immunoblotting in human fetal and adult autopsy brain tissue. Of the 20 cases with sufficient tissue preservation, 18 (90%) showed positive staining for S214 (pSer214), with the majority also positive for CP13 (pSer202), and PHF-1 (pSer396/pSer404). AT8 (pSer202/pThr205) and RZ3 (pThr231) were largely negative while PG5 (pSer409) was negative in all cases. Immunoblotting showed tau monomers with a similar staining pattern. We also observed phospho-tau aggregates in the fetal molecular layer, staining positively for S214, CP13, and PHF1 and negative for thioflavin S. These corresponded to high-molecular weight (∼150 kD) bands seen on Western blots probed with S214, PHF1, and PG5. We therefore conclude that fetal phosphorylation overlaps with AD in some residues, while others (e.g. T231, S409) appear to be unique to AD, and that tau is capable of forming nontoxic aggregates in the developing brain. These findings suggest that the fetal brain is resilient to formation of toxic aggregates, the mechanism for which may yield insights into the pathogenesis of tau aggregation and toxicity in the aging brain.