Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration.

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.

Acetylated tau inhibits chaperone-mediated autophagy and promotes tau pathology propagation in mice.

Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression.

A MAC2-positive progenitor-like microglial population is resistant to CSF1R inhibition in adult mouse brain.

Microglia are the resident myeloid cells in the central nervous system (CNS). The majority of microglia rely on CSF1R signaling for survival. However, a small subset of microglia in mouse brains can survive without CSF1R signaling and reestablish the microglial homeostatic population after CSF1R signaling returns. Using single-cell transcriptomic analysis, we characterized the heterogeneous microglial populations under CSF1R inhibition, including microglia with reduced homeostatic markers and elevated markers of inflammatory chemokines and proliferation. Importantly, MAC2/Lgals3 was upregulated under CSF1R inhibition, and shared striking similarities with microglial progenitors in the yolk sac and immature microglia in early embryos. Lineage-tracing studies revealed that these MAC2+ cells were of microglial origin. MAC2+ microglia were also present in non-treated adult mouse brains and exhibited immature transcriptomic signatures indistinguishable from those that survived CSF1R inhibition, supporting the notion that MAC2+ progenitor-like cells are present among adult microglia.

A Comprehensive Resource for Induced Pluripotent Stem Cells from Patients with Primary Tauopathies.

Primary tauopathies are characterized neuropathologically by inclusions containing abnormal forms of the microtubule-associated protein tau (MAPT) and clinically by diverse neuropsychiatric, cognitive, and motor impairments. Autosomal dominant mutations in the MAPT gene cause heterogeneous forms of frontotemporal lobar degeneration with tauopathy (FTLD-Tau). Common and rare variants in the MAPT gene increase the risk for sporadic FTLD-Tau, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We generated a collection of fibroblasts from 140 MAPT mutation/risk variant carriers, PSP, CBD, and cognitively normal controls; 31 induced pluripotent stem cell (iPSC) lines from MAPT mutation carriers, non-carrier family members, and autopsy-confirmed PSP patients; 33 genome engineered iPSCs that were corrected or mutagenized; and forebrain neural progenitor cells (NPCs). Here, we present a resource of fibroblasts, iPSCs, and NPCs with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for tauopathies.

Pathogenic Tau Impairs Axon Initial Segment Plasticity and Excitability Homeostasis.

Dysregulation of neuronal excitability underlies the pathogenesis of tauopathies, including frontotemporal dementia (FTD) with tau inclusions. A majority of FTD-causing tau mutations are located in the microtubule-binding domain, but how these mutations alter neuronal excitability is largely unknown. Here, using CRISPR/Cas9-based gene editing in human pluripotent stem cell (iPSC)-derived neurons and isogenic controls, we show that the FTD-causing V337M tau mutation impairs activity-dependent plasticity of the cytoskeleton in the axon initial segment (AIS). Extracellular recordings by multi-electrode arrays (MEAs) revealed that the V337M tau mutation in human neurons leads to an abnormal increase in neuronal activity in response to chronic depolarization. Stochastic optical reconstruction microscopy of human neurons with this mutation showed that AIS plasticity is impaired by the abnormal accumulation of end-binding protein 3 (EB3) in the AIS submembrane region. These findings expand our understanding of how FTD-causing tau mutations dysregulate components of the neuronal cytoskeleton, leading to network dysfunction.

SIRT1 Deacetylates Tau and Reduces Pathogenic Tau Spread in a Mouse Model of Tauopathy.

