PQBP3 prevents senescence by suppressing PSME3-mediated proteasomal Lamin B1 degradation.

Senescence of nondividing neurons remains an immature concept, with especially the regulatory molecular mechanisms of senescence-like phenotypes and the role of proteins associated with neurodegenerative diseases in triggering neuronal senescence remaining poorly explored. In this study, we reveal that the nucleolar polyglutamine binding protein 3 (PQBP3; also termed NOL7), which has been linked to polyQ neurodegenerative diseases, regulates senescence as a gatekeeper of cytoplasmic DNA leakage. PQBP3 directly binds PSME3 (proteasome activator complex subunit 3), a subunit of the 11S proteasome regulator complex, decreasing PSME3 interaction with Lamin B1 and thereby preventing Lamin B1 degradation and senescence. Depletion of endogenous PQBP3 causes nuclear membrane instability and release of genomic DNA from the nucleus to the cytosol. Among multiple tested polyQ proteins, ataxin-1 (ATXN1) partially sequesters PQBP3 to inclusion bodies, reducing nucleolar PQBP3 levels. Consistently, knock-in mice expressing mutant Atxn1 exhibit decreased nuclear PQBP3 and a senescence phenotype in Purkinje cells of the cerebellum. Collectively, these results suggest homologous roles of the nucleolar protein PQBP3 in cellular senescence and neurodegeneration.

Mutant α-synuclein propagates via the lymphatic system of the brain in the monomeric state.

Prion-like protein propagation is considered a common pathogenic mechanism in neurodegenerative diseases. Here we investigate the in vivo propagation pattern and aggregation state of mutant α-synuclein by injecting adeno-associated viral (AAV)-α-synuclein-A53T-EGFP into the mouse olfactory cortex. Comparison of aggregation states in various brain regions at multiple time points after injection using western blot analyses shows that the monomeric state of the mutant/misfolded protein propagates to remote brain regions by 2 weeks and that the propagated proteins aggregate in situ after being incorporated into neurons. Moreover, injection of Alexa 488-labeled α-synuclein-A53T confirms the monomeric propagation at 2 weeks. Super-resolution microscopy shows that both α-synuclein-A53T proteins propagate via the lymphatic system, penetrate perineuronal nets, and reach the surface of neurons. Electron microscopy shows that the propagated mutant/misfolded monomer forms fibrils characteristic of Parkinson's disease after its incorporation into neurons. These findings suggest a mode of propagation different from that of aggregate-dependent propagation.

PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions.

Polyglutamine binding protein 5 (PQBP5), also called nucleolar protein 10 (NOL10), binds to polyglutamine tract sequences and is expressed in the nucleolus. Using dynamic imaging of high-speed atomic force microscopy, we show that PQBP5/NOL10 is an intrinsically disordered protein. Super-resolution microscopy and correlative light and electron microscopy method show that PQBP5/NOL10 makes up the skeletal structure of the nucleolus, constituting the granule meshwork in the granular component area, which is distinct from other nucleolar substructures, such as the fibrillar center and dense fibrillar component. In contrast to other nucleolar proteins, which disperse to the nucleoplasm under osmotic stress conditions, PQBP5/NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins. Droplet and thermal shift assays show that the biophysical features of PQBP5/NOL10 remain stable under stress conditions, explaining the spatial role of this protein. PQBP5/NOL10 can be functionally depleted by sequestration with polyglutamine disease proteins in vitro and in vivo, leading to the pathological deformity or disappearance of the nucleolus. Taken together, these findings indicate that PQBP5/NOL10 is an essential protein needed to maintain the structure of the nucleolus.

Tau activates microglia via the PQBP1-cGAS-STING pathway to promote brain inflammation.

Brain inflammation generally accompanies and accelerates neurodegeneration. Here we report a microglial mechanism in which polyglutamine binding protein 1 (PQBP1) senses extrinsic tau 3R/4R proteins by direct interaction and triggers an innate immune response by activating a cyclic GMP-AMP synthase (cGAS)-Stimulator of interferon genes (STING) pathway. Tamoxifen-inducible and microglia-specific depletion of PQBP1 in primary culture in vitro and mouse brain in vivo shows that PQBP1 is essential for sensing-tau to induce nuclear translocation of nuclear factor κB (NFκB), NFκB-dependent transcription of inflammation genes, brain inflammation in vivo, and eventually mouse cognitive impairment. Collectively, PQBP1 is an intracellular receptor in the cGAS-STING pathway not only for cDNA of human immunodeficiency virus (HIV) but also for the transmissible neurodegenerative disease protein tau. This study characterises a mechanism of brain inflammation that is common to virus infection and neurodegenerative disorders.

HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer's disease pathology.

