Mutant Human Tau Research Articles

MAIT cell deficiency exacerbates neuroinflammation in P301S human tau transgenic mice.

The role of immune cells in neurodegeneration remains incompletely understood. Our recent study revealed the presence of mucosal-associated invariant T (MAIT) cells in the meninges, where they express antioxidant molecules to maintain meningeal barrier integrity. Accumulation of misfolded tau proteins are a hallmark of neurodegenerative diseases. The role of MAIT cells in tau-related neuroinflammation and neurodegeneration, however, remains unclear. Here we report that the meninges of P301 mutant human tau transgenic mice had increased numbers of MAIT cells, which retained their expression of antioxidant molecules. Mr1 -/- P301S mice that lacked MAIT cells exhibited increased tau pathology and hippocampus atrophy compared to control Mr1 +/+ P301S mice. Adoptive transfer of MAIT cells reduced tau pathology and hippocampus atrophy in Mr1 -/- P301S mice. Meningeal barrier integrity was compromised in Mr/ -/- P301S mice, but not in control Mr1 +/+ P301S mice. A distinctive microglia subset with proinflammatory gene expression profile (M-inflammatory) was enriched in the hippocampus of Mr1 -/- P301S mice. The transcriptomes of the remaining microglia in these mice also shifted towards a proinflammatory state, with increased expression of inflammatory cytokines, chemokines, and genes related with ribosome biogenesis and immune responses to toxic substances. The transfer of MAIT cells restored meningeal barrier integrity and suppressed microglial inflammation in the Mr1 -/- P301S mice. Together, our data indicate an important role for MAIT cells in regulating tau-pathology-related neuroinflammation and neurodegeneration.

Long-term exposure to excessive norepinephrine in the brain induces tau aggregation, neuronal death, and cognitive deficits in early tau transgenic mice.

Alzheimer's disease (AD) is marked by the presence of intraneuronal neurofibrillary tangles (NFTs), which are primarily composed of hyperphosphorylated tau protein. The locus coeruleus (LC), the brain's main source of norepinephrine (NE), is one of the earliest regions to develop NFTs and experience neurodegeneration in AD. While LC-derived NE plays beneficial roles in cognition, emotion, locomotion, and the sleep-wake cycle, its impact on tau pathology is unclear. To explore this relationship, we administered intraperitoneal injections of either N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), a selective neurotoxin for noradrenergic neurons, or reboxetine (RBX), a norepinephrine reuptake inhibitor, to decrease or increase NE levels, respectively, in early tau transgenic mice expressing mutant human P301L tau (ADLPTau) for two months. Only the RBX-treated mice exhibited cognitive deficits, as evidenced by their performance in the Y-maze, novel object recognition, and contextual fear conditioning tests. Immunohistochemical analysis revealed increased hyperphosphorylated tau aggregates in the LC and hippocampus of the RBX-treated mice. Furthermore, neuronal apoptosis was observed in the hippocampal CA1 region of these mice. Western blotting showed that RBX injections led to the overactivation of tau kinases PKA and GSK3β, resulting in hyperphosphorylated tau, neuronal loss, and cognitive impairments. Consistent with these findings, human brain organoids exposed to higher NE concentrations also displayed elevated hyperphosphorylated tau and increased activity of the same tau kinases. These findings suggest that excessive NE exposure accelerates tau pathology by overactivating the tau kinases. Thus, modulating NE levels in the brain via the LC-NE system could be a potential therapeutic strategy for tau-related AD.

Severe neurodegeneration in brains of transgenic rats producing human tau prions

Both wild-type and mutant tau proteins can misfold into prions and self-propagate in the central nervous system of animals and people. To extend the work of others, we investigated the molecular basis of tau prion–mediated neurodegeneration in transgenic (Tg) rats expressing mutant human tau (P301S); this line of Tg rats is denoted Tg12099. We used the rat Prnp promoter to drive the overexpression of mutant tau (P301S) in the human 0N4R isoform. In Tg12099(+/+) rats homozygous for the transgene, ubiquitous expression of mutant human tau resulted in the progressive accumulation of phosphorylated tau inclusions, including silver-positive tangles in the frontal cortices and limbic system. Signs of central nervous system dysfunction were found in terminal Tg12099(+/+) rats exhibiting severe neurodegeneration and profound atrophy of the amygdala and piriform cortex. The greatest increases in tau prion activity were found in the corticolimbic structures. In contrast to the homozygous Tg12099(+/+) rats, we found lower levels of mutant tau in the hemizygous rats, resulting in few neuropathologic changes up to 2 years of age. Notably, these hemizygous rats could be infected by intracerebral inoculation with recombinant tau fibrils or precipitated tau prions from the brain homogenates of sick, aged homozygous Tg12099(+/+) rats. Our studies argue that the regional propagation of tau prions and neurodegeneration in the Tg12099 rats resembles that found in human primary tauopathies. These findings seem likely to advance our understanding of human tauopathies and may lead to effective therapeutics for Alzheimer’s disease and other tau prion disorders.

