Alzheimer's disease-linked risk alleles elevate microglial cGAS-associated senescence and neurodegeneration in a tauopathy model.

The strongest risk factors for late-onset sporadic Alzheimer's disease (AD) include the ε4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H (R47H) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting detrimental disease mechanisms. We find that R47H induces neurodegeneration in 9- to 10-month-old female APOE4 tauopathy mice. The combination of APOE4 and R47H (APOE4-R47H) worsened hyperphosphorylated tau pathology in the frontal cortex and amplified tauopathy-induced microglial cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling and downstream interferon response. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties.

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

APOE3-R136S mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition.

The Christchurch mutation (R136S) on the APOE3 (E3S/S) gene is associated with low tau pathology and slowdown of cognitive decline despite the causal PSEN1 mutation and high levels of amyloid beta pathology in the carrier1. However, the molecular effects enabling E3S/S mutation to confer protection remain unclear. Here, we replaced mouse Apoe with wild-type human E3 or E3S/S on a tauopathy background. The R136S mutation markedly mitigated tau load and protected against tau-induced synaptic loss, myelin loss, and spatial learning. Additionally, the R136S mutation reduced microglial interferon response to tau pathology both in vivo and in vitro, suppressing cGAS-STING activation. Treating tauopathy mice carrying wild-type E3 with cGAS inhibitor protected against tau-induced synaptic loss and induced similar transcriptomic alterations to those induced by the R136S mutation across brain cell types. Thus, cGAS-STING-IFN inhibition recapitulates the protective effects of R136S against tauopathy.

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

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

Alzheimer's disease-linked risk alleles elevate microglial cGAS-associated senescence and neurodegeneration in a tauopathy model.

The strongest risk factors for Alzheimer's disease (AD) include the χ4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H ( R47H ) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting disease-causing mechanisms. We find that the R47H variant induces neurodegeneration in female APOE4 mice without impacting hippocampal tau load. The combination of APOE4 and R47H amplified tauopathy-induced cell-autonomous microglial cGAS-STING signaling and type-I interferon response, and interferon signaling converged across glial cell types in the hippocampus. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties.

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. We recently reported a family with a paternally inherited intragenic ASTN2 duplication with a range of neurodevelopmental disorders, including autism spectrum disorder (ASD), learning difficulties, and speech and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity and repetitive behaviors, altered social behaviors, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors were also prominent in Astn2 cKO animals. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrates a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

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

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to lack of appropriate human models. Current human induced pluripotent stem cell (hiPSC)-derived neurons express very low levels of 4-repeat (4R)-tau isoforms that are normally expressed in adult brain. Here, we engineered new iPSC lines to express 4R-tau and 4R-tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes, including shared transcriptomic signatures, autophagic body accumulation, and impaired neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of Tau-seeding-induced Tau propagation, including retromer VPS29 and the UFMylation cascade as top modifiers. In AD brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade suppressed seeding-induced Tau propagation. This model provides a powerful platform to identify novel therapeutic strategies for 4R tauopathy.

Tau activation of microglial cGAS-IFN reduces MEF2C-mediated cognitive resilience.

Pathological hallmarks of Alzheimer's disease (AD) precede clinical symptoms by years, indicating a period of cognitive resilience before the onset of dementia. Here, we report that activation of cyclic GMP-AMP synthase (cGAS) diminishes cognitive resilience by decreasing the neuronal transcriptional network of myocyte enhancer factor 2c (MEF2C) through type I interferon (IFN-I) signaling. Pathogenic tau activates cGAS and IFN-I responses in microglia, in part mediated by cytosolic leakage of mitochondrial DNA. Genetic ablation of Cgas in mice with tauopathy diminished the microglial IFN-I response, preserved synapse integrity and plasticity and protected against cognitive impairment without affecting the pathogenic tau load. cGAS ablation increased, while activation of IFN-I decreased, the neuronal MEF2C expression network linked to cognitive resilience in AD. Pharmacological inhibition of cGAS in mice with tauopathy enhanced the neuronal MEF2C transcriptional network and restored synaptic integrity, plasticity and memory, supporting the therapeutic potential of targeting the cGAS-IFN-MEF2C axis to improve resilience against AD-related pathological insults.

The Purkinje-myocardial junction is the anatomic origin of ventricular arrhythmia in CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

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.

AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation.

The hemizygous R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific gene in the brain, increases risk for late-onset Alzheimer's disease (AD). Using transcriptomic analysis of single nuclei from brain tissues of patients with AD carrying the R47H mutation or the common variant (CV)­TREM2, we found that R47H-associated microglial subpopulations had enhanced inflammatory signatures reminiscent of previously identified disease-associated microglia (DAM) and hyperactivation of AKT, one of the signaling pathways downstream of TREM2. We established a tauopathy mouse model with heterozygous knock-in of the human TREM2 with the R47H mutation or CV and found that R47H induced and exacerbated TAU-mediated spatial memory deficits in female mice. Single-cell transcriptomic analysis of microglia from these mice also revealed transcriptomic changes induced by R47H that had substantial overlaps with R47H microglia in human AD brains, including robust increases in proinflammatory cytokines, activation of AKT signaling, and elevation of a subset of DAM signatures. Pharmacological AKT inhibition with MK-2206 largely reversed the enhanced inflammatory signatures in primary R47H microglia treated with TAU fibrils. In R47H heterozygous tauopathy mice, MK-2206 treatment abolished a tauopathy-dependent microglial subcluster and rescued tauopathy-induced synapse loss. By uncovering disease-enhancing mechanisms of the R47H mutation conserved in human and mouse, our study supports inhibitors of AKT signaling as a microglial modulating strategy to treat AD.

Parallel ascending spinal pathways for affective touch and pain.