Hyperacetylation of tau has been implicated in neurodegeneration and cognitive decline in tauopathy brains. The nicotinamide adenosine dinucleotide-dependent class-III protein deacetylase SIRT1 is one of the major enzymes involved in removal of acetyl groups from tau in vitro However, whether SIRT1 regulates acetylation of pathogenic tau and ameliorates tau-mediated pathogenesis remains unclear. Here, we report deacetylating activity of SIRT1 for acetylated Lys174 (K174) of tau in tauP301S transgenic mice with a brain-specific SIRT1 deletion. We show that SIRT1 deficiency leads to exacerbation of premature mortality, synapse loss, and behavioral disinhibition in tauP301S transgenic mice of both sexes. By contrast, SIRT1 overexpression by stereotaxic delivery of adeno-associated virus that encodes SIRT1 into the hippocampus reduces acetylated K174 tau. Furthermore, SIRT1 overexpression significantly attenuates the spread of tau pathology into anatomically connected brain regions of tauP301S transgenic mice of both sexes. These findings suggest the functional importance of SIRT1 in regulating pathogenic tau acetylation and in suppressing the spread of tau pathology in vivoSIGNIFICANCE STATEMENT In neurodegenerative disorders with inclusions of microtubule-associated protein tau, aberrant lysine acetylation of tau plays critical roles in promoting tau accumulation and toxicity. Identifying strategies to deacetylate tau could interfere with disease progression; however, little is known about how pathogenic tau is deacetylated in vivo Here we show that the protein deacetylase SIRT1 reduces tau acetylation in a mouse model of neurodegeneration. SIRT1 deficiency in the brain aggravates synapse loss and behavioral disinhibition, and SIRT1 overexpression ameliorates propagation of tau pathology.

Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening.

Lowering total tau levels is an attractive therapeutic strategy for Alzheimer's disease and other tauopathies. High-throughput screening in neurons derived from human induced pluripotent stem cells (iPSCs) is a powerful tool to identify tau-targeted therapeutics. However, such screens have been hampered by heterogeneous neuronal production, high cost and low yield, and multi-step differentiation procedures. We engineered an isogenic iPSC line that harbors an inducible neurogenin 2 transgene, a transcription factor that rapidly converts iPSCs to neurons, integrated at the AAVS1 locus. Using a simplified two-step protocol, we differentiated these iPSCs into cortical glutamatergic neurons with minimal well-to-well variability. We developed a robust high-content screening assay to identify tau-lowering compounds in LOPAC and identified adrenergic receptors agonists as a class of compounds that reduce endogenous human tau. These techniques enable the use of human neurons for high-throughput screening of drugs to treat neurodegenerative disease.

Acetylated tau destabilizes the cytoskeleton in the axon initial segment and is mislocalized to the somatodendritic compartment.

BACKGROUND: Neurons are highly polarized cells in which asymmetric axonal-dendritic distribution of proteins is crucial for neuronal function. Loss of polarized distribution of the axonal protein tau is an early sign of Alzheimer's disease (AD) and other neurodegenerative disorders. The cytoskeletal network in the axon initial segment (AIS) forms a barrier between the axon and the somatodentritic compartment, contributing to axonal retention of tau. Although perturbation of the AIS cytoskeleton has been implicated in neurological disorders, the molecular triggers and functional consequence of AIS perturbation are incompletely understood. RESULTS: Here we report that tau acetylation and consequent destabilization of the AIS cytoskeleton promote the somatodendritic mislocalization of tau. AIS cytoskeletal proteins, including ankyrin G and ßIV-spectrin, were downregulated in AD brains and negatively correlated with an increase in tau acetylated at K274 and K281. AIS proteins were also diminished in transgenic mice expressing tauK274/281Q, a tau mutant that mimics K274 and K281 acetylation. In primary neuronal cultures, the tauK274/281Q mutant caused hyperdynamic microtubules (MTs) in the AIS, shown by live-imaging of MT mobility and fluorescence recovery after photobleaching. Using photoconvertible tau constructs, we found that axonal tauK274/281Q was missorted into the somatodendritic compartment. Stabilizing MTs with epothilone D to restore the cytoskeletal barrier in the AIS prevented tau mislocalization in primary neuronal cultures. CONCLUSIONS: Together, these findings demonstrate that tau acetylation contributes to the pathogenesis of neurodegenerative disease by compromising the cytoskeletal sorting machinery in the AIS.

Acetylated Tau Obstructs KIBRA-Mediated Signaling in Synaptic Plasticity and Promotes Tauopathy-Related Memory Loss.

