Psychobiotics and the Microbiota-Gut-Brain Axis: Where Do We Go from Here?

The bidirectional relationship between the gut microbiota and the nervous system is known as the microbiota-gut-brain axis (MGBA). The MGBA controls the complex interactions between the brain, the enteric nervous system, the gut-associated immune system, and the enteric neuroendocrine systems, regulating key physiological functions such as the immune response, sleep, emotions and mood, food intake, and intestinal functions. Psychobiotics are considered tools with the potential to modulate the MGBA through preventive, adjunctive, or curative approaches, but their specific mechanisms of action on many aspects of health are yet to be characterized. This narrative review and perspectives article highlights the key paradigms needing attention as the scope of potential probiotics applications in human health increases, with a growing body of evidence supporting their systemic beneficial effects. However, there are many limitations to overcome before establishing the extent to which we can incorporate probiotics in the management of neuropsychiatric disorders. Although this article uses the term probiotics in a general manner, it remains important to study probiotics at the strain level in most cases.

PIKFYVE inhibition mitigates disease in models of diverse forms of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.

Genetic Interactions of Progranulin Across the ALS-FTD Spectrum and Beyond.

Progranulin (PGRN) is a growth factor in which mutations are one of the leading causes of frontotemporal dementia (FTD), and has been implicated in an assortment of neurodegenerative diseases. Conversely, higher levels of the protein have shown potential as a general neuronal protective factor. While examining its neuroprotective applications on a broader scale would be unfeasible in mammalian models, we turned to the nematode C. elegans to map the interactions of PGRN across multiple genetic models of neurodegenerative diseases. Our results indicate that while the overexpression of PGRN appears to be protective across all models tested, the loss of PGRN exacerbated the disease phenotypes of all but three of the models tested. Given the ease of genetic analysis in nematodes, we propose this model organism as an efficient tool to build a comprehensive map of PGRN's genetic interactions.

Genetic and pharmacological PARP inhibition reduces axonal degeneration in C. elegans models of ALS.

Axonal degeneration is observed in early stages of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). This degeneration generally precedes apoptosis and therefore may be a promising therapeutic target. An increasing number of genes have been identified to actively regulate axonal degeneration and regeneration; however, only a few potential therapeutic targets have been identified in the context of neurodegenerative diseases. Here we investigate DLK-1, a major axonal regeneration pathway and its contribution to axonal degeneration phenotypes in several Caenorhabditis elegans ALS models. From this pathway, we identified the poly (ADP-ribose) (PAR) polymerases (PARP) PARP-1 and PARP-2 as the most consistent modifiers of axonal degeneration in our models of ALS. Genetic and pharmacological inhibition of PARP-1 and PARP-2 reduces axonal degeneration and improves related motor phenotypes.

Phosphoglycolate phosphatase homologs act as glycerol-3-phosphate phosphatase to control stress and healthspan in C. elegans.

Metabolic stress due to nutrient excess and lipid accumulation is at the root of many age-associated disorders and the identification of therapeutic targets that mimic the beneficial effects of calorie restriction has clinical importance. Here, using C. elegans as a model organism, we study the roles of a recently discovered enzyme at the heart of metabolism in mammalian cells, glycerol-3-phosphate phosphatase (G3PP) (gene name Pgp) that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol. We identify three Pgp homologues in C. elegans (pgph) and demonstrate in vivo that their protein products have G3PP activity, essential for glycerol synthesis. We demonstrate that PGPH/G3PP regulates the adaptation to various stresses, in particular hyperosmolarity and glucotoxicity. Enhanced G3PP activity reduces fat accumulation, promotes healthy aging and acts as a calorie restriction mimetic at normal food intake without altering fertility. Thus, PGP/G3PP can be considered as a target for age-related metabolic disorders.

A single copy transgenic mutant FUS strain reproduces age-dependent ALS phenotypes in C. elegans.

Mutations in the human DNA/RNA binding protein FUS are associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration, including some aggressive and juvenile onset forms. Cytoplasmic inclusions of human FUS proteins are observed in various neurodegenerative disorders, such as Huntington's disease or spinocerebellar ataxia, suggesting that FUS proteinopathy may be a key player in neurodegeneration. To better understand the pathogenic mechanisms of FUS, we created single copy transgenic Caenorhabditis elegans strains expressing full-length, untagged human FUS in the worm's GABAergic neurons. These transgenic worms expressing human mutant FUS (mFUS) display the same ALS-associated phenotypes than our previous multiple copy transgenic model, including adult-onset age-dependent loss of motility, progressive paralysis and GABAergic neurodegeneration. These phenotypes are distinct from the transgenic worms expressing human wild-type FUS (wtFUS). We introduce here our C. elegans single copy transgenic for human mutant FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.