The anterolateral pathway consists of ascending spinal tracts that convey pain, temperature and touch information from the spinal cord to the brain1-4. Projection neurons of the anterolateral pathway are attractive therapeutic targets for pain treatment because nociceptive signals emanating from the periphery are channelled through these spinal projection neurons en route to the brain. However, the organizational logic of the anterolateral pathway remains poorly understood. Here we show that two populations of projection neurons that express the structurally related G-protein-coupled receptors (GPCRs) TACR1 and GPR83 form parallel ascending circuit modules that cooperate to convey thermal, tactile and noxious cutaneous signals from the spinal cord to the lateral parabrachial nucleus of the pons. Within this nucleus, axons of spinoparabrachial (SPB) neurons that express Tacr1 or Gpr83 innervate distinct sets of subnuclei, and strong optogenetic stimulation of the axon terminals induces distinct escape behaviours and autonomic responses. Moreover, SPB neurons that  express Gpr83 are highly sensitive to cutaneous mechanical stimuli and receive strong synaptic inputs from both high- and low-threshold primary mechanosensory neurons. Notably, the valence associated with activation of SPB neurons that express Gpr83 can be either positive or negative, depending on stimulus intensity. These findings reveal anatomically, physiologically and functionally distinct subdivisions of the SPB tract that underlie affective aspects of touch and pain.

Growth of Saccharomyces cerevisiae and Preparation of DNA.

This protocol describes methods for isolation of total DNA from a strain of Sacchromyces cerevisiae carrying a recombinant yeast artificial chromosome (YAC). This method is appropriate for preparing DNA that will be subjected to regular agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, polymerase chain reaction (PCR), or other methods that do not require intact high-molecular-weight DNA. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. Drop dialysis should be used to exchange buffers. The expected yield from a 10-mL culture is 2-4 µg of yeast DNA.

Working with Bacterial Artificial Chromosomes (BACs) and Other High-Capacity Vectors.

Genetic targeting of specific cell types is fundamentally important for modern molecular-genetic studies. The development of simple methods to engineer high-capacity vectors-in particular, bacterial artificial chromosomes (BACs)-for the preparation of transgenic lines that accurately express a gene of interest has resulted in commonplace usage of transgenic techniques in a wide variety of experimental systems. Here we provide a brief description of each of the four major types of large-capacity vectors, with a focus on the use of BAC vectors.

Small-Scale Preparations of Yeast DNA.

In this protocol, yeast DNA is prepared by digestion of the cell wall and lysis of the resulting spheroplasts with SDS. This method reproducibly yields several micrograms of yeast DNA that can be efficiently cleaved by restriction enzymes and used as a template in polymerase chain reaction (PCR). Note that yeast colonies can also be used directly in PCR, without purifying yeast DNA.

One-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of Plasmids.

In the one-step approach to bacterial artificial chromosome (BAC) modification, two plasmids are introduced into the BAC host cells. The shuttle pLD53.SC2, carrying the EFGP reporter sequence and requiring the π protein to replicate, must be grown in PIR1- or PIR2-competent Escherichia coli Our preference for these vectors is PIR1, because these cells are able to maintain about 250 copies of the donor vector. This small-sized vector is stable in PIR1. The RecA plasmid pSV1.RecA has a temperature-sensitive origin of replication and can be grown in most competent bacteria at 30°C; here we use DH5α competent cells. This protocol describes preparation of the vector DNAs. The shuttle-reporter vector DNA is subsequently digested for introduction of one homology arm (typically the A-box).

One-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of the A Homology Arm (A-Box).

The one-step approach to bacterial artificial chromosome (BAC) modification requires that only one homology arm be cloned into the shuttle vector (in the example presented here, we use the "A-box"). The homology arm, which in this case lies upstream of the ATG start codon, is amplified by polymerase chain reaction (PCR) using purified BAC DNA as template. The resulting amplification product is then digested with the appropriate restriction endonuclease to render it suitable for cloning into the shuttle vector.

One-Step Bacterial Artificial Chromosome (BAC) Modification: Transformation of the BAC Host with the RecA Vector.

This protocol outlines the steps for introducing the RecA plasmid into bacterial artificial chromosome (BAC) host cells, and their preparation for subsequent transformation with the reporter plasmid for one-step BAC modification. BAC host cells are rendered chemically competent and transformed with the RecA plasmid, and transformants are selected for tetracycline resistance to ensure the presence of the RecA marker.

One-Step Bacterial Artificial Chromosome (BAC) Modification: Cloning of the A Homology Arm into Reporter-Shuttle Vector.

In this protocol, the homology arm sequence for one-step bacterial artificial chromosome (BAC) modification is introduced by ligation into the shuttle vector carrying the reporter sequence to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector. To provide further assurance that the homology box has been successfully integrated into the plasmid, the enzyme digestion pattern of the modified plasmid is compared with that of the unmodified plasmid.

One-Step Bacterial Artificial Chromosome (BAC) Modification: Transfer of the Reporter Vector into BAC/RecA Cells and Selection of Co-Integrates.

In one-step bacterial artificial chromosome (BAC) modification, recombination of the reporter vector with the BAC-leading to the modified BAC-is facilitated by the presence of RecA. Recombinants are selected for by growth in the presence of chloramphenicol, ampicillin, and tetracycline. Only bacteria containing correctly modified BACs and copies of pSV1.RecA will be selected. Unmodified BACs (i.e., those lacking a pLD53.SC2/A-box insert) are eliminated by exposure to ampicillin. Free reporter plasmid remaining in the BAC host bacteria will also be eliminated, because this vector requires the π protein to replicate. The co-integrates are selected by growth at high temperature, thereby eliminating the RecA plasmid. Successful modification of the BAC-formation of the co-integrate-is confirmed in separate amplification reactions.