A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling.

Protein Tyrosine Phosphatase Non-receptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 post-translational regulation. Here we characterize a phosphorylation site at Ser325 situated C-terminal to the catalytic domain of PTPN22, and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by Glycogen Synthase Kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9 mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p and Moira (SWIRM) domain binding proteins, and next-generation sequencing (NGS) revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by Deuterium Exchange Mass Spectrometry (DX/MS) and Nuclear Magnetic Resonance (NMR) spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22. Significance statement The tyrosine phosphatase PTPN22 serves as a negative regulator in T cells, and its phosphorylation is a major regulatory process for controlling its function. Here, we uncovered a novel phosphorylation site at Ser325 on PTPN22 that allosterically regulates its activity leading to impaired TCR-dependent pathways. Biophysical methods identify multiple regions affected upon Ser325 phosphorylation, which can be the basis for future mechanistic studies of PTPN22 activators or inhibitors.

A bacterial sialidase mediates early-life colonization by a pioneering gut commensal.

The early microbial colonization of the gastrointestinal tract can have long-term impacts on development and health. Keystone species, including Bacteroides spp., are prominent in early life and play crucial roles in maintaining the structure of the intestinal ecosystem. However, the process by which a resilient community is curated during early life remains inadequately understood. Here, we show that a single sialidase, NanH, in Bacteroides fragilis mediates stable occupancy of the intestinal mucosa in early life and regulates a commensal colonization program. This program is triggered by sialylated glycans, including those found in human milk oligosaccharides and intestinal mucus. NanH is required for vertical transmission from dams to pups and promotes B. fragilis dominance during early life. Furthermore, NanH facilitates commensal resilience and recovery after antibiotic treatment in a defined microbial community. Collectively, our study reveals a co-evolutionary mechanism between the host and microbiota mediated through host-derived glycans to promote stable colonization.

Dysregulation of Human Juvenile Huntington's Disease Brain Proteomes in Cortex and Putamen Involves Mitochondrial and Neuropeptide Systems.

BACKGROUND: Huntington's disease (HD) is a genetic neurodegenerative disease caused by trinucleotide repeat CAG expansions in the human HTT gene. Early onset juvenile HD (JHD) in children is the most severe form of the disease caused by high CAG repeat numbers of the HTT gene. OBJECTIVE: To gain understanding of human HD mechanisms hypothesized to involve dysregulated proteomes of brain regions that regulate motor and cognitive functions, this study analyzed the proteomes of human JHD cortex and putamen brain regions compared to age-matched controls. METHODS: JHD and age-matched control brain tissues were assessed for CAG repeat numbers of HTT by PCR. Human brain JHD brain cortex regions of BA4 and BA6 with the putamen region (n = 5) were analyzed by global proteomics, compared to age-matched controls (n = 7). Protein interaction pathways were assessed by gene ontology (GO), STRING-db, and KEGG bioinformatics. RESULTS: JHD brain tissues were heterozygous for one mutant HTT allele containing 60 to 120 CAG repeats, and one normal HTT allele with 10 to 19 CAG repeats. Proteomics data for JHD brain regions showed dysregulated mitochondrial energy pathways and changes in synaptic systems including peptide neurotransmitters. JHD compared to control proteomes of cortex and putamen displayed (a) proteins present only in JHD, (b) proteins absent in JHD, and (c) proteins that were downregulated or upregulated. CONCLUSIONS: Human JHD brain cortex and putamen regions display significant dysregulation of proteomes representing deficits in mitochondrial and synaptic neurotransmission functions. These findings advance understanding of JHD brain molecular mechanisms associated with HD disabilities.

A bacterial sialidase mediates early life colonization by a pioneering gut commensal.

