Parkinson's disease (PD) is commonly associated with dysfunctional mitochondrial homeostasis. PINK1, a S/T kinase mutated in early-onset PD, generates phosphoserine 65 ubiquitin (pS65Ub) on damaged mitochondria facilitating their removal. Here, we show that pS65Ub translocates into the nucleus after generation at damaged mitochondria and is directly attached to substrates by resident E3 ligases. Histone H2A is a major substrate and is modified at lysine 119 (H2AK119) by the polycomb silencer, E3 ligase RING1B. At nucleosomes, pS65Ub simultaneously suppresses RING1B and potentiates H2A deubiquitinases USP16 and USP21. Epigenetic profiling and RNA sequencing reveal that pS65Ub is enriched at the promoters of poorly expressed yet dynamically regulated genes and is associated with H2AK119ub depletion. Functionally, we show that pS65Ub enrichment drives polycomb target gene expression, which accelerates the maturation of dopaminergic neurons. Importantly, post-mortem PD brains exhibit elevated nuclear pS65Ub, potentially linking nuclear pS65Ub accumulation with disease pathogenesis. Together, these data indicate that pS65Ub generated at damaged mitochondria regulates fundamental cellular processes at distant sites.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by alpha-(α)-Synuclein neuronal aggregation and loss of dopaminergic (DA) neurons. Developing animal models that replicate PD’s neuropathological phenotypes is critical for understanding its pathophysiology and evaluating potential therapeutic targets. While intrastriatal PFF injection in M83 has been widely utilized, they often exhibit slower disease progression and limited midbrain neurodegeneration. In this study, we show that direct unilateral injection of human α-Synuclein PFFs into the Substantia Nigra (SN) of mutant A53T α-synuclein overexpressing mice induce bilateral phosphorylated α-Synuclein (pS129) pathology in the SN. This pathology spreads to the striatum, cerebral cortex, and midbrain within 60 days and is accompanied by neuroinflammation in the midbrain and cerebral cortex. Additionally, we observed synuclein-dependent neurodegeneration, with a 50

Hereditary spastic paraplegia type 11 (SPG11-HSP) is a neurodegenerative disorder caused by mutations in SPG11, which encodes the large scaffolding protein spatacsin, involved in lysosomal and autophagosomal trafficking. A portion of patients with SPG11 mutations present with parkinsonism features. While spatacsin dysfunction is linked to neurodegeneration, the underlying cellular mechanisms, especially in the midbrain, remain largely unclear. Here, we demonstrate that loss of Spg11 in mice results in neuroinflammation and lipid accumulation in myeloid cells. Bulk RNA sequencing revealed a strong upregulation of microglial genes in the midbrain of Spg11 knockouts, supported by increased CD68 and CLEC7A expression and morphological changes consistent with microglial activation. Spg11 depletion in two in vivo models of synucleinopathy revealed no enhancement of phosphorylated α-synuclein-positive inclusions or dopaminergic neuron loss; however, the mice did exhibit Spg11-dependent microglial reactivity. Further in vitro studies using primary bone-derived macrophages revealed increased phagocytic capacity and neutral lipid accumulation under basal and stress conditions. These findings support a model where SPG11 is a critical regulator of microglial activation and myeloid lipid metabolism, contributing to neurodegeneration through pathways distinct from α-synuclein-mediated pathology. Spg11 knockout activates microglia and induces lipid accumulation. Pathology observed in Spg11 knockout persists in mouse models of synucleinopathy. RNA-seq shows robust upregulation of microglial genes in Spg11 knockout midbrains. SPG11 deficiency alters microglial morphology and increases CD68 expression. Macrophages lacking SPG11 display enhanced phagocytosis and lipid storage.

