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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-β 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. Eight genome-wide CRISPR screens identify genes required for substrate-specific phagocytosis. The study highlights roles for NHLRC2 in filopodia formation, very-long-chain fatty acids in substrate-specific phagocytosis and TM2D3 in uptake of amyloid-β aggregates.

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.

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. A genome-wide CRISPR screen for suppressors and enhancers of C9ORF72 dipeptide-repeat protein toxicity identifies candidate genes involved in nucleocytoplasmic transport and other pathways including RNA processing and chromatin modification.

Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence 1 , 2 . The gut microbiota contribute to social activity in mice 3 , 4 , but the gut–brain connections that regulate this complex behaviour and its underlying neural basis are unclear 5 , 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. The gut microbiota in mice can modulate social behaviour by influencing activity in stress-related brain areas.

Background Lower limb venous disease can cause significant pain, loss of mobility, and can be detrimental to an individual’s quality of life. Manifestations of venous disease often pose a substantially negative impact on patients and place a high demand on finite healthcare resources. Whilst this problem is internationally recognised, most research and discourse has predominantly focussed on treatment of leg ulceration and prevention of recurrence. Prevention of lower limb venous disease progression to the first ulceration has received far less attention. Overall, the care of this condition appears to rest in the domain of medicine and nursing yet podiatry, a profession with responsibility for the lower limb and foot, is conspicuous by its absence from the literature.  Methods An ethnographic approach was used to gather data from 26 participants through observation, semi-structured interviews, and a focus group interview. Qualitative analysis was conducted using the framework approach. Results The findings revealed an identity crisis within the podiatry profession. Evidence emerged of ritual and routine practices that did not include lower limb venous disease. External control over practice limited the professional autonomy of podiatrists determining their own activities. Inter-professional relationships with nursing, and perceptions of boundaries that venous disease was a nursing role were also found to be limiting factors. Conclusions This research revealed that podiatry does not occupy a substantive role in contributing to the early identification and prevention of lower limb venous disease. Policy, education, research and practice changes are all required to enhance the contribution of podiatry to reduce the burden of this disease.

The rich, diverse community of microorganisms in the gastrointestinal tract of animals, or gut microbiota, regulates aspects of host metabolism, immunity, and neural function, with resulting effects on the expression of complex behaviors, including feeding.In this thesis, we sought to characterize gut microbiota influences on the behavioral response to palatable foods in mice. We discover that binge-like consumption of palatable foods, including high-sucrose pellets and a high-fat diet, is exacerbated in mice in the absence of a gut microbiota. Furthermore, using automated feeding dispensers and video analysis, we find that microbiota depletion with oral antibiotics results in elongated feeding bouts and conserved changes in the dynamics of palatable food intake. We show the hyperphagic phenotype of antibiotic-treated mice is reversible upon microbiota reconstitution via fecal microbiota transplant. Operant conditioning tests reveal that the motivation to pursue high-sucrose rewards is augmented in microbiota-depleted mice. The mesolimbic brain region activity induced upon high-sucrose pellet consumption is elevated in antibiotic-treated mice. Gut bacteria from the family S24-7 and genus Lactobacillus were identified by differential antibiotic treatment and fecal microbiota transplants as correlating with reduction of high-sucrose pellet consumption. Indeed, colonization of vancomycin-treated mice with a mixture of S24-7 and Lactobacillus johnsoniireduces overconsumption of high-sucrose pellets in a limited-access bingeeating model. The work in this thesis comprehensively demonstrates that the gut microbiota regulates feeding induced in response to palatable foods in mice.

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.

The gut microbiome is altered in several neurologic disorders including Parkinson’s disease (PD). 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. 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 datasets from two previous studies in Germany and China to determine generalizable microbiome features. The gut microbiome in PD shows significant alterations in community composition. Robust taxonomic alterations include depletion of putative “beneficial” gut commensals Faecalibacterium prausnitzii and Eubacterium and Roseburia species, and increased abundance of Akkermansia muciniphila and Bifidobacterium species. Pathway enrichment analysis and metabolic potential, constructed from microbial gene abundance, revealed disruptions in microbial carbohydrate and lipid metabolism and increased amino acid and nucleotide metabolism. These global gene-level signatures indicate an increased response to oxidative stress, decreased cellular growth and microbial motility, and disrupted inter-community signaling. A metagenomic meta-analysis of PD shows consistent and novel alterations in taxonomic representation, functional metabolic potential, and microbial gene abundance across four independent studies from three continents. These data reveal stereotypic changes in the gut microbiome are a consistent feature of PD, highlighting potential diagnostic and therapeutic avenues for future research.

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.

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (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. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from model systems. We also uncovered new potent modifiers of DPR toxicity whose gene products function in the endoplasmic reticulum (ER), proteasome, RNA processing pathways, and in chromatin modification. Since regulators of ER stress emerged prominently from the screens, we further investigated one such modifier, TMX2, which we identified as a modulator of the ER-stress signature elicited by C9orf72 DPRs in neurons. Together, this work identifies novel suppressors of DPR toxicity that represent potential therapeutic targets and demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases.