TDP-43 knockdown in mouse model of ALS leads to dsRNA deposition, gliosis, and neurodegeneration in the spinal cord.

Transactive response DNA binding protein 43 kilodaltons (TDP-43) is a DNA and RNA binding protein associated with severe neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), primarily affecting motor neurons in the brain and spinal cord. Partial knockdown of TDP-43 expression in a mouse model (the amiR-TDP-43 mice) leads to progressive, age-related motor dysfunction, as observed in ALS patients. Work in Caenorhabditis elegans suggests that TDP-43 dysfunction can lead to deficits in chromatin processing and double-stranded RNA (dsRNA) accumulation, potentially activating the innate immune system and promoting neuroinflammation. To test this hypothesis, we used immunostaining to investigate dsRNA accumulation and other signs of CNS pathology in the spinal cords of amiR-TDP-43 mice. Compared with wild-type controls, TDP-43 knockdown animals show increases in dsRNA deposition in the dorsal and ventral horns of the spinal cord. Additionally, animals with heavy dsRNA expression show markedly increased levels of astrogliosis and microgliosis. Interestingly, areas of high dsRNA expression and microgliosis overlap with regions of heavy neurodegeneration, indicating that activated microglia could contribute to the degeneration of spinal cord neurons. This study suggests that loss of TDP-43 function could contribute to neuropathology by increasing dsRNA deposition and subsequent innate immune system activation.

α-Sheet secondary structure in amyloid ß-peptide drives aggregation and toxicity in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the deposition of ß-sheet-rich, insoluble amyloid ß-peptide (Aß) plaques; however, plaque burden is not correlated with cognitive impairment in AD patients; instead, it is correlated with the presence of toxic soluble oligomers. Here, we show, by a variety of different techniques, that these Aß oligomers adopt a nonstandard secondary structure, termed "α-sheet." These oligomers form in the lag phase of aggregation, when Aß-associated cytotoxicity peaks, en route to forming nontoxic ß-sheet fibrils. De novo-designed α-sheet peptides specifically and tightly bind the toxic oligomers over monomeric and fibrillar forms of Aß, leading to inhibition of aggregation in vitro and neurotoxicity in neuroblastoma cells. Based on this specific binding, a soluble oligomer-binding assay (SOBA) was developed as an indirect probe of α-sheet content. Combined SOBA and toxicity experiments demonstrate a strong correlation between α-sheet content and toxicity. The designed α-sheet peptides are also active in vivo where they inhibit Aß-induced paralysis in a transgenic Aß Caenorhabditis elegans model and specifically target and clear soluble, toxic oligomers in a transgenic APPsw mouse model. The α-sheet hypothesis has profound implications for further understanding the mechanism behind AD pathogenesis.

Amyloid beta acts synergistically as a pro-inflammatory cytokine.

The amyloid beta (Aß) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aß are incompletely understood. Here, we report that nanomolar concentrations of Aß act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aß can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aß have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aß at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aß often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aß and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome.

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein-protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein-protein and RNA-RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA-RNA assembly in cells could lead to disease.

TDP-43 knockdown causes innate immune activation via protein kinase R in astrocytes.

TAR-DNA binding protein 43 (TDP-43) is a multifunctional RNA binding protein directly implicated in the etiology of amyotrophic lateral sclerosis (ALS). Previous studies have demonstrated that loss of TDP-43 function leads to intracellular accumulation of non-coding repetitive element transcripts and double-stranded RNA (dsRNA). These events could cause immune activation and contribute to the neuroinflammation observed in ALS, but this possibility has not been investigated. Here, we knock down TDP-43 in primary rat astrocytes via siRNA, and we use RNA-seq, immunofluorescence, and immunoblotting to show that this results in: 1) accumulation of repetitive element transcripts and dsRNA; and 2) pro-inflammatory gene and protein expression consistent with innate immune signaling and astrocyte activation. We also show that both chemical inhibition and siRNA knockdown of protein kinase R (PKR), a dsRNA-activated kinase implicated in the innate immune response, block the expression of all activation markers assayed. Based on these findings, we suggest that intracellular accumulation of endogenous dsRNA may be a novel and important mechanism underlying the pathogenesis of ALS (and perhaps other neurodegenerative diseases), and that PKR inhibitors may have the potential to prevent reactive astrocytosis in ALS.