DNA damage is increased in Alzheimer's disease (AD), while the underlying mechanisms are unknown. Here, we employ comprehensive phosphoproteome analysis, and identify abnormal phosphorylation of 70 kDa subunit of Ku antigen (Ku70) at Ser77/78, which prevents Ku70-DNA interaction, in human AD postmortem brains. The abnormal phosphorylation inhibits accumulation of Ku70 to the foci of DNA double strand break (DSB), impairs DNA damage repair and eventually causes transcriptional repression-induced atypical cell death (TRIAD). Cells under TRIAD necrosis reveal senescence phenotypes. Extracellular high mobility group box 1 (HMGB1) protein, which is released from necrotic or hyper-activated neurons in AD, binds to toll-like receptor 4 (TLR4) of neighboring neurons, and activates protein kinase C alpha (PKCα) that executes Ku70 phosphorylation at Ser77/78. Administration of human monoclonal anti-HMGB1 antibody to post-symptomatic AD model mice decreases neuronal DSBs, suppresses secondary TRIAD necrosis of neurons, prevents escalation of neurodegeneration, and ameliorates cognitive symptoms. TRIAD shares multiple features with senescence. These results discover the HMGB1-Ku70 axis that accounts for the increase of neuronal DNA damage and secondary enhancement of TRIAD, the cell death phenotype of senescence, in AD.

Prediction and verification of the AD-FTLD common pathomechanism based on dynamic molecular network analysis.

Multiple gene mutations cause familial frontotemporal lobar degeneration (FTLD) while no single gene mutations exists in sporadic FTLD. Various proteins aggregate in variable regions of the brain, leading to multiple pathological and clinical prototypes. The heterogeneity of FTLD could be one of the reasons preventing development of disease-modifying therapy. We newly develop a mathematical method to analyze chronological changes of PPI networks with sequential big data from comprehensive phosphoproteome of four FTLD knock-in (KI) mouse models (PGRNR504X-KI, TDP43N267S-KI, VCPT262A-KI and CHMP2BQ165X-KI mice) together with four transgenic mouse models of Alzheimer's disease (AD) and with APPKM670/671NL-KI mice at multiple time points. The new method reveals the common core pathological network across FTLD and AD, which is shared by mouse models and human postmortem brains. Based on the prediction, we performed therapeutic intervention of the FTLD models, and confirmed amelioration of pathologies and symptoms of four FTLD mouse models by interruption of the core molecule HMGB1, verifying the new mathematical method to predict dynamic molecular networks.

DNA damage in embryonic neural stem cell determines FTLDs' fate via early-stage neuronal necrosis.

The early-stage pathologies of frontotemporal lobal degeneration (FTLD) remain largely unknown. In VCPT262A-KI mice carrying VCP gene mutation linked to FTLD, insufficient DNA damage repair in neural stem/progenitor cells (NSCs) activated DNA-PK and CDK1 that disabled MCM3 essential for the G1/S cell cycle transition. Abnormal neural exit produced neurons carrying over unrepaired DNA damage and induced early-stage transcriptional repression-induced atypical cell death (TRIAD) necrosis accompanied by the specific markers pSer46-MARCKS and YAP. In utero gene therapy expressing normal VCP or non-phosphorylated mutant MCM3 rescued DNA damage, neuronal necrosis, cognitive function, and TDP43 aggregation in adult neurons of VCPT262A-KI mice, whereas similar therapy in adulthood was less effective. The similar early-stage neuronal necrosis was detected in PGRNR504X-KI, CHMP2BQ165X-KI, and TDPN267S-KI mice, and blocked by embryonic treatment with AAV-non-phospho-MCM3. Moreover, YAP-dependent necrosis occurred in neurons of human FTLD patients, and consistently pSer46-MARCKS was increased in cerebrospinal fluid (CSF) and serum of these patients. Collectively, developmental stress followed by early-stage neuronal necrosis is a potential target for therapeutics and one of the earliest general biomarkers for FTLD.

YAP-dependent necrosis occurs in early stages of Alzheimer's disease and regulates mouse model pathology.

The timing and characteristics of neuronal death in Alzheimer's disease (AD) remain largely unknown. Here we examine AD mouse models with an original marker, myristoylated alanine-rich C-kinase substrate phosphorylated at serine 46 (pSer46-MARCKS), and reveal an increase of neuronal necrosis during pre-symptomatic phase and a subsequent decrease during symptomatic phase. Postmortem brains of mild cognitive impairment (MCI) rather than symptomatic AD patients reveal a remarkable increase of necrosis. In vivo imaging reveals instability of endoplasmic reticulum (ER) in mouse AD models and genome-edited human AD iPS cell-derived neurons. The level of nuclear Yes-associated protein (YAP) is remarkably decreased in such neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta (Aß) aggregates, supporting the feature of YAP-dependent necrosis. Suppression of early-stage neuronal death by AAV-YAPdeltaC reduces the later-stage extracellular Aß burden and cognitive impairment, suggesting that preclinical/prodromal YAP-dependent neuronal necrosis represents a target for AD therapeutics.