Study of the neuroprotective properties of the heteroreceptor EPOR/CD131 agonist of peptide structure in tau-proteinopathy modeling

Introduction: Tau proteinopathy is a pathology associated with the activation of post-translational modifications and interactions of pathophysiological cascades of neuroinflammation with hyperphosphorylation of Tau aggregates. Therefore, preference is given to agents that have properties in reducing or slowing down the processes of neuroinflammation and post-translational modifications in the brain. Materials and Methods: The study was conducted on male and female homozygous individuals of a transgenic murine line with overexpression of mutant human Tau gene (P301S) and a background wild mouse line C57Bl/6J. To assess the progression of Tau proteinopathy, behavioral tests were used at two control time points, and the last one measured the level of neuroinflammation markers and tau-proteinopathy. Results and Discussion: In the group of P301S mice treated with ARA-290, an improvement in the phenotypic picture of Tau proteinopathy was demonstrated compared with intact animals. In the Barnes circular maze test, mice showed a decrease in the total distance traveled and the latent time spent on the platform, which indicates a rapid entry into the shelter. In the O-shaped maze test, the group maintained a fairly high level of spontaneous exploratory behavior. In the vertical rod test, the animals recorded the best time indicators that they needed to turn and maintain balance compared to the intact group. A statistically significant decrease in the level of GSK-3β and an increase in CDK5 and PP2A were revealed, which indicates a dephosphorylating effect on Tau protein, as well as markers of neuroinflammation. NF-KB and TNF-α were significantly reduced by 57% and 32%, respectively, compared to the intact group. Conclusion: In the model of transgenic P301S murine line with overexpression of the mutant human Tau gene, the peptide agonist of the EPOR/CD 131 heteroreceptor demonstrated neuroprotective properties, which were confirmed by indicators of behavioral tests and markers of neuroinflammation and tau-proteinopathy.

Cytoplasmic aggregation of TDP43 and topographic correlation with tau and α-synuclein accumulation in the rTg4510 mouse model of tauopathy.

In patients with TDP43 proteinopathy, phosphorylated TDP43 (p-TDP43) accumulates in the cytoplasm of neurons. The accumulation of p-TDP43 has also been reported in patients with tauopathy and α-synucleinopathy. We investigated spatiotemporal changes in p-TDP43 accumulation in the brains of rTg4510 mice that overexpressed human mutant tau (P301L) and exhibited hyperphosphorylated tau (hp-tau) and phosphorylated αSyn (p-αSyn) accumulation. Immunohistochemically, p-TDP43 aggregates were observed in the cytoplasm of neurons, which increased with age. A significant positive correlation was observed between the number of cells with p-TDP43 aggregates and hp-tau and p-αSyn aggregates. Suppression of the human mutant tau (P301L) expression by doxycycline treatment reduces the accumulation of p-TDP43, hp-tau, and p-αSyn. Proteinase K-resistant p-TDP43 aggregates were found in regions with high hp-tau, and p-αSyn accumulation. Western blotting of the sarkosyl-insoluble fraction revealed bands of monomeric TDP43 and p-TDP43. These results indicate that the accumulation of mouse p-TDP43 is associated with the accumulation of human mutant tau (P301L) in rTg4510 mouse brains. The accumulation of hp-tau and p-αSyn may promote sarkosyl-insoluble p-TDP43 aggregates that are resistant to proteinase K. The synergistic effects of tau, TDP43, and αSyn may be involved in the pathology of proteinopathies, leading to the accumulation of multiple abnormal proteins.

Cholesterol 25-hydroxylase mediates neuroinflammation and neurodegeneration in a mouse model of tauopathy.

Alzheimer's disease (AD) is characterized by amyloid plaques and neurofibrillary tangles, in addition to neuroinflammation and changes in brain lipid metabolism. 25-Hydroxycholesterol (25-HC), a known modulator of both inflammation and lipid metabolism, is produced by cholesterol 25-hydroxylase encoded by Ch25h expressed as a "disease-associated microglia" signature gene. However, whether Ch25h influences tau-mediated neuroinflammation and neurodegeneration is unknown. Here, we show that in the absence of Ch25h and the resultant reduction in 25-HC, there is strikingly reduced age-dependent neurodegeneration and neuroinflammation in the hippocampus and entorhinal/piriform cortex of PS19 mice, which express the P301S mutant human tau transgene. Transcriptomic analyses of bulk hippocampal tissue and single nuclei revealed that Ch25h deficiency in PS19 mice strongly suppressed proinflammatory signaling in microglia. Our results suggest a key role for Ch25h/25-HC in potentiating proinflammatory signaling to promote tau-mediated neurodegeneration. Ch25h may represent a novel therapeutic target for primary tauopathies, AD, and other neuroinflammatory diseases.