TDP-43 stabilizes G3BP1 mRNA: relevance to amyotrophic lateral sclerosis/frontotemporal dementia.

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. Ras-GAP SH3-domain-binding protein 1 (G3BP1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3' untranslated region. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data indicate that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

Chemical and genetic rescue of in vivo progranulin-deficient lysosomal and autophagic defects.

In 2006, GRN mutations were first linked to frontotemporal dementia (FTD), the leading cause of non-Alzheimer dementias. While much research has been dedicated to understanding the genetic causes of the disease, our understanding of the mechanistic impacts of GRN deficiency has only recently begun to take shape. With no known cure or treatment available for GRN-related FTD, there is a growing need to rapidly advance genetic and/or small-molecule therapeutics for this disease. This issue is complicated by the fact that, while lysosomal dysfunction seems to be a key driver of pathology, the mechanisms linking a loss of GRN to a pathogenic state remain unclear. In our attempt to address these key issues, we have turned to the nematode, Caenorhabditis elegans, to model, study, and find potential therapies for GRN-deficient FTD. First, we show that the loss of the nematode GRN ortholog, pgrn-1, results in several behavioral and molecular defects, including lysosomal dysfunction and defects in autophagic flux. Our investigations implicate the sphingolipid metabolic pathway in the regulation of many of the in vivo defects associated with pgrn-1 loss. Finally, we utilized these nematodes as an in vivo tool for high-throughput drug screening and identified two small molecules with potential therapeutic applications against GRN/pgrn-1 deficiency. These compounds reverse the biochemical, cellular, and functional phenotypes of GRN deficiency. Together, our results open avenues for mechanistic and therapeutic research into the outcomes of GRN-related neurodegeneration, both genetic and molecular.

Chromatin remodeller CHD7 is required for GABAergic neuron development by promoting PAQR3 expression.

Mutations in the chromatin remodeller-coding gene CHD7 cause CHARGE syndrome (CS). CS features include moderate to severe neurological and behavioural problems, clinically characterized by intellectual disability, attention-deficit/hyperactivity disorder and autism spectrum disorder. To investigate the poorly characterized neurobiological role of CHD7, we here generate a zebrafish chd7-/- model. chd7-/- mutants have less GABAergic neurons and exhibit a hyperactivity behavioural phenotype. The GABAergic neuron defect is at least in part due to downregulation of the CHD7 direct target gene paqr3b, and subsequent upregulation of MAPK/ERK signalling, which is also dysregulated in CHD7 mutant human cells. Through a phenotype-based screen in chd7-/- zebrafish and Caenorhabditis elegans, we show that the small molecule ephedrine restores normal levels of MAPK/ERK signalling and improves both GABAergic defects and behavioural anomalies. We conclude that chd7 promotes paqr3b expression and that this is required for normal GABAergic network development. This work provides insight into the neuropathogenesis associated with CHD7 deficiency and identifies a promising compound for further preclinical studies.

Small Molecule Rescue of ATXN3 Toxicity in C. elegans via TFEB/HLH-30.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a polyglutamine expansion disease arising from a trinucleotide CAG repeat expansion in exon 10 of the gene ATXN3. There are no effective pharmacological treatments for MJD, thus the identification of new pathogenic mechanisms, and the development of novel therapeutics is urgently needed. In this study, we performed a comprehensive, blind drug screen of 3942 compounds (many FDA approved) and identified small molecules that rescued the motor-deficient phenotype in transgenic ATXN3 Caenorhabditis elegans strain. Out of this screen, five lead compounds restoring motility, protecting against neurodegeneration, and increasing the lifespan in ATXN3-CAG89 mutant worms were identified. These compounds were alfacalcidol, chenodiol, cyclophosphamide, fenbufen, and sulfaphenazole. We then investigated how these molecules might exert their neuroprotective properties. We found that three of these compounds, chenodiol, fenbufen, and sulfaphenazole, act as modulators for TFEB/HLH-30, a key transcriptional regulator of the autophagy process, and require this gene for their neuroprotective activities. These genetic-chemical approaches, using genetic C. elegans models for MJD and the screening, are promising tools to understand the mechanisms and pathways causing neurodegeneration, leading to MJD. Positively acting compounds may be promising candidates for investigation in mammalian models of MJD and preclinical applications in the treatment of this disease.