Repetitive element transcripts are elevated in the brain of C9orf72 ALS/FTLD patients.

Significant transcriptome alterations are detected in the brain of patients with amyotrophic lateral sclerosis (ALS), including carriers of the C9orf72 repeat expansion and C9orf72-negative sporadic cases. Recently, the expression of repetitive element transcripts has been associated with toxicity and, while increased repetitive element expression has been observed in several neurodegenerative diseases, little is known about their contribution to ALS. To assess whether aberrant expression of repetitive element sequences are observed in ALS, we analysed RNA sequencing data from C9orf72-positive and sporadic ALS cases, as well as healthy controls. Transcripts from multiple classes and subclasses of repetitive elements (LINEs, endogenous retroviruses, DNA transposons, simple repeats, etc.) were significantly increased in the frontal cortex of C9orf72 ALS patients. A large collection of patient samples, representing both C9orf72 positive and negative ALS, ALS/FTLD, and FTLD cases, was used to validate the levels of several repetitive element transcripts. These analyses confirmed that repetitive element expression was significantly increased in C9orf72-positive compared to C9orf72-negative or control cases. While previous studies suggest an important link between TDP-43 and repetitive element biology, our data indicate that TDP-43 pathology alone is insufficient to account for the observed changes in repetitive elements in ALS/FTLD. Instead, we found that repetitive element expression positively correlated with RNA polymerase II activity in postmortem brain, and pharmacologic modulation of RNA polymerase II activity altered repetitive element expression in vitro. We conclude that increased RNA polymerase II activity in ALS/FTLD may lead to increased repetitive element transcript expression, a novel pathological feature of ALS/FTLD.

DLK-1, SEK-3 and PMK-3 Are Required for the Life Extension Induced by Mitochondrial Bioenergetic Disruption in C. elegans.

Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, we find that dlk-1, sek-3 and pmk-3 are all required for their life extension.

TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA.

Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans.

At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington's disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ~0.9 in as little as six-weeks with a mean firing rate of ~13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of sporadic Alzheimer's disease by mimicking blood-brain barrier breakdown using a human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Toxic role of K+ channel oxidation in mammalian brain.

Potassium (K(+)) channels are essential to neuronal signaling and survival. Here we show that these proteins are targets of reactive oxygen species in mammalian brain and that their oxidation contributes to neuropathy. Thus, the KCNB1 (Kv2.1) channel, which is abundantly expressed in cortex and hippocampus, formed oligomers upon exposure to oxidizing agents. These oligomers were ∼10-fold more abundant in the brain of old than young mice. Oxidant-induced oligomerization of wild-type KCNB1 enhanced apoptosis in neuronal cells subject to oxidative insults. Consequently, a KCNB1 variant resistant to oxidation, obtained by mutating a conserved cysteine to alanine, (C73A), was neuroprotective. The fact that oxidation of KCNB1 is toxic, argues that this mechanism may contribute to neuropathy in conditions characterized by high levels of oxidative stress, such as Alzheimer's disease (AD). Accordingly, oxidation of KCNB1 channels was exacerbated in the brain of a triple transgenic mouse model of AD (3xTg-AD). The C73A variant protected neuronal cells from apoptosis induced by incubation with ß-amyloid peptide (Aß(1-42)). In an invertebrate model (Caenorhabditis elegans) that mimics aspects of AD, a C73A-KCNB1 homolog (C113S-KVS-1) protected specific neurons from apoptotic death induced by ectopic expression of human Aß(1-42). Together, these data underscore a novel mechanism of toxicity in neurodegenerative disease.

The reverse transcriptase inhibitor 3TC protects against age-related cognitive dysfunction.