Tau toxicity has been implicated in the emergence of synaptic dysfunction in Alzheimer's disease (AD), but the mechanism by which tau alters synapse physiology and leads to cognitive decline is unclear. Here we report abnormal acetylation of K274 and K281 on tau, identified in AD brains, promotes memory loss and disrupts synaptic plasticity by reducing postsynaptic KIdney/BRAin (KIBRA) protein, a memory-associated protein. Transgenic mice expressing human tau with lysine-to-glutamine mutations to mimic K274 and K281 acetylation (tauKQ) exhibit AD-related memory deficits and impaired hippocampal long-term potentiation (LTP). TauKQ reduces synaptic KIBRA levels and disrupts activity-induced postsynaptic actin remodeling and AMPA receptor insertion. The LTP deficit was rescued by promoting actin polymerization or by KIBRA expression. In AD patients with dementia, we found enhanced tau acetylation is linked to loss of KIBRA. These findings suggest a novel mechanism by which pathogenic tau causes synaptic dysfunction and cognitive decline in AD pathogenesis.

Critical role of acetylation in tau-mediated neurodegeneration and cognitive deficits.

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer's disease (AD), are neurodegenerative diseases in which tau fibrils accumulate. Recent evidence supports soluble tau species as the major toxic species. How soluble tau accumulates and causes neurodegeneration remains unclear. Here we identify tau acetylation at Lys174 (K174) as an early change in AD brains and a critical determinant in tau homeostasis and toxicity in mice. The acetyl-mimicking mutant K174Q slows tau turnover and induces cognitive deficits in vivo. Acetyltransferase p300-induced tau acetylation is inhibited by salsalate and salicylate, which enhance tau turnover and reduce tau levels. In the PS19 transgenic mouse model of FTD, administration of salsalate after disease onset inhibited p300 activity, lowered levels of total tau and tau acetylated at K174, rescued tau-induced memory deficits and prevented hippocampal atrophy. The tau-lowering and protective effects of salsalate were diminished in neurons expressing K174Q tau. Targeting tau acetylation could be a new therapeutic strategy against human tauopathies.

SIRT1 deficiency in microglia contributes to cognitive decline in aging and neurodegeneration via epigenetic regulation of IL-1ß.

Aging is the predominant risk factor for neurodegenerative diseases. One key phenotype as the brain ages is an aberrant innate immune response characterized by proinflammation. However, the molecular mechanisms underlying aging-associated proinflammation are poorly defined. Whether chronic inflammation plays a causal role in cognitive decline in aging and neurodegeneration has not been established. Here we report a mechanistic link between chronic inflammation and aging microglia and a causal role of aging microglia in neurodegenerative cognitive deficits. We showed that SIRT1 is reduced with the aging of microglia and that microglial SIRT1 deficiency has a causative role in aging- or tau-mediated memory deficits via IL-1ß upregulation in mice. Interestingly, the selective activation of IL-1ß transcription by SIRT1 deficiency is likely mediated through hypomethylating the specific CpG sites on IL-1ß proximal promoter. In humans, hypomethylation of IL-1ß is strongly associated with chronological age and with elevated IL-1ß transcription. Our findings reveal a novel epigenetic mechanism in aging microglia that contributes to cognitive deficits in aging and neurodegenerative diseases.

Argyrophilic grain disease differs from other tauopathies by lacking tau acetylation.

Post-translational modifications play a key role in tau protein aggregation and related neurodegeneration. Because hyperphosphorylation alone does not necessarily cause tau aggregation, other post-translational modifications have been recently explored. Tau acetylation promotes aggregation and inhibits tau's ability to stabilize microtubules. Recent studies have shown co-localization of acetylated and phosphorylated tau in AD and some 4R tauopathies. We developed a novel monoclonal antibody against acetylated tau at lysine residue 274, which recognizes both 3R and 4R tau, and used immunohistochemistry and immunofluorescence to probe 22 cases, including AD and another eight familial or sporadic tauopathies. Acetylated tau was identified in all tauopathies except argyrophilic grain disease (AGD). AGD is an age-associated, common but atypical 4R tauopathy, not always associated with clinical progression. Pathologically, AGD is characterized by neuropil grains, pre-neurofibrillary tangles, and oligodendroglial coiled bodies, all recognized by phospho-tau antibodies. The lack of acetylated tau in these inclusions suggests that AGD represents a distinctive tauopathy. Our data converge with previous findings to raise the hypothesis that AGD could play a protective role against the spread of AD-related tau pathology. Tau acetylation as a key modification for the propagation tau toxicity deserves further investigation.