Genetic variants affecting microglia can cause early-onset neurodegeneration or elevate Alzheimer’s disease risk. To nominate regulators of relevant signaling pathways, we developed a genome-wide CRISPR screen in primary macrophages focused on survival. We identified Ptpn6, which encodes the inhibitory phosphatase SHP-1, as a crucial regulator for macrophage survival under reduced CSF1R signaling conditions in vitro. Deletion of Ptpn6 from adult microglia in vivo enhanced survival and decreased neuritic dystrophy around amyloid plaques in the TauPS2APP model of Alzheimer’s disease. However, deletion also dysregulated homeostasis in normal white matter and exacerbated neurodegeneration in disease. Heterozygous deletion revealed a differential gene-dosage sensitivity for beneficial and detrimental effects, exhibiting reduced neuritic damage near plaques without white-matter harm. Single-cell RNA sequencing uncovered multiple disease-associated microglia (DAM)-like transcriptional states, with Lgals3+ microglia emerging alongside neurodegeneration after Ptpn6 deletion. In all, these findings reveal both the protective and latent degenerative potential of microglia held in check by Ptpn6.

Abstract Addison’s disease (AD) results in glucocorticoid and mineralocorticoid deficiencies and is often immune-mediated. While AD is uncommon in dogs, Nova Scotia Duck Tolling Retrievers (NSDTRs) exhibit increased incidence, suggesting genetic predisposition. Detailed clinical evaluation of 24 juvenile-onset cases revealed that while all dogs presented with adrenal insufficiency, at least 10 dogs (41.7%) had concurrent autoimmune conditions. This suggests juvenile-onset AD in NSDTRs represents part of a broader multiple autoimmune syndrome (MAS) with variable expressivity. Strikingly, NSDTRs affected by juvenile-onset AD had severely decreased lifespans, with a median survival of 2 years despite appropriate treatment. Genome-wide association identified a significant association on chromosome 27 (chr27:29,724,286, p = 6.96 × 10− 13). Whole-genome, short-read sequencing identified a recessive missense variant in RESF1 (Chr27:29,736,795). The variant exhibited 76% penetrance for early-onset disease, and the decreased penetrance was not attributable to differences in Dog Leukocyte Antigen (DLA) haplotypes. Immunohistochemistry confirmed T cell infiltration in the adrenal cortex of two unrelated affected dogs, with necropsy findings including severe bilateral lymphocytic adrenalitis, multisystemic granulomatous inflammation, and lymphoplasmacytic conjunctivitis supporting autoimmune pathogenesis. This study identifies RESF1 as a novel gene associated with autoimmune disease in NSDTRs, ranging from isolated juvenile-onset AD to multi-organ autoimmune manifestations. The findings represent a rare example of monogenic autoimmune disease and establish RESF1 as a candidate gene for further investigation of immune tolerance mechanisms.

Aberrant activation of the classical complement pathway in the brain is implicated in contributing to synapse loss and neurodegeneration in various neurodegenerative conditions. Given that C3aR is a druggable target in the complement pathway, we evaluated the potential of C3aR knockout (KO) to rescue neurodegeneration in a tauopathy model and neuroinflammatory responses in an acute endotoxemia model. We found that C3aR KO did not rescue Tau pathology, microglia activation markers, neurodegeneration, or behavioral abnormalities in tauopathy model mice. While we found that endotoxemia resulted in numerous transcriptional changes, including distinct alterations in subpopulations of microglia, astrocytes, and oligodendrocytes, C3aR KO did not impact these alterations. Together, our results suggest that the beneficial effects of blocking the complement classical pathway in neurodegeneration models are likely independent of C3aR activation and raise questions about the rationale for therapeutically targeting C3aR for neurodegenerative disease.

Abstract Neurodegenerative disease is largely driven by pathological protein states, which cannot be fully inferred from transcript levels. Yet, joint measurement of cytoplasmic proteins and RNA in single cells from archival human brain tissue remains challenging. Here we present Soma-seq, a method integrating transcriptome and antibody-based intracellular protein measurements in single cells from frozen human brain tissue. Applying Soma-seq to Alzheimer’s disease cortex, we quantified multiple intracellular proteins, including hyperphosphorylated Tau, alongside RNA. Soma-seq resolves a continuous trajectory of pathological progression based on multiplexed protein measurements and reveals associated gene programs. CRISPR perturbation in human iPSC-derived neurons validates Soma-seq-derived candidates and identifies protective factors to Tau aggregation.

Supplementary Figure S2: Ex vivo RIT1 cell lines have Trp53 genetic knockout and therapeutic vulnerabilities include MAPK/PI3K inhibitors and statins.