Aging is the primary risk factor for most neurodegenerative diseases, including Alzheimer's disease. Major hallmarks of brain aging include neuroinflammation/immune activation and reduced neuronal health/function. These processes contribute to cognitive dysfunction (a key risk factor for Alzheimer's disease), but their upstream causes are incompletely understood. Age-related increases in transposable element (TE) transcripts might contribute to reduced cognitive function with brain aging, as the reverse transcriptase inhibitor 3TC reduces inflammation in peripheral tissues and TE transcripts have been linked with tau pathology in Alzheimer's disease. However, the effects of 3TC on cognitive function with aging have not been investigated. Here, in support of a role for TE transcripts in brain aging/cognitive decline, we show that 3TC: (a) improves cognitive function and reduces neuroinflammation in old wild-type mice; (b) preserves neuronal health with aging in mice and Caenorhabditis elegans; and (c) enhances cognitive function in a mouse model of tauopathy. We also provide insight on potential underlying mechanisms, as well as evidence of translational relevance for these observations by showing that TE transcripts accumulate with brain aging in humans, and that these age-related increases intersect with those observed in Alzheimer's disease. Collectively, our results suggest that TE transcript accumulation during aging may contribute to cognitive decline and neurodegeneration, and that targeting these events with reverse transcriptase inhibitors like 3TC could be a viable therapeutic strategy.

Neurotoxic effects of TDP-43 overexpression in C. elegans.

RNA-binding protein TDP-43 has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have engineered pan-neuronal expression of human TDP-43 protein in Caenorhabditis elegans, with the goal of generating a convenient in vivo model of TDP-43 function and neurotoxicity. Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses. Caenorhabditis elegans contains a single putative ortholog of TDP-43, designated TDP-1, which we show can support alternative splicing of CFTR in a cell-based assay. Neuronal overexpression of TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses. By using the uncoordinated phenotype as a read-out of TDP-43 overexpression neurotoxicty, we have investigated the contribution of specific TDP-43 domains and subcellular localization to toxicity. Full-length (wild-type) human TDP-43 expressed in C. elegans is localized to the nucleus. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. Interestingly, fusion of TDP-1 C-terminal sequences to TDP-43 missing its C-terminal domain restores normal subnuclear localization and toxicity in C. elegans and CFTR splicing in cell-based assays. Overexpression of wild-type, full-length TDP-43 in mammalian cells (differentiated M17 cells) can also result in cell toxicity. Our results demonstrate that in vivo TDP-43 neurotoxicity can result from nuclear activity of overexpressed full-length protein.

Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity.

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a approximately 25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.

Identifying an Optimal Neuroinflammation Treatment Using a Nanoligomer Discovery Engine.

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leading to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and it is causally linked to a range of neurodegenerative diseases such as Alzheimer's diseases (AD), Multiple Sclerosis (MS), Parkinson's disease (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier, making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer Discovery Engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1ß or IL-1ß, tumor necrosis factor-alpha or TNF-α, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-κß) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and artificial intelligence-based ranking method to design sequence-specific peptide molecules to up- or downregulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-κß (SB.201.17D.8_NF-κß1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low dose (5 mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.

Multiple neurodegenerative diseases are causally linked to aggregation-prone proteins. Cellular mechanisms involving protein turnover may be key defense mechanisms against aggregating protein disorders. We have used a transgenic Caenorhabditis elegans Alzheimer's disease model to identify cellular responses to proteotoxicity resulting from expression of the human beta amyloid peptide (Abeta). We show up-regulation of aip-1 in Abeta-expressing animals. Mammalian homologues of AIP-1 have been shown to associate with, and regulate the function of, the 26S proteasome, leading us to hypothesize that induction of AIP-1 may be a protective cellular response directed toward modulating proteasomal function in response to toxic protein aggregation. Using our transgenic model, we show that overexpression of AIP-1 protected against, while RNAi knockdown of AIP-1 exacerbated, Abeta toxicity. AIP-1 overexpression also reduced accumulation of Abeta in this model, which is consistent with AIP-1 enhancing protein degradation. Transgenic expression of one of the two human aip-1 homologues (AIRAPL), but not the other (AIRAP), suppressed Abeta toxicity in C. elegans, which advocates the biological relevance of the data to human biology. Interestingly, AIRAPL and AIP-1 contain a predicted farnesylation site, which is absent from AIRAP. This farnesylation site was shown by others to be essential for an AIP-1 prolongevity function. Consistent with this, we show that an AIP-1 mutant lacking the predicted farnesylation site failed to protect against Abeta toxicity. Our results implicate AIP-1 in the regulation of protein turnover and protection against Abeta toxicity and point at AIRAPL as the functional mammalian homologue of AIP-1.

