Integrated Multi-Cohort Analysis of the Parkinson's Disease Gut Metagenome.

BACKGROUND: The gut microbiome is altered in several neurologic disorders, including Parkinson's disease (PD). OBJECTIVES: The aim is to profile the fecal gut metagenome in PD for alterations in microbial composition, taxon abundance, metabolic pathways, and microbial gene products, and their relationship with disease progression. METHODS: Shotgun metagenomic sequencing was conducted on 244 stool donors from two independent cohorts in the United States, including individuals with PD (n = 48, n = 47, respectively), environmental household controls (HC, n = 29, n = 30), and community population controls (PC, n = 41, n = 49). Microbial features consistently altered in PD compared to HC and PC subjects were identified. Data were cross-referenced to public metagenomic data sets from two previous studies in Germany and China to determine generalizable microbiome features. RESULTS: We find several significantly altered taxa between PD and controls within the two cohorts sequenced in this study. Analysis across global cohorts returns consistent changes only in Intestinimonas butyriciproducens. Pathway enrichment analysis reveals disruptions in microbial carbohydrate and lipid metabolism and increased amino acid and nucleotide metabolism in PD. Global gene-level signatures indicate an increased response to oxidative stress, decreased cellular growth and microbial motility, and disrupted intercommunity signaling. CONCLUSIONS: A metagenomic meta-analysis of PD shows consistent and novel alterations in functional metabolic potential and microbial gene abundance across four independent studies from three continents. These data reveal that stereotypic changes in the functional potential of the gut microbiome are a consistent feature of PD, highlighting potential diagnostic and therapeutic avenues for future research. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Gut microbiota suppress feeding induced by palatable foods.

Feeding behaviors depend on intrinsic and extrinsic factors including genetics, food palatability, and the environment.1,2,3,4,5 The gut microbiota is a major environmental contributor to host physiology and impacts feeding behavior.6,7,8,9,10,11,12 Here, we explored the hypothesis that gut bacteria influence behavioral responses to palatable foods and reveal that antibiotic depletion (ABX) of the gut microbiota in mice results in overconsumption of several palatable foods with conserved effects on feeding dynamics. Gut microbiota restoration via fecal transplant into ABX mice is sufficient to rescue overconsumption of high-sucrose pellets. Operant conditioning tests found that ABX mice exhibit intensified motivation to pursue high-sucrose rewards. Accordingly, neuronal activity in mesolimbic brain regions, which have been linked with motivation and reward-seeking behavior,3 was elevated in ABX mice after consumption of high-sucrose pellets. Differential antibiotic treatment and functional microbiota transplants identified specific gut bacterial taxa from the family S24-7 and the genus Lactobacillus whose abundances associate with suppression of high-sucrose pellet consumption. Indeed, colonization of mice with S24-7 and Lactobacillus johnsonii was sufficient to reduce overconsumption of high-sucrose pellets in an antibiotic-induced model of binge eating. These results demonstrate that extrinsic influences from the gut microbiota can suppress the behavioral response toward palatable foods in mice.

Microbiota regulate social behaviour via stress response neurons in the brain.

Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence1,2. The gut microbiota contribute to social activity in mice3,4, but the gut-brain connections that regulate this complex behaviour and its underlying neural basis are unclear5,6. Here we show that the microbiome modulates neuronal activity in specific brain regions of male mice to regulate canonical stress responses and social behaviours. Social deviation in germ-free and antibiotic-treated mice is associated with elevated levels of the stress hormone corticosterone, which is primarily produced by activation of the hypothalamus-pituitary-adrenal (HPA) axis. Adrenalectomy, antagonism of glucocorticoid receptors, or pharmacological inhibition of corticosterone synthesis effectively corrects social deficits following microbiome depletion. Genetic ablation of glucocorticoid receptors in specific brain regions or chemogenetic inactivation of neurons in the paraventricular nucleus of the hypothalamus that produce corticotrophin-releasing hormone (CRH) reverse social impairments in antibiotic-treated mice. Conversely, specific activation of CRH-expressing neurons in the paraventricular nucleus induces social deficits in mice with a normal microbiome. Via microbiome profiling and in vivo selection, we identify a bacterial species, Enterococcus faecalis, that promotes social activity and reduces corticosterone levels in mice following social stress. These studies suggest that specific gut bacteria can restrain the activation of the HPA axis, and show that the microbiome can affect social behaviours through discrete neuronal circuits that mediate stress responses in the brain.

Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72.

Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.

Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens.

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-ß aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.

Endoplasmic reticulum-resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2.

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).