The early microbial colonization of the gastrointestinal tract can lead to long-term impacts in development and overall human health. Keystone species, including Bacteroides spp ., play a crucial role in maintaining the structure, diversity, and function of the intestinal ecosystem. However, the process by which a defined and resilient community is curated and maintained during early life remains inadequately understood. Here, we show that a single sialidase, NanH, in Bacteroides fragilis mediates stable occupancy of the intestinal mucosa and regulates the commensal colonization program during the first weeks of life. This program is triggered by sialylated glycans, including those found in human milk oligosaccharides and intestinal mucus. After examining the dynamics between pioneer gut Bacteroides species in the murine gut, we discovered that NanH enables vertical transmission from dams to pups and promotes B. fragilis dominance during early life. Furthermore, we demonstrate that NanH facilitates commensal resilience and recovery after antibiotic treatment in a defined microbial community. Collectively, our study reveals a co-evolutionary mechanism between the host and the microbiota mediated through host-derived glycans to promote stable intestinal colonization.

Amplification of Inflammation by Lubricin Deficiency Implicated in Incident, Erosive Gout Independent of Hyperuricemia.

OBJECTIVE: In gout, hyperuricemia promotes urate crystal deposition, which stimulates the NLRP3 inflammasome and interleukin-1ß (IL-1ß)-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis in a young female patient with normouricemia diagnosed as having sufficient and weighted classification criteria for gout according to the American College of Rheumatology (ACR)/EULAR gout classification criteria (the proband). METHODS: We conducted whole-genome sequencing, quantitative proteomics, whole-blood RNA-sequencing analysis using serum samples from the proband. We used a mouse model of IL-1ß-induced knee synovitis to characterize proband candidate genes, biomarkers, and pathogenic mechanisms of gout. RESULTS: Lubricin level was attenuated in human proband serum and associated with elevated acute-phase reactants and inflammatory whole-blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of inter-α trypsin inhibitor heavy chain 3, an inhibitor of lubricin-degrading cathepsin G. Changes in the proband's serum protein interactome network supported enhanced lubricin degradation, with cathepsin G activity increased relative to its inhibitors, SERPINB6 and thrombospondin 1. Activation of Toll-like receptor 2 (TLR-2) suppressed levels of lubricin mRNA and lubricin release in cultured human synovial fibroblasts (P < 0.01). Lubricin blunted urate crystal precipitation and IL-1ß induction of xanthine oxidase and urate in cultured macrophages (P < 0.001). In lubricin-deficient mice, injection of IL-1ß in knees increased xanthine oxidase-positive synovial resident M1 macrophages (P < 0.05). CONCLUSION: Our findings linked normouricemic erosive gout to attenuated lubricin, with impaired control of cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization and blunted IL-1ß-induced increases in xanthine oxidase and urate in macrophages. The collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated cathepsin G, and synovial fibroblast TLR-2 signaling.

Mutations in protein kinase Cγ promote spinocerebellar ataxia type 14 by impairing kinase autoinhibition.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca2+-regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.

DEPDC5-dependent mTORC1 signaling mechanisms are critical for the anti-seizure effects of acute fasting.

Caloric restriction and acute fasting are known to reduce seizures but through unclear mechanisms. mTOR signaling has been suggested as a potential mechanism for seizure protection from fasting. We demonstrate that brain mTORC1 signaling is reduced after acute fasting of mice and that neuronal mTORC1 integrates GATOR1 complex-mediated amino acid and tuberous sclerosis complex (TSC)-mediated growth factor signaling. Neuronal mTORC1 is most sensitive to withdrawal of leucine, arginine, and glutamine, which are dependent on DEPDC5, a component of the GATOR1 complex. Metabolomic analysis reveals that Depdc5 neuronal-specific knockout mice are resistant to sensing significant fluctuations in brain amino acid levels after fasting. Depdc5 neuronal-specific knockout mice are resistant to the protective effects of fasting on seizures or seizure-induced death. These results establish that acute fasting reduces seizure susceptibility in a DEPDC5-dependent manner. Modulation of nutrients upstream of GATOR1 and mTORC1 could offer a rational therapeutic strategy for epilepsy treatment.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

Dysregulation of Neuropeptide and Tau Peptide Signatures in Human Alzheimer's Disease Brain.