Life-span extension by dietary restriction is mediated by NLP-7 signaling and coelomocyte endocytosis in C. elegans.

Recent studies have shown that the rate of aging can be modulated by diverse interventions. Dietary restriction is the most widely used intervention to promote longevity; however, the mechanisms underlying the effect of dietary restriction remain elusive. In a previous study, we identified two novel genes, nlp-7 and cup-4, required for normal longevity in Caenorhabditis elegans. nlp-7 is one of a set of neuropeptide-like protein genes; cup-4 encodes an ion-channel involved in endocytosis by coelomocytes. Here, we assess whether nlp-7 and cup-4 mediate longevity increases by dietary restriction. RNAi of nlp-7 or cup-4 significantly reduces the life span of the eat-2 mutant, a genetic model of dietary restriction, but has no effect on the life span of long-lived mutants resulting from reduced insulin/IGF-1 signaling or dysfunction of the mitochondrial electron transport chain. The life-span extension observed in wild-type N2 worms by dietary restriction using bacterial dilution is prevented significantly in nlp-7 and cup-4 mutants. RNAi knockdown of genes encoding candidate receptors of NLP-7 and genes involved in endocytosis by coelomocytes also specifically shorten the life span of the eat-2 mutant. We conclude that two novel pathways, NLP-7 signaling and endocytosis by coelomocytes, are required for life extension under dietary restriction in C. elegans.

Is There a Brain Microbiome?

Numerous studies have identified microbial sequences or epitopes in pathological and non-pathological human brain samples. It has not been resolved if these observations are artifactual, or truly represent population of the brain by microbes. Given the tempting speculation that resident microbes could play a role in the many neuropsychiatric and neurodegenerative diseases that currently lack clear etiologies, there is a strong motivation to determine the "ground truth" of microbial existence in living brains. Here I argue that the evidence for the presence of microbes in diseased brains is quite strong, but a compelling demonstration of resident microbes in the healthy human brain remains to be done. Dedicated animal models studies may be required to determine if there is indeed a "brain microbiome."

The gut microbiome-derived metabolite trimethylamine N-oxide modulates neuroinflammation and cognitive function with aging.

Aging is associated with declines in cognitive performance, which are mediated in part by neuroinflammation, characterized by astrocyte activation and higher levels of pro-inflammatory cytokines; however, the upstream drivers are unknown. We investigated the potential role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in modulating neuroinflammation and cognitive function with aging. Study 1: In middle-aged and older humans (65 ± 7 years), plasma TMAO levels were inversely related to performance on NIH Toolbox Cognition Battery tests of memory and fluid cognition (both r2 = 0.07, p < 0.05). Study 2: In mice, TMAO concentrations in plasma and the brain increased in parallel with aging (r2 = 0.60), suggesting TMAO crosses the blood-brain barrier. The greater TMAO concentrations in old mice (27 months) were associated with higher brain pro-inflammatory cytokines and markers of astrocyte activation vs. young adult mice (6 months). Study 3: To determine if TMAO independently induces an "aging-like" decline in cognitive function, young mice (6 months) were supplemented with TMAO in chow for 6 months. Compared with controls, TMAO-supplemented mice performed worse on the novel object recognition test, indicating impaired memory and learning, and had increased neuroinflammation and markers of astrocyte activation. Study 4: Human astrocytes cultured with TMAO vs. control media exhibited changes in cellular morphology and protein markers consistent with astrocyte activation, indicating TMAO directly acts on these cells. Our results provide translational insight into a novel pathway that modulates neuroinflammation and cognitive function with aging, and suggest that TMAO might be a promising target for prevention of neuroinflammation and cognitive decline with aging.