Ataxin-2 (Atxn2), a ubiquitously expressed RNA-binding protein, has been implicated in ALS risk, and its silencing represents a promising therapeutic strategy to extend life span and ameliorate symptoms in ALS. Although neuroprotective effects have been shown in several ALS preclinical models, the molecular mechanisms underlying ataxin-2 downregulation neuroprotective effects remain poorly understood. Starting with a global proteomic profiling of the yeast and mouse TDP-43 models, we uncovered that pbp1/ataxin-2 downregulation rewires metabolism to activate alternative cellular pathways for energy production under stress. By combining proteomic profiling insights and functional studies in neural cultures, we further show that ataxin-2 downregulation adjusts metabolism in both cell-autonomous and non cell-autonomous pathways. In neurons, ataxin-2 downregulation activates glycolysis and reductive glutamine carboxylation, while astrocytes provide enhanced support by increasing cholesterol synthesis. Collectively, our data characterize novel cellular pathways to overcome TDP-43 toxicity and establish ataxin-2 as a regulator of adaptive response in brain cells under the conditions of stress. ### Competing Interest Statement All authors are employees of Genentech.

Neurofibrillary tangles (NFTs) are pathological hallmarks of Alzheimer's Disease (AD), consisting of aggregated tau protein. Tau is frequently used as a biomarker in AD clinical trials, as it provides diagnostic and prognostic information and serves as a pharmacodynamic marker to assess effects of tau-targeting treatments. Tau PET tracers specifically bind to NFTs, allowing for tau pathology imaging. However, PET imaging requires specialized infrastructure and is costly, limiting its widespread use in trials. Our study assessed the relationship between cerebrospinal fluid (CSF) soluble tau species and tau PET imaging. The aim was to identify fluid biomarkers that could serve as more accessible alternatives for assessing tau pathology in clinical trials. CSF tau species were measured at baseline in a subset of 53 prodromal-to-moderate AD participants enrolled in two anti-Tau Ph II trials ( n  = 26, n  = 27). Tau peptides were measured by data-independent acquisition mass spectrometry (DIA-MS). Total tau, pTau181, and pTau205 were measured using Elecsys immunoassays. N-term and Mid-domain tau peptides were measured by targeted LC-MS. pTau217 was measured using a Simoa assay and later with an Elecsys assay. Trial participants also completed [18F]GTP1 imaging. Standardized uptake value ratios (SUVR) were reported from the whole cortical gray matter and meta temporal regions using inferior cerebellar gray matter as the reference area. 46 pathology confirmed AD brains from the Arizona Study of Aging and Neurodegenerative Disorders at Banner Sun Health Research Institute were used to determine correlations between tau peptides and NFT burden in the fusiform gyrus. Tau peptides were measured by DIA-MS; NFT burden was measured by quantifying AT8-positive gray matter areas of adjacent brain sections. CSF and brain Tau species were correlated to [18F]GTP1 SUVR and %AT8-positive area, respectively. The analysis demonstrated CSF pTau205, pTau217, and a proteomic peptide from the 2N4R isoform MTBR region had highest correlations with [18F]GTP1 SUVR across both target regions. The same MTBR peptide was highly correlated with the %AT8 positive area in the fusiform gyrus. A proteomic peptide from the MTBR region was identified to be the highest correlated tau peptide in AD CSF and in the brain with tau pathology.

Abstract Ataxin-2 (ATXN2) is a genetic modifier of TDP-43 toxicity and a promising therapeutic target in amyotrophic lateral sclerosis (ALS). However, the mechanisms underlying its neuroprotective effects remain poorly understood. Here we show that ataxin-2 reduction confers neuroprotection by engaging adaptive metabolic programs in both neuronal and glial cells. Using global proteomic profiling in yeast and mouse TDP-43 models, we establish that pbp1/ataxin-2 (pbp1 is the yeast ataxin-2 ortholog) activates orthogonal stress-adaptive programs that enable alternative energy production and augment trophic support, rather than simply reversing TDP-43-induced damage. In neurons, ataxin-2 downregulation activates glycolysis and reductive glutamine carboxylation driven by IDH1, restoring ATP production independently of impaired mitochondria. In astrocytes, ataxin-2 downregulation upregulates cholesterol biosynthesis via HMGCS1, enhancing trophic support to neurons in a non-cell-autonomous manner. Full neuroprotection requires both mechanisms: neuronal survival is only completely rescued when ataxin-2 is reduced in the context of neuron-astrocyte co-cultures. Importantly, ataxin-2 downregulation protects against both TDP-43 gain-of-function and loss-of-function toxicity, broadening its therapeutic relevance. Collectively, our findings reveal novel mechanisms by which ataxin-2 orchestrates adaptive metabolic resilience and establish ataxin-2 as a regulator of stress-adaptive response in brain cells under the conditions of stress.