Overexpression of FKH-2/FOXG1 is neuroprotective in a C. elegans model of Machado-Joseph disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is the most common form of dominantly inherited ataxia worldwide. This disease is caused by an expanded CAG repeat in the coding region of ATXN3. Due to our incomplete understanding of mechanisms and molecular pathways related to this disease, there are no therapies that successfully treat core MJD patients. Therefore, the identification of new candidate targets related to this disease is needed. In this study, we performed a large-scale RNA interference (RNAi) screen of 387 transcription factor genes leading to the identification of several modifiers (suppressors and enhancers) of impaired motility phenotypes in a mutant ATXN3 transgenic C. elegans model. We showed that inactivation of one particular gene, fkh-2/FOXG1, enhanced the motility defect, neurodegeneration and reduced longevity in our MJD models. Opposite to genetic inactivation, the overexpression of fkh-2 rescued the impaired motility, shortened-lifespan, and neurodegeneration phenotypes of mutant ATXN3 transgenics. We found that overexpression of FKH-2/FOXG1 in ATXN3 mutant worms is neuroprotective. Using our transgenic ATXN3 C. elegans models and the screening of an RNAi library, we gained insights into the pathways contributing to neurodegeneration, and found that FKH-2/FOXG1 has neuroprotective activity. These findings may aid the development of novel therapeutic interventions for MJD.

Modulating the endoplasmic reticulum stress response attenuates neurodegeneration in a Caenorhabditiselegans model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating autosomal recessive neuromuscular disease resulting in muscle atrophy and neurodegeneration, and is the leading genetic cause of infant death. SMA arises when there are homozygous deletion mutations in the human SMN1 gene, leading to a decrease in corresponding SMN1 protein. Although SMN1 is expressed across multiple tissue types, much of the previous research into SMA focused on the neuronal aspect of the disease, overlooking many of the potential non-neuronal aspects of the disease. Therefore, we sought to address this gap in knowledge by modeling SMA in the nematode Caenorhabditis elegans We mutated a previously uncharacterized allele, which resulted in the onset of mild SMA-like phenotypes, allowing us to monitor the onset of phenotypes at different stages. We observed that these mutant animals recapitulated many key features of the human disease, and most importantly, we observed that muscle dysfunction preceded neurodegeneration. Furthermore, we tested the therapeutic efficacy of targeting endoplasmic reticulum (ER) stress in non-neuronal cells and found it to be more effective than targeting ER stress in neuronal cells. We also found that the most potent therapeutic potential came from a combination of ER- and neuromuscular junction-targeted drugs. Together, our results suggest an important non-neuronal component of SMA pathology and highlight new considerations for therapeutic intervention.

Pleiotropic Effects of mTOR and Autophagy During Development and Aging.

Autophagy as a ubiquitous catabolic process causes degradation of cytoplasmic components and is generally considered to have beneficial effects on health and lifespan. In contrast, inefficient autophagy has been linked with detrimental effects on the organism and various diseases, such as Parkinson's disease. Previous research, however, showed that this paradigm is far from being black and white. For instance, it has been reported that increased levels of autophagy during development can be harmful, but become advantageous in the aging cell or organism, causing enhanced healthspan and even longevity. The antagonistic pleiotropy hypothesis postulates that genes, which control various traits in an organism, can be fitness-promoting in early life, but subsequently trigger aging processes later. Autophagy is controlled by the mechanistic target of rapamycin (mTOR), a key player of nutrient sensing and signaling and classic example of a pleiotropic gene. mTOR acts upstream of transcription factors such as FOXO, NRF, and TFEB, controlling protein synthesis, degradation, and cellular growth, thereby regulating fertility as well as aging. Here, we review recent findings about the pleiotropic role of autophagy during development and aging, examine the upstream factors, and contemplate specific mechanisms leading to disease, especially neurodegeneration.

The Novel Small Molecule TRVA242 Stabilizes Neuromuscular Junction Defects in Multiple Animal Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder in which the neuromuscular junction progressively degenerates, leading to movement difficulties, paralysis, and eventually death. ALS is currently being treated by only two FDA-approved drugs with modest efficacy in slowing disease progression. Often, the translation of preclinical findings to bedside terminates prematurely as the evaluation of potential therapeutic compounds focuses on a single study or a single animal model. To circumscribe these issues, we screened 3,765 novel small molecule derivatives of pimozide, a recently identified repurposed neuroleptic for ALS, in Caenorhabditis elegans, confirmed the hits in zebrafish and validated the most active compounds in mouse genetic models. Out of the 27 small molecules identified from the high-throughput screen in worms, 4 were found to recover locomotor defects in C. elegans and genetic zebrafish models of ALS. TRVA242 was identified as the most potent compound as it significantly improved efficiency in rescuing locomotor, motorneuron, and neuromuscular junction synaptic deficits in a C. elegans TDP-43 model and in multiple zebrafish genetic (TDP-43, SOD1, and C9ORF72) models of ALS. The actions of TRVA242 were also conserved in a mammalian model as it also stabilized neuromuscular junction deficits in a mouse SOD1 model of ALS. Compounds such as TRVA242 therefore represent new potential therapeutics for the treatment of ALS.