Synaptic dysfunction and loss occur in Alzheimer's disease (AD) brains, which results in cognitive deficits and brain neurodegeneration. Neuropeptides comprise the major group of synaptic neurotransmitters in the nervous system. This study evaluated neuropeptide signatures that are hypothesized to differ in human AD brain compared to age-matched controls, achieved by global neuropeptidomics analysis of human brain cortex synaptosomes. Neuropeptidomics demonstrated distinct profiles of neuropeptides in AD compared to controls consisting of neuropeptides derived from chromogranin A (CHGA) and granins, VGF (nerve growth factor inducible), cholecystokinin, and others. The differential neuropeptide signatures indicated differences in proteolytic processing of their proneuropeptides. Analysis of cleavage sites showed that dibasic residues at the N-termini and C-termini of neuropeptides were the main sites for proneuropeptide processing, and data also showed that the AD group displayed differences in preferred residues adjacent to the cleavage sites. Notably, tau peptide signatures differed in the AD compared to age-matched control human brain cortex synaptosomes. Unique tau peptides were derived from the tau protein through proteolysis using similar and differential cleavage sites in the AD brain cortex compared to the control. Protease profiles differed in the AD compared to control, indicated by proteomics data. Overall, these results demonstrate that dysregulation of neuropeptides and tau peptides occurs in AD brain cortex synaptosomes compared to age-matched controls, involving differential cleavage site properties for proteolytic processing of precursor proteins. These dynamic changes in neuropeptides and tau peptide signatures may be associated with the severe cognitive deficits of AD.

The Host-Microbiome Response to Hyperbaric Oxygen Therapy in Ulcerative Colitis Patients.

BACKGROUND & AIMS: Hyperbaric oxygen therapy (HBOT) is a promising treatment for moderate-to-severe ulcerative colitis. However, our current understanding of the host and microbial response to HBOT remains unclear. This study examined the molecular mechanisms underpinning HBOT using a multi-omic strategy. METHODS: Pre- and post-intervention mucosal biopsies, tissue, and fecal samples were collected from HBOT phase 2 clinical trials. Biopsies and fecal samples were subjected to shotgun metaproteomics, metabolomics, 16s rRNA sequencing, and metagenomics. Tissue was subjected to bulk RNA sequencing and digital spatial profiling (DSP) for single-cell RNA and protein analysis, and immunohistochemistry was performed. Fecal samples were also used for colonization experiments in IL10-/- germ-free UC mouse models. RESULTS: Proteomics identified negative associations between HBOT response and neutrophil azurophilic granule abundance. DSP identified an HBOT-specific reduction of neutrophil STAT3, which was confirmed by immunohistochemistry. HBOT decreased microbial diversity with a proportional increase in Firmicutes and a secondary bile acid lithocholic acid. A major source of the reduction in diversity was the loss of mucus-adherent taxa, resulting in increased MUC2 levels post-HBOT. Targeted database searching revealed strain-level associations between Akkermansia muciniphila and HBOT response status. Colonization of IL10-/- with stool obtained from HBOT responders resulted in lower colitis activity compared with non-responders, with no differences in STAT3 expression, suggesting complementary but independent host and microbial responses. CONCLUSIONS: HBOT reduces host neutrophil STAT3 and azurophilic granule activity in UC patients and changes in microbial composition and metabolism in ways that improve colitis activity. Intestinal microbiota, especially strain level variations in A muciniphila, may contribute to HBOT non-response.

Multi-omics of human plasma reveals molecular features of dysregulated inflammation and accelerated aging in schizophrenia.

Schizophrenia is a devastating psychiatric illness that detrimentally affects a significant portion of the worldwide population. Aging of schizophrenia patients is associated with reduced longevity, but the potential biological factors associated with aging in this population have not yet been investigated in a global manner. To address this gap in knowledge, the present study assesses proteomics and metabolomics profiles in the plasma of subjects afflicted with schizophrenia compared to non-psychiatric control patients over six decades of life. Global, unbiased analyses of circulating blood plasma can provide knowledge of prominently dysregulated molecular pathways and their association with schizophrenia, as well as features of aging and gender in this disease. The resulting data compiled in this study represent a compendium of molecular changes associated with schizophrenia over the human lifetime. Supporting the clinical finding of schizophrenia's association with more rapid aging, both schizophrenia diagnosis and age significantly influenced the plasma proteome in subjects assayed. Schizophrenia was broadly associated with prominent dysregulation of inflammatory and metabolic system components. Proteome changes demonstrated increased abundance of biomarkers for risk of physiologic comorbidities of schizophrenia, especially in younger individuals. These findings advance our understanding of the molecular etiology of schizophrenia and its associated comorbidities throughout the aging process.