Optimized Calcium-Phosphate-Based Co-transfection of Tau and tdTomato into Human iPSC-Derived Neurons for the Study of Intracellular Distribution of Wild-type and Mutant Human Tau.

The study of Tau protein in disease-relevant neuronal cells in culture requires efficient delivery systems for transfection of exogenous Tau and also modulators and interactors of Tau. Transfection of cultivated cells using calcium phosphate precipitation is a simple and cost-effective approach, also for difficult-to-transfect and sensitive cells such as primary neurons. Because of its low cell toxicity and ease of use, the Ca2+-phosphate transfection method is one of the most widely used gene transfer procedures in neuroscience. However, Ca2+-phosphate transfection efficacy in neurons is poor, often in the range of 1-5%, limiting its use in functional investigations. Here, we outline our improved Ca2+-phosphate transfection methodology for human iPSC-derived neurons that yields a reasonable efficiency (20-30% for bright volume markers) without apparent effects on cell health. We have used it to introduce wild-type and mutant human Tau with and without co-transfection of a volume marker (used here: tdTomato). In sum, our procedure can deliver neuronal genes (e.g., MAPT) using typical eukaryotic expression vectors (e.g., using CMV promoter) and is optimized for transfection of human iPSC-derived neurons.

Inhibiting tau-induced elevated nSMase2 activity and ceramides is therapeutic in an Alzheimer’s disease mouse model

BackgroundCognitive decline in Alzheimer’s disease (AD) is associated with hyperphosphorylated tau (pTau) propagation between neurons along synaptically connected networks, in part via extracellular vesicles (EVs). EV biogenesis is triggered by ceramide enrichment at the plasma membrane from neutral sphingomyelinase2 (nSMase2)-mediated cleavage of sphingomyelin. We report, for the first time, that human tau expression elevates brain ceramides and nSMase2 activity.MethodsTo determine the therapeutic benefit of inhibiting this elevation, we evaluated PDDC, the first potent, selective, orally bioavailable, and brain-penetrable nSMase2 inhibitor in the transgenic PS19 AD mouse model. Additionally, we directly evaluated the effect of PDDC on tau propagation in a mouse model where an adeno-associated virus (AAV) encoding P301L/S320F double mutant human tau was stereotaxically-injected unilaterally into the hippocampus. The contralateral transfer of the double mutant human tau to the dentate gyrus was monitored. We examined ceramide levels, histopathological changes, and pTau content within EVs isolated from the mouse plasma.ResultsSimilar to human AD, the PS19 mice exhibited increased brain ceramide levels and nSMase2 activity; both were completely normalized by PDDC treatment. The PS19 mice also exhibited elevated tau immunostaining, thinning of hippocampal neuronal cell layers, increased mossy fiber synaptophysin immunostaining, and glial activation, all of which were pathologic features of human AD. PDDC treatment reduced these changes. The plasma of PDDC-treated PS19 mice had reduced levels of neuronal- and microglial-derived EVs, the former carrying lower pTau levels, compared to untreated mice. In the tau propagation model, PDDC normalized the tau-induced increase in brain ceramides and significantly reduced the amount of tau propagation to the contralateral side.ConclusionsPDDC is a first-in-class therapeutic candidate that normalizes elevated brain ceramides and nSMase2 activity, leading to the slowing of tau spread in AD mice.

