Cell non-autonomous control of autophagy and metabolism by glial cells.

Glia are the protectors of the nervous system, providing neurons with support and protection from cytotoxic insults. We previously discovered that four astrocyte-like glia can regulate organismal proteostasis and longevity in C. elegans. Expression of the UPRER transcription factor, XBP-1s, in these glia increases stress resistance, and longevity, and activates the UPRER in intestinal cells via neuropeptides. Autophagy, a key regulator of metabolism and aging, has been described as a cell autonomous process. Surprisingly, we find that glial XBP-1s enhances proteostasis and longevity by cell non-autonomously reprogramming organismal lipid metabolism and activating autophagy. Glial XBP-1s regulates the activation of another transcription factor, HLH-30/TFEB, in the intestine. HLH-30 activates intestinal autophagy, increases intestinal lipid catabolism, and upregulates a robust transcriptional program. Our study reveals a novel role for glia in regulating peripheral lipid metabolism, autophagy, and organellar health through peripheral activation of HLH-30 and autophagy.

Glia of C. elegans coordinate a protective organismal heat shock response independent of the neuronal thermosensory circuit.

Aging organisms lose the ability to induce stress responses, becoming vulnerable to protein toxicity and tissue damage. Neurons can signal to peripheral tissues to induce protective organelle-specific stress responses. Recent work shows that glia can independently induce such responses. Here, we show that overexpression of heat shock factor 1 (hsf-1) in the four astrocyte-like cephalic sheath cells of Caenorhabditis elegans induces a non-cell-autonomous cytosolic unfolded protein response, also known as the heat shock response (HSR). These animals have increased lifespan and heat stress resistance and decreased protein aggregation. Glial HSR regulation is independent of canonical thermosensory circuitry and known neurotransmitters but requires the small clear vesicle release protein UNC-13. HSF-1 and the FOXO transcription factor DAF-16 are partially required in peripheral tissues for non-cell-autonomous HSR, longevity, and thermotolerance. Cephalic sheath glial hsf-1 overexpression also leads to pathogen resistance, suggesting a role for this signaling pathway in immune function.

Aging alters the metabolic flux signature of the ER-unfolded protein response in vivo in mice.

Age is a risk factor for numerous diseases, including neurodegenerative diseases, cancers, and diabetes. Loss of protein homeostasis is a central hallmark of aging. Activation of the endoplasmic reticulum unfolded protein response (UPRER ) includes changes in protein translation and membrane lipid synthesis. Using stable isotope labeling, a flux "signature" of the UPRER in vivo in mouse liver was developed by inducing ER stress with tunicamycin and measuring rates of both proteome-wide translation and de novo lipogenesis. Several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared with young mice. Binding immunoglobulin protein (BiP) synthesis rates and mRNA levels were increased more in aged than young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was seen only in the triglyceride fraction. These data indicate that aged mice have an exaggerated metabolic flux response to ER stress, which may indicate that aging renders the UPRER less effective in resolving proteotoxic stress.

ER Unfolded Protein Response in Liver In Vivo Is Characterized by Reduced, Not Increased, De Novo Lipogenesis and Cholesterol Synthesis Rates with Uptake of Fatty Acids from Adipose Tissue: Integrated Gene Expression, Translation Rates and Metabolic Fluxes.

The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases. Here, we characterize UPRER-induced metabolic changes in mouse livers in vivo through metabolic labeling and mass spectrometric analysis of lipid and proteome-wide fluxes. We induced UPRER by tunicamycin administration and measured synthesis rates of proteins, fatty acids and cholesterol, as well as RNA-seq. Contrary to reports in isolated cells, hepatic de novo lipogenesis and cholesterogenesis were markedly reduced, as were mRNA levels and synthesis rates of lipogenic proteins. H&E staining showed enrichment with lipid droplets while electron microscopy revealed ER morphological changes. Interestingly, the pre-labeling of adipose tissue prior to UPRER induction resulted in the redistribution of labeled fatty acids from adipose tissue to the liver, with replacement by unlabeled glycerol in the liver acylglycerides, indicating that the liver uptake was of free fatty acids, not whole glycerolipids. The redistribution of adipose fatty acids to the liver was not explicable by altered plasma insulin, increased fatty acid levels (lipolysis) or by reduced food intake. Synthesis of most liver proteins was suppressed under UPRER conditions, with the exception of BiP, other chaperones, protein disulfide isomerases, and proteins of ribosomal biogenesis. Protein synthesis rates generally, but not always, paralleled changes in mRNA. In summary, this combined approach, linking static changes with fluxes, revealed an integrated reduction of lipid and cholesterol synthesis pathways, from gene expression to translation and metabolic flux rates, under UPRER conditions. The reduced lipogenesis does not parallel human fatty liver disease. This approach provides powerful tools to characterize metabolic processes underlying hepatic UPRER in vivo.

Measurements of Physiological Stress Responses in C. Elegans.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Four glial cells regulate ER stress resistance and longevity via neuropeptide signaling in C. elegans.