Supplementary Table S1: Assessment of immunohistochemistry staining intensity on lungs from RIT1 mice. Pathological findings are summarized for all tumors within each animal. Localization of CD3-positive T cells is described. IHC staining intensity is qualitatively scored as +++; strongly positive, +/-; mixed and description is included, -; negative.

Supplementary Figure S1: RIT1 tumors have heterogenous pERK expression.

RIT1 is a RAS-family guanosine triphosphatase that is mutated in 2.4% and amplified in up to 14% of patients with lung adenocarcinoma. Yet the oncogenic potential of RIT1 in the lungs has not been fully established. Consequently, patients with RIT1 alterations are considered "oncogene-negative" and are not eligible for any targeted therapy in the clinic. The role of RIT1 in cancer has been historically understudied due to the lack of in vitro and in vivo models harboring RIT1 alterations. In this study, we generated a murine model of RIT1M90I-mutant lung cancer. RIT1M90I expression induced tumorigenesis in the lungs, and the tumors displayed histopathologic features similar to lung adenocarcinoma in humans. An unbiased chemical compound screen leveraging this model revealed a sensitivity to inhibitors of the MAPK, PI3K, and cholesterol biosynthesis pathways in RIT1-mutant cell lines. The SHP2 inhibitor, migoprotafib, in combination with other MAPK pathway-targeted therapies, effectively suppressed the growth of RIT1-mutant cells ex vivo and in vivo. Finally, RIT1M90I drove resistance to the KRASG12C inhibitor, divarasib, and the combination with migoprotafib reverted this phenotype. Together, these data show that RIT1M90I is a bona fide oncogenic driver of lung cancer and a mediator of targeted therapy resistance as a co-occurring mutation and suggest that patients with RIT1-altered cancer may benefit from combination treatments with an SHP2 inhibitor. SIGNIFICANCE:Development of a mouse model of RIT1M90I-altered non-small cell lung cancer reveals that RIT1M90I is a driver of lung tumorigenesis and that RIT1-mutated tumors are sensitive to MAPK pathway inhibitors. See related commentary by Wu and Vaishnavi, p. 3186 See related article by Mozzarelli et al., p. 3196.

The integrated stress response (ISR) is a cellular signaling pathway that reduces protein synthesis in the face of cellular stress, including viral infection. Two eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and general control nonderepressible 2 (GCN2), are commonly activated during viral infections. Mouse adenovirus type 1 (MAV-1) infection leads to a steep reduction of PKR levels by proteasomal degradation. We assayed whether GCN2, a sensor of amino acid starvation and UV damage, plays a role in the ISR to MAV-1 infection. There was more phosphorylated GCN2 in MAV-1-infected cells, and its activation was dependent on virus replication since UV-inactivated virus was not able to increase the phosphorylation of GCN2. Infected Eif2ak4tm1.2Dron mice (designated here Gcn2-/- mice) had lower survival than wild-type (WT) mice, but results indicated that this was not due to increased viral replication. Both Gcn2-/- and WT mice developed multifocal brain parenchymal microhemorrhages during infection. While Gcn2-/- animals had more lesions, their higher mortality is likely not due to the microhemorrhages alone. Cytokine RNA and protein assays of WT and Gcn2-/- mice only showed a difference for IL- β levels, which were higher in Gcn2-/- mice. Our results also indicate that of the two eIF2α kinases, PKR and GCN2, GCN2 is the primary inducer of phosphorylated-eIF2α during MAV-1 infection. GCN2 is thus antiviral and contributes to the host response to MAV-1 infection.IMPORTANCECells often respond to viral infection by activation of the host protein kinase R (PKR), part of the integrated stress response (ISR). We show that a second host protein kinase, general control nonderepressible 2 (GCN2), is activated by phosphorylation in response to mouse adenovirus type 1 (MAV-1) infection. Our results indicate GCN2 is antiviral: without it, the mortality in MAV-1-infected mouse is higher. Furthermore, the data show that GCN2, rather than PKR, is the main inducer of eIf2α phosphorylation (and thus the ISR) upon MAV-1 infection. This is consistent with PKR exerting antiviral effects in MAV-1 infections through a pathway independent of eIf2α phosphorylation.