Dysregulation of memory‐induced protein synthesis in tauopathies

AbstractBackgroundProtein synthesis is a vital biological process, important for many neuronal and cognitive processes such as synaptic plasticity and the formation, updating, and extinction of long‐term memories. Recent studies have identified dysregulated translation as a molecular hallmark of many neurodegenerative diseases, including tauopathies such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), and corticobasal degeneration (CBD) (Evans et al., EMBO J, 2019; Evans et al., Acta Neuropathoc Comms, 2021; Elder et al., Commun Biol. 2021). This dysregulation of protein synthesis is thought to be driven in part by pathogenic alterations to tau, a neuronally enriched microtubule binding protein.MethodHere, we have utilized a series of de novo proteomic analyses, including in vivo non‐canonical amino acid (NCAA) tagging, surface sensing of translation‐based ribosome speed of elongation (SunRiSE), and polysome profiling, to explore both in vitro and in vivo how mutant human tau alters translation in a variety of tauopathies models, including the PS19 mouse strain, human neurons from a FTD patient‐derived inducible pluripotent stem cells (iPSCs) line, as well as in transfected HEK293 cells.ResultUsing these techniques, we have found that both P301S and V337M mutant human tau can severely impair protein synthesis, even when protein degradation is inhibited. Our data also suggests that the dysregulated translation is caused, at least in part, by impaired elongation, with SunRiSE assays showing that P301S and V337M mutant human tau both slow elongation rates. We also have demonstrated that FTD‐mutant tau impairs protein synthesis in CA1 hippocampal neurons in PS19 mice and are determining whether learning‐induced increases in hippocampal protein synthesis following contextual fear conditioning are impaired in these mice.ConclusionTogether, our results demonstrate that FTD‐mutant tau can severely impact several important components of the cellular translational machinery. Furthermore, our results also contribute to the growing evidence that impairments in protein synthesis are a feature of neurodegenerative diseases. This work was supported by the Alzheimer’s Association (H.T.E), Leon Levy foundation (H.T.E.), the Rainwater foundation (H.T.E.) and NIH grant NS121786 (E.K.).

The role of immunological signaling in disrupted proteostasis in the gut‐brain axis

AbstractBackgroundThe pathophysiological mechanism underlying the progressive neurodegenerative process in Alzheimer’s disease (AD) is not well understood. Hence, there is no disease‐modifying treatment. It is then imperative to identify the triggering molecular factors of the disease and the mechanisms supporting molecular spreading of toxic assemblies. Our search for these key clues in the periphery is founded on the fact that peripheral dysfunction, particularly in the gastrointestinal (GI) system, can arise decades before the onset of the classic symptoms in neurodegenerative disorders. In fact, the risk of AD increases significantly in patients with inflammatory bowel disease (IBD) when compared with the non‐IBD population. Furthermore, Tau accumulates in Crohn’s Disease guts and in sigmoid colon of AD and FTD patients. Thus, we are investigating the physiological and molecular mechanisms that support Tau spreading in the prodromal stages, particularly, along the gut‐brain axis.MethodTo test whether corrupted proteins expressed in the gut will spread their pathology to the brain, we employed the Gal4/UAS system and expressed wild‐type or mutant human Tau in selected populations of gut epithelium cells. We then monitor behavior on aging flies expressing Tau in the gut epithelium using Drosophila activity monitors and analyzed their circadian patterns of activity along with several sleep parameters.ResultWe observed that flies selectively expressing full‐length‐2N4R human Tau in enteroendocrine cells of the gut exhibited a significant impairment in their daily activity pattern that specifically affected their behavior during the light phase (day) but not during the dark period (night). Interestingly, these flies also showed an increased sleepiness during the day without affecting their nocturnal sleep pattern.ConclusionOur results support the presence of spreading mechanisms for Tau from gut epithelial cells to the brain; hence, affecting the neuronal physiology that sustain daily activity patterns. We are now investigating the pathways and mechanisms that pathological Tau in the gut employs to target these brain regions responsible for the behavioral phenotypes: transsynaptic spreading or humoral signaling via the innate immune system. We are dissecting the 3 major pathways of innate immunity in Drosophila (Imd‐Toll‐TNF) for their role in disease progression and the involvement of their target antimicrobial peptides.