The ability of the nervous system to sense cellular stress and coordinate protein homeostasis is essential for organismal health. Unfortunately, stress responses that mitigate disturbances in proteostasis, such as the unfolded protein response of the endoplasmic reticulum (UPRER), become defunct with age. In this work, we expressed the constitutively active UPRER transcription factor, XBP-1s, in a subset of astrocyte-like glia, which extended the life span in Caenorhabditis elegans Glial XBP-1s initiated a robust cell nonautonomous activation of the UPRER in distal cells and rendered animals more resistant to protein aggregation and chronic ER stress. Mutants deficient in neuropeptide processing and secretion suppressed glial cell nonautonomous induction of the UPRER and life-span extension. Thus, astrocyte-like glial cells play a role in regulating organismal ER stress resistance and longevity.

The UPRER: Sensor and Coordinator of Organismal Homeostasis.

Life is stressful. Organisms are repeatedly exposed to stressors that disrupt protein homeostasis (proteostasis), resulting in protein misfolding and aggregation. To sense and respond to proteotoxic perturbations, cells have evolved compartment-specific stress responses, such as the unfolded protein response of the endoplasmic reticulum (UPRER). However, UPRER function is impaired with age, which, we propose, creates a permissive environment for protein aggregation, unresolved ER stress, and chronic inflammation. Understanding age-related changes to the UPRER will provide new avenues for therapeutic intervention in metabolic disease, neurodegeneration, and aging.

Additive amelioration of ALS by co-targeting independent pathogenic mechanisms.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which glia are central mediators of motor neuron (MN) death. Since multiple cell types are involved in disease pathogenesis, the objective of this study was to determine the benefit of co-targeting independent pathogenic mechanisms in a familial ALS mouse model. METHODS: Recently, our laboratory identified that ALS microglia induce MN death in an NF-κB-dependent mechanism. We also demonstrated that a single, post-natal, intravenous injection of adeno-associated viral vector serotype 9 encoding a shRNA against mutant SOD1 is able to traverse the blood-brain barrier of ALS mice and reduce SOD1-expression in astrocytes and MNs. Reducing mutant SOD1 in MNs and astrocytes led to a robust increase in survival. To evaluate the benefit of co-targeting multiple cell types in ALS, we combined microglial NF-κB suppression with SOD1 reduction in astrocytes and MNs. RESULTS: Targeting both astrocytes and microglia resulted in an additive increase in survival and motor function by delaying both onset and progression. Strikingly, targeting all three cell types (astrocytes, motor neurons [MNs], and microglia) resulted in an additive increase in lifespan and motor function, with maximum survival reaching 204 days, 67 days longer than the mean survival of untreated control animals. INTERPRETATION: Our data suggest that a combinatorial approach co-targeting different pathogenic mechanisms in independent cell types is a beneficial therapeutic strategy for ALS.

rAAV Gene Therapy in a Canavan's Disease Mouse Model Reveals Immune Impairments and an Extended Pathology Beyond the Central Nervous System.

Aspartoacylase (AspA) gene mutations cause the pediatric lethal neurodegenerative Canavan disease (CD). There is emerging promise of successful gene therapy for CD using recombinant adeno-associated viruses (rAAVs). Here, we report an intracerebroventricularly delivered AspA gene therapy regime using three serotypes of rAAVs at a 20-fold reduced dose than previously described in AspA(-/-) mice, a bona-fide mouse model of CD. Interestingly, central nervous system (CNS)-restricted therapy prolonged survival over systemic therapy in CD mice but failed to sustain motor functions seen in systemically treated mice. Importantly, we reveal through histological and functional examination of untreated CD mice that AspA deficiency in peripheral tissues causes morphological and functional abnormalities in this heretofore CNS-defined disorder. We demonstrate for the first time that AspA deficiency, possibly through excessive N-acetyl aspartic acid accumulation, elicits both a peripheral and CNS immune response in CD mice. Our data establish a role for peripheral tissues in CD pathology and serve to aid the development of more efficacious and sustained gene therapy for this disease.

Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis.

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.

An NF-κB--EphrinA5-Dependent Communication between NG2(+) Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates.

Skeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2(+) interstitial cells and myoblasts. NF-κB in NG2(+) cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2(+) cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth.

Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis.

Neuroinflammation is one of the most striking hallmarks of amyotrophic lateral sclerosis (ALS). Nuclear factor-kappa B (NF-κB), a master regulator of inflammation, is upregulated in spinal cords of ALS patients and SOD1-G93A mice. In this study, we show that selective NF-κB inhibition in ALS astrocytes is not sufficient to rescue motor neuron (MN) death. However, the localization of NF-κB activity and subsequent deletion of NF-κB signaling in microglia rescued MNs from microglial-mediated death in vitro and extended survival in ALS mice by impairing proinflammatory microglial activation. Conversely, constitutive activation of NF-κB selectively in wild-type microglia induced gliosis and MN death in vitro and in vivo. Taken together, these data provide a mechanism by which microglia induce MN death in ALS and suggest a novel therapeutic target that can be modulated to slow the progression of ALS and possibly other neurodegenerative diseases by which microglial activation plays a role.