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-β (Aβ) pathology (PS2APP) or combined Aβ and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.

ABSTRACT Protein kinase R (PKR) is an interferon-induced antiviral protein activated by autophosphorylation in response to double strand DNA (dsRNA) and other stimuli. Activated PKR causes translation inhibition and apoptosis, and it contributes to proinflammatory responses, cell growth, and differentiation. Mouse adenovirus type 1 (MAV-1) counteracts PKR by causing its degradation via a viral protein, early region 4 open reading frame 6 (E4orf6). Degradation is dependent on E4orf6 binding to Cullin 2, a component of the MAV-1 E4orf6 ubiquitin ligase. We investigated the importance of E4orf6 for induction of PKR degradation by exploiting the ability to infect the natural host with the adenovirus MAV-1. First, we used a new PKR-deficient mouse strain, PKR-TKO. PKR-TKO mouse embryo fibroblasts (MEFs) produced higher levels of MAV-1 upon infection than did wild-type (WT) MEFs. PKR-TKO mice had significantly reduced survival, and MAV-1 had a lower LD 50 than in WT control mice. However, virus loads in brains and spleens, key organs infected by MAV-1, were similar between PKR-TKO and WT mice. Second, we constructed a virus, E4orf6TMC2, that has three amino acid changes in the E4orf6 domain involved in Cullin 2 binding. In cell culture infection, compared to WT virus, E4orf6TMC2 resulted in reduced PKR degradation, but its growth was equivalent to WT virus. However, E4orf6TMC2 was avirulent in three mouse strains, including the PKR-TKO mice. The results indicate that PKR is an essential antiviral protein that protects against MAV-1 infection. We confirmed that the viral E4orf6 protein is a virulence protein important for PKR degradation during virus infection, and our results suggest its function is not limited to PKR degradation. IMPORTANCE Protein kinase R (PKR) is a host protein that is central to many aspects of the cellular stress response. PKR protects against viral infection by inhibiting viral and host protein synthesis. Most animal viruses have developed ways to circumvent PKR effects by at least one of a variety of means, including inducing its degradation. A new mouse strain knocked out for PKR expression has enabled us to show the importance of PKR for protection from mouse adenovirus type 1 infection in the natural host, which is not possible for human adenoviruses. Mouse adenovirus type 1 induces degradation of PKR through an interaction with host protein Cullin 2. We generated a mutant virus that is defective in its ability to interact with Cullin 2 and showed that the virus does not cause pathogenesis in mice. This work provides critical evidence from mouse studies supporting the importance of PKR for adenovirus pathogenesis.

Multiple lines of evidence indicate that mitochondrial dysfunction occurs in demyelinating diseases, such as multiple sclerosis (MS). Failure of remyelination is thought to be caused in part by a block of oligodendrocyte progenitor cell (OPC) differentiation into oligodendrocytes, which generate myelin sheaths around axons. The process of OPC differentiation requires a substantial amount of energy and high demand for ATP which is supplied through the mitochondria. In this study, we highlight mitochondrial gene expression changes during OPC differentiation in two murine models of remyelination and in human postmortem MS brains. Given these transcriptional alterations, we then investigate whether genetic alteration of USP30, a mitochondrial deubiquitinase, enhances OPC differentiation and myelination. By genetic knockout of USP30, we observe increased OPC differentiation and myelination without affecting OPC proliferation and survival in in vitro and ex vivo assays. We also find that OPC differentiation is accelerated in vivo following focal demyelination in USP30 knockout mice. The promotion of OPC differentiation and myelination observed is associated with increased oxygen consumption rates in USP30 knockout OPCs. Together, these data indicate a role for mitochondrial function and USP30 in OPC differentiation and myelination.

INTRODUCTION:Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS:We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS:We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION:CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS:CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.