Consequences of Pathogenic Tau Expression in Mouse Locus Coeruleus

AbstractBackgroundDuring the early stages of Alzheimer’s disease (AD), patients often suffer from prodromal symptoms such as anxiety, impulsivity, depression, agitation, and sleep disturbances prior to the onset of cognitive impairment. Incidentally, the brainstem noradrenergic locus coeruleus (LC) is the first structure to develop hyperphosphorylated ‘pretangle’ tau pathology in the human brain. Furthermore, norepinephrine (NE) influences several physiological processes such as sleep/wake cycle, arousal, stress, mood, and attention which have been implicated in the prodromal behavioral abnormalities. While clinical studies show significant correlations between LC dysfunction and neuropsychiatric symptoms, a causal relationship between early tau accumulation in the LC and the manifestations of prodromal behaviors remains to be resolved. To explore this, we developed a translationally‐relevant tau mouse model that recapitulates the ‘LC first’ phenomena commonly observed in humans.MethodAdult male and female tyrosine hydroxylase (TH)‐Cre mice aged 4 months underwent intra‐LC infusions with a Cre‐dependent adeno‐associated virus (AAV) expressing mCherry, wild‐type (WT) human tau or P301S mutant human tau (n = 3/group). 3 months post‐infusion, mice underwent a panel of behavioral tests to assess sleep latency, locomotor activity, compulsivity, stress‐induced anxiety‐like behavior, association memory deficits, impulsivity, and attentional impairments. Following behavioral tests, brain sections comprising the LC, hippocampus and prefrontal cortex (PFC) were immunostained with TH to assess for LC integrity, AT8 to detect pretangle tau, NE transporter (NET) to evaluate NE fiber intensity in projection regions, and GFAP and IBA1 to identify neuroinflammatory markers. Immunoreactivity (IR) will be calculated as a measure of fluorescence via ImageJ.ResultIt has been proposed that tau accumulation in the LC may provoke mild to moderate damage in LC neurons, triggering compensatory mechanisms such as LC hyperactivity. It is hypothesized that at 3 months, LC‐specific tau pathology will result in hyperarousal, compulsivity, anxiety‐like behavior, and impulsivity. In tandem, human tau‐expressing mice are expected to display increased TH, reflecting LC hyperactivity, neuroinflammatory markers and reductions in NE fibers in LC‐projecting regions. These findings will be presented in July 2023.ConclusionThis research will elucidate the role of LC in governing prodromal symptoms. Further studies will be needed to assess molecular mechanisms underlying tau‐mediated LC dysfunction in early AD.

SUMOylation impact on synaptotoxicity of tau oligomers

AbstractBackgroundTau oligomers (oTau) secreted by neurons and astrocytes are linked to the propagation of pathology and negatively affect neuronal activity and viability. oTau‐mediated synaptic loss leads to cognitive deficits and manifests the clinical outcomes of Alzheimer’s disease and related tauopathies. Small Ubiquitin‐like MOdifier (SUMO) proteins regulate multiple cellular events and SUMOylation plays pivotal roles in synaptic biology. Changes in SUMO conjugation and function are also linked to AD and related neurodegenerative disorders such as Huntington’s and Parkinson’s disease. The goal of this investigation is to determine the impact of the two main SUMO isoforms, SUMO1 and SUMO2, on oTau related synaptotoxicity.MethodSUMO1 and SUMO2 transgenic mice were generated with expression regulated by the neuron‐specific prion cos‐tet promoter. These in vivo models were used to generate double transgenic mice expressing human SUMO proteins and P301S mutant Tau. Changes in synaptic density and activity and the effects of oTau pathology were investigated with respect to cognitive deficits and long term potentiation (LTP).ResultElevated expression of human SUMO1 lead to impaired synaptic development and increased tau aggregation. SUMO1 transgenics crossed to Tau mutant mice resulted in an accelerated synaptic loss and a more severe disease phenotype as evidenced by LTP impairments and rapid cognitive decline. Conversely, SUMO2 conjugation levels were decreased in the presence of oTau and phospho‐tau pathology indicating a down‐regulation of its function. Enhanced SUMO2 expression in a mouse model of Tau pathology reversed this process and resulted in a significant reduction in oTau‐mediated synaptotoxicity and the associated LTP and cognitive impairments.ConclusionCumulatively, our findings indicate that SUMO1 negative impacts and exacerbates oTau‐related synaptic dysfunction. In contrast, SUMO2 confers substantial neuroprotection and represents a potential therapeutic avenue to counteract oTau‐induced synaptotoxicity in AD.

TREM2 protects against complement‐mediated synaptic loss via binding to C1q in neurodegeneration

AbstractBackgroundThe triggering receptor expressed on myeloid cells 2 (TREM2) is recognized as the strongest immune‐specific genetic risk factor for Alzheimer’s disease (AD), since its heterozygous R47H variant confers a 3‐4‐fold increased risk of AD. It has been widely acknowledged that TREM2 plays versatile roles in regulating microglial functions by interacting with a variety of ligands.MethodWe performed affinity‐purification coupled to mass spectrometry with the TREM2‐Fc fusion construct to identify new binding partners for TREM2. The binding of TREM2 to new partners was further confirmed by a solid‐phase binding assay, surface plasmon resonance assay, dot blot binding assay and proximity ligation assay. Next, the functional effects of TREM2 binding to new partners were determined both in vitro and in vivo. We further performed serial truncations to determine the amino acid sequence in TREM2 responsible for binding to new partners. Finally, the functional effects of TREM2 peptide binding to new partners were examined in both the physiological and pathological contexts.ResultHere we show that C1q protein, the initiator of the classical complement pathway, is a binding partner for TREM2. TREM2 specifically attenuated the activation of classical complement cascade via high‐affinity binding to C1q in vitro. The formation of TREM2‐C1q complexes was also detected in human AD brain. Remarkably, the increased density of TREM2‐C1q complexes was associated with lower deposition of C3 but higher density of PSD95, pinpointing to a protective role of TREM2 against complement activity in AD brain. In mice expressing mutant human tau, Trem2 haploinsufficiency increased complement‐mediated microglial engulfment of synapses and accelerated synaptic loss with negligible effects on microglial density and tau pathology. We further identified a 41 amino acid sequence in TREM2 responsible for binding to C1q. In vivo administration of this short peptide reduced synapse removal by microglia and rescued synapse density in both the amyloidosis and tauopathy mouse models of AD.ConclusionTogether, our results demonstrate a critical role for microglial TREM2 in restricting complement‐mediated synaptic elimination in neurodegeneration, providing new mechanistic insights into the protective roles of TREM2 against AD pathogenesis.