Factors influencing the acute pentylenetetrazole-induced seizure paradigm and a literature review.

OBJECTIVE: To confirm the critical factors affecting seizure susceptibility in acute pentylenetetrazole (PTZ) mouse epilepsy models and evaluate the prior literature for these factors. METHODS: Serial cohorts of wild-type mice administered intraperitoneal (IP)-PTZ were aggregated and analyzed by multivariate logistic regression for the effect of sex, age, background strain, dose, and physiologic stress (i.e., EEG implantation and/or single-housing) on seizure response. We assessed the reporting of these factors in a comprehensive literature review over the last 10 years (2010-2020). RESULTS: We conducted aggregated analysis of pooled data of 307 mice (220 C57BL/6J mice and 87 mixed background mice; 202 males, 105 females) with median age of 10 weeks (range: 6-49 weeks) with acute PTZ injection (dose range 40-65 mg/kg). Significance in multivariate analysis was found between seizures and increased PTZ dose (odds ratio (OR) 1.149, 95% confidence interval (CI) 1.102-1.205), older age (OR 1.1, 95% CI 1.041-1.170), physiologic stress (OR 17.36, 95% CI 7.349-44.48), and mixed background strain (OR 0.4725, 95% CI 0.2315-0.9345). Literature review identified 97 papers using acute PTZ-seizure models. Age, housing, sex, and background were omitted by 61% (59/97), 51% (49/97), 18% (17/97), and 8% (8/97) papers, respectively. Only 17% of publications specified all four factors (16/97). INTERPRETATION: Our analysis and literature review demonstrate a critical gap in standardization of acute PTZ-induced seizure paradigm in mice. We recommend that future studies specify and control for age, background strain, sex, and housing conditions of experimental animals.

Chronic mTORC1 inhibition rescues behavioral and biochemical deficits resulting from neuronal Depdc5 loss in mice.

DEPDC5 is now recognized as one of the genes most often implicated in familial/inherited focal epilepsy and brain malformations. Individuals with pathogenic variants in DEPDC5 are at risk for epilepsy, associated neuropsychiatric comorbidities and sudden unexplained death in epilepsy. Depdc5flox/flox-Syn1Cre (Depdc5cc+) neuronal-specific Depdc5 knockout mice exhibit seizures and neuronal mTORC1 hyperactivation. It is not known if Depdc5cc+ mice have a hyperactivity/anxiety phenotype, die early from terminal seizures or whether mTOR inhibitors rescue DEPDC5-related seizures and associated comorbidities. Herein, we report that Depdc5cc+ mice were hyperactive in open-field testing but did not display anxiety-like behaviors on the elevated-plus maze. Unlike many other mTOR-related models, Depdc5cc+ mice had minimal epileptiform activity and rare seizures prior to seizure-induced death, as confirmed by video-EEG monitoring. Treatment with the mTORC1 inhibitor rapamycin starting after 3 weeks of age significantly prolonged the survival of Depdc5cc+ mice and partially rescued the behavioral hyperactivity. Rapamycin decreased the enlarged brain size of Depdc5cc+ mice with corresponding decrease in neuronal soma size. Loss of Depdc5 led to a decrease in the other GATOR1 protein levels (NPRL2 and NPRL3). Rapamycin failed to rescue GATOR1 protein levels but rather rescued downstream mTORC1 hyperactivity as measured by phosphorylation of S6. Collectively, our data provide the first evidence of behavioral alterations in mice with Depdc5 loss and support mTOR inhibition as a rational therapeutic strategy for DEPDC5-related epilepsy in humans.