Nicotinamide-N-methyltransferase controls behavior, neurodegeneration and lifespan by regulating neuronal autophagy.

Nicotinamide N-methyl-transferase (NNMT) is an essential contributor to various metabolic and epigenetic processes, including the regulating of aging, cellular stress response, and body weight gain. Epidemiological studies show that NNMT is a risk factor for psychiatric diseases like schizophrenia and neurodegeneration, especially Parkinson's disease (PD), but its neuronal mechanisms of action remain obscure. Here, we describe the role of neuronal NNMT using C. elegans. We discovered that ANMT-1, the nematode NNMT ortholog, competes with the methyltransferase LCMT-1 for methyl groups from S-adenosyl methionine. Thereby, it regulates the catalytic capacities of LCMT-1, targeting NPRL-2, a regulator of autophagy. Autophagy is a core cellular, catabolic process for degrading cytoplasmic material, but very little is known about the regulation of autophagy during aging. We report an important role for NNMT in regulation of autophagy during aging, where high neuronal ANMT-1 activity induces autophagy via NPRL-2, which maintains neuronal function in old wild type animals and various disease models, also affecting longevity. In younger animals, however, ANMT-1 activity disturbs neuronal homeostasis and dopamine signaling, causing abnormal behavior. In summary, we provide fundamental insights into neuronal NNMT/ANMT-1 as pivotal regulator of behavior, neurodegeneration, and lifespan by controlling neuronal autophagy, potentially influencing PD and schizophrenia risk in humans.

Methods to Investigate the Molecular Basis of Progranulin Action on Neurons In Vivo Using Caenorhabditis elegans.

The C. elegans nematode is a powerful genetic tool for the study of aging, developmental biology, and neurodegenerative diseases. They are a small and simple model, but its well-known genome and presence of many human orthologs has made it an ideal model to study the effects of PGRN in vivo. Here, we will describe the protocols used by our laboratory to study how PGRN affects C. elegans neuronal morphology and locomotion behavior through the use of a loss-of-function model of the nematode otholog of the GRN gene, pgrn-1. Although these protocols have been used by us to specifically study the pgrn-1 gene, the experiments are applicable to any of the animal's genes.

Worms on the spectrum - C. elegans models in autism research.

The small non-parasitic nematode Caenorhabditis elegans is widely used in neuroscience thanks to its well-understood development and lineage of the nervous system. Furthermore, C. elegans has been used to model many human developmental and neurological conditions to better understand disease mechanisms and identify potential therapeutic strategies. Autism spectrum disorder (ASD) is the most prevalent of all neurodevelopmental disorders, and the C. elegans system may provide opportunities to learn more about this complex disorder. Since basic cell biology and biochemistry of the C. elegans nervous system is generally very similar to mammals, cellular or molecular phenotypes can be investigated, along with a repertoire of behaviours. For instance, worms have contributed greatly to the understanding of mechanisms underlying mutations in genes coding for synaptic proteins such as neuroligin and neurexin. Using worms to model neurodevelopmental disorders like ASD is an emerging topic that harbours great, untapped potential. This review summarizes the numerous contributions of C. elegans to the field of neurodevelopment and introduces the nematode system as a potential research tool to study essential roles of genes associated with ASD.

Rescue of ATXN3 neuronal toxicity in Caenorhabditiselegans by chemical modification of endoplasmic reticulum stress.

Polyglutamine expansion diseases are a group of hereditary neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders, such as Huntington's disease, dentatorubral-pallidoluysian atrophy, spinobulbar muscular atrophy and multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited SCA is the type 3 (SCA3), also known as Machado-Joseph disease (MJD), which is an autosomal dominant, progressive neurological disorder. The gene causatively associated with MJD is ATXN3 Recent studies have shown that this gene modulates endoplasmic reticulum (ER) stress. We generated transgenic Caenorhabditiselegans strains expressing human ATXN3 genes in motoneurons, and animals expressing mutant ATXN3-CAG89 alleles showed decreased lifespan, impaired movement, and rates of neurodegeneration greater than wild-type ATXN3-CAG10 controls. We tested three neuroprotective compounds (Methylene Blue, guanabenz and salubrinal) believed to modulate ER stress and observed that these molecules rescued ATXN3-CAG89 phenotypes. Furthermore, these compounds required specific branches of the ER unfolded protein response (UPRER), reduced global ER and oxidative stress, and polyglutamine aggregation. We introduce new C. elegans models for MJD based on the expression of full-length ATXN3 in a limited number of neurons. Using these models, we discovered that chemical modulation of the UPRER reduced neurodegeneration and warrants investigation in mammalian models of MJD.