Basic Science and Pathogenesis - Part 2.

Traumatic Brain Injury (TBI) is a known risk factor for developing a neurodegenerative disease later in life, including Alzheimer's Disease (AD) and other forms of tauopathy such as chronic traumatic encephalopathy (CTE) and frontal-temporal dementia (FTD). Although several studies highlight the relationship between the severity and frequency of a TBI and the development of these tauopathies, the mechanism behind this association remains unknown. Proper meningeal lymphatic function is required for clearance of waste products and protein aggregates such as amyloid beta and tau from the brain parenchyma. While post-TBI inflammation is beneficial for clearing debris and promoting tissue repair, prolonged glial activation and decreased meningeal lymphatic drainage have been linked to worsened cognitive function. A mild TBI (mTBI) was delivered to three-month-old young adult mice overexpressing P301S mutant human tau (PS19) through the skull on the right inferior temporal lobe of the brain. Sham and TBI injured mice were tested on behavioral tests at 5 months of age. At 7 months of age, brains were examined for tau pathology and neuroinflammation using confocal microscopy. Four months after mTBI, the brains of TBI PS19 mice exhibit increased levels of microglia and astrocytes, and increased P-S202/T-205-Tau in the cortex and thalamus of the ipsilateral hemisphere compared to the contralateral side and to Sham PS19 controls. These findings correlate with our behavior study showing increased hyperactivity, risk-taking behavior, and impaired short term working memory. PS19 TBI mice also present larger ventricles than their uninjured controls suggesting a potential loss of white matter in the cortex and/or hippocampus. P-S202/T-205-Tau orginated from the cortex near the injury site can aggregate and promote the formation of more toxic tau that spreads to deeper regions of the cortex and in the thalamus primarily through the thalamocortical circuit. Also, microglia and astrocyte activation and production of inflammatory mediators has been associated with synapse pruning and engulfment of damaged neurons expressing 'eat me signals' leading to neurodegeneration.

Inhibition of EV biogenesis reduces tau propagation in Alzheimer’s Disease mouse models

AbstractBackgroundMounting evidence correlates the propagation of hyperphosphorylated tau (pTau) along synaptically connected networks in the brain with progressive cognitive decline in Alzheimer’s Disease (AD). Recent findings have highlighted extracellular vesicle (EV)s in enabling transcellular transmission of pathological tau and identified the partial inhibition of EV biogenesis via small‐molecule inhibitors of nSMase2 as a potential therapeutic avenue. However, there are no suitable compounds for clinical development so far.MethodThrough high‐throughput screening, we identified PDDC, a highly selective and potent nSMase2 inhibitor with excellent brain penetration and oral bioavailability. To evaluate PDDC’s effect on tau propagation in vivo, we chronically administered PDDC‐containing chow to PS19 transgenic mice and measured brain ceramides, nSMase2 activity, tau levels, glial activation, hippocampal neuronal cell layer thickness, and mossy fiber synaptophysin staining. In addition, neuronally‐derived EVs from plasma were isolated and characterized. To directly monitor the effect of PDDC on tau propagation, we developed a murine model where an AAV encoding for P301L/S320F double mutant human tau was stereotaxically‐injected unilaterally into the hippocampus and transfer to the contralateral dentate gyrus (DG) was monitored ± PDDC treatment.ResultPS19 mice exhibited robust elevation of multiple ceramide species and nSMase2 enzymatic activity in the brain, both of which were normalized by PDDC treatment. PS19 mice treated with PDDC had significantly reduced hippocampal total tau and Thr181 pTau, reduced glial activation, protected synapses, and improved pyramidal and granule cell layer thickness. Plasma NEVs of treated mice were fewer in number and had lower p181‐Tau levels than the untreated group; FCA confirmed the decrease of NEVs carrying p262‐Tau at the single EV level. Similarly, the AAV‐hTau‐seeded mice treated with PDDC had reduced tau staining intensity in the contralateral DG.ConclusionData in two AD models using PDDC provides strong preclinical support for using nSMase2 inhibition as a therapeutic strategy to slow tau propagation in AD.

Modulation of cannabinoid receptor‐2 alters brain clearance of human tau

AbstractBackgroundMultiple studies have demonstrated a strong neuroinflammatory response in the presence of Tau pathology, which is strongly associated with the clinical symptoms and cognitive decline found in Alzheimer’s disease (AD). The correlation between AD progression and tau pathologies, rather than Ab accumulation, suggests that targeting pathological tau may be a more effective therapeutic approach. Microglia have been implicated in tauopathies as they may contribute to tau phosphorylation and aggregation and the uptake and spread of seed‐competent tau to healthy neurons due to dysfunctional degradation. Cannabinoid type 2 receptors (CB2) are highly expressed in immune cells and upregulated in activated microglia under conditions of neurologic disease, such as AD. The pharmacological modulation of CB2 have demonstrated a reduction in inflammation and plaque deposition, suggesting a role of CB2 on the immune system to influence AD‐related pathologies and phenotypes. However, there are limited findings on the CB2 regulation of tau accumulation.MethodWe have generated organotypic brain slice cultures from CB2‐EGFP/f/f mice and transduced with rAAV‐P301L/S320F‐human tau, which previously has shown to accumulate total tau and develop insoluble tau species. Slices were treated with CB2‐selective compounds and evaluated for tau pathology and microglial phenotypes using real‐time imaging and biochemical analyses.ResultPrevious investigation in our lab of a synucleinopathy animal model revealed the capacity of CB2 signaling to alter the function of immune cells and reduce accumulation of phosphorylated human alpha‐synuclein. Furthermore, we have determined that treatment with a CB2 inverse agonist will increase phagocytosis of pHrodo E. coli bioparticles in microglia BV2 cultures stimulated by LPS. Our preliminary work in brain slices over‐expressing mutant human tau show a trend toward reduced ratio of phosphorylated tau (Ser202/Thr205) to total tau in the soluble protein fraction (p = 0.0549) in slices treated with CB2 inverse agonist SR144528 compared to vehicle. We are repeating the experiment and performing further evaluations in organotypic brain slice cultures from CB2‐KO mice.ConclusionThese studies will advance our understanding of the role of brain CB2 in pathologic tau removal and potentially highlight CB2 as a therapeutic target against tauopathies such as AD.

Neuronal tau pathology worsens late-phase white matter degeneration after traumatic brain injury in transgenic mice.

Traumatic brain injury (TBI) causes diffuse axonal injury which can produce chronic white matter pathology and subsequent post-traumatic neurodegeneration with poor patient outcomes. Tau modulates axon cytoskeletal functions and undergoes phosphorylation and mis-localization in neurodegenerative disorders. The effects of tau pathology on neurodegeneration after TBI are unclear. We used mice with neuronal expression of human mutant tau to examine effects of pathological tau on white matter pathology after TBI. Adult male and female hTau.P301S (Tg2541) transgenic and wild-type (Wt) mice received either moderate single TBI (s-TBI) or repetitive mild TBI (r-mTBI; once daily × 5), or sham procedures. Acutely, s-TBI produced more extensive axon damage in the corpus callosum (CC) as compared to r-mTBI. After s-TBI, significant CC thinning was present at 6weeks and 4months post-injury in Wt and transgenic mice, with homozygous tau expression producing additional pathology of late demyelination. In contrast, r-mTBI did not produce significant CC thinning except at the chronic time point of 4months in homozygous mice, which exhibited significant CC atrophy (- 29.7%) with increased microgliosis. Serum neurofilament light quantification detected traumatic axonal injury at 1 day post-TBI in Wt and homozygous mice. At 4months, high tau and neurofilament in homozygous mice implicated tau in chronic axon pathology. These findings did not have sex differences detected. Conclusions: Neuronal tau pathology differentially exacerbated CC pathology based on injury severity and chronicity. Ongoing CC atrophy from s-TBI became accompanied by late demyelination. Pathological tau significantly worsened CC atrophy during the chronic phase after r-mTBI.

TREM2 receptor protects against complement-mediated synaptic loss by binding to complement C1q during neurodegeneration

Triggering receptor expressed on myeloid cells 2 (TREM2) is strongly linked to Alzheimer's disease (AD) risk, but its functions are not fully understood. Here, we found that TREM2 specifically attenuated the activation of classical complement cascade via high-affinity binding to its initiator C1q. In the human AD brains, the formation of TREM2-C1q complexes was detected, and the increased density of the complexes was associated with lower deposition of C3 but higher amounts of synaptic proteins. In mice expressing mutant human tau, Trem2 haploinsufficiency increased complement-mediated microglial engulfment of synapses and accelerated synaptic loss. Administration of a 41-amino-acid TREM2 peptide, which we identified to be responsible for TREM2 binding to C1q, rescued synaptic impairments in AD mouse models. We thus demonstrate a critical role for microglial TREM2 in restricting complement-mediated synaptic elimination during neurodegeneration, providing mechanistic insights into the protective roles of TREM2 against AD pathogenesis.

Humanized APOE genotypes influence lifespan independently of tau aggregation in the P301S mouse model of tauopathy

Apolipoprotein (APOE) E4 isoform is a major risk factor of Alzheimer’s disease and contributes to metabolic and neuropathological abnormalities during brain aging. To provide insights into whether APOE4 genotype is related to tau-associated neurodegeneration, we have generated human P301S mutant tau transgenic mice (PS19) that carry humanized APOE alleles (APOE2, APOE3 or APOE4). In aging mice that succumbed to paralysis, PS19 mice homozygous for APOE3 had the longest lifespan when compared to APOE4 and APOE2 homozygous mice (APOE3 > APOE4 ~ APOE2). Heterozygous mice with one human APOE and one mouse Apoe allele did not show any variations in lifespan. At end-stage, PS19 mice homozygous for APOE3 and APOE4 showed equivalent levels of phosphorylated tau burden, inflammation levels and ventricular volumes. Compared to these cohorts, PS19 mice homozygous for APOE2 showed lower induction of phosphorylation on selective epitopes, though the effect sizes were small and variable. In spite of this, the APOE2 cohort showed shorter lifespan relative to APOE3 homozygous mice. None of the cohorts accumulated appreciable levels of phosphorylated tau compartmentalized in the insoluble cell fraction. RNAseq analysis showed that the induction of immune gene expression was comparable across all the APOE genotypes in PS19 mice. Notably, the APOE4 homozygous mice showed additional induction of transcripts corresponding to the Alzheimer’s disease-related plaque-induced gene signature. In human Alzheimer’s disease brain tissues, we found no direct correlation between higher burden of phosphorylated tau and APOE4 genotype. As expected, there was a strong correlation between phosphorylated tau burden with amyloid deposition in APOE4-positive Alzheimer’s disease cases. Overall, our results indicate that APOE3 genotype may confer some resilience to tauopathy, while APOE4 and APOE2 may act through multiple pathways to increase the pathogenicity in the context of tauopathy.

Effect of Nrf2 loss on senescence and cognition of tau-based P301S mice.

Cellular senescence may contribute to chronic inflammation involved in the progression of age-related diseases such as Alzheimer's disease (AD), and its removal prevents cognitive impairment in a model of tauopathy. Nrf2, the major transcription factor for damage response pathways and regulators of inflammation, declines with age. Our previous work showed that silencing Nrf2 gives rise to premature senescence in cells and mice. Others have shown that Nrf2 ablation can exacerbate cognitive phenotypes of some AD models. In this study, we aimed to understand the relationship between Nrf2 elimination, senescence, and cognitive impairment in AD, by generating a mouse model expressing a mutant human tau transgene in an Nrf2 knockout (Nrf2KO) background. We assessed senescent cell burden and cognitive decline of P301S mice in the presence and absence of Nrf2. Lastly, we administered 4.5-month-long treatments with two senotherapeutic drugs to analyze their potential to prevent senescent cell burden and cognitive decline: the senolytic drugs dasatinib and quercetin (DQ) and the senomorphic drug rapamycin. Nrf2 loss accelerated the onset of hind-limb paralysis in P301S mice. At 8.5months of age, P301S mice did not exhibit memory deficits, while P301S mice without Nrf2 were significantly impaired. However, markers of senescence were not elevated by Nrf2 ablation in any of tissues that we examined. Neither drug treatment improved cognitive performance, nor did it reduce expression of senescence markers in brains of P301S mice. Contrarily, rapamycin treatment at the doses used delayed spatial learning and led to a modest decrease in spatial memory. Taken together, our data suggests that the emergence of senescence may be causally associated with onset of cognitive decline in the P301S model, indicate that Nrf2 protects brain function in a model of AD through mechanisms that may include, but do not require the inhibition of senescence, and suggest possible limitations for DQ and rapamycin as therapies for AD.