GTP energy dependence of endocytosis and autophagy in the aging brain and Alzheimer's disease.

Increased interest in the aging and Alzheimer's disease (AD)-related impairments in autophagy in the brain raise important questions about regulation and treatment. Since many steps in endocytosis and autophagy depend on GTPases, new measures of cellular GTP levels are needed to evaluate energy regulation in aging and AD. The recent development of ratiometric GTP sensors (GEVALS) and findings that GTP levels are not homogenous inside cells raise new issues of regulation of GTPases by the local availability of GTP. In this review, we highlight the metabolism of GTP in relation to the Rab GTPases involved in formation of early endosomes, late endosomes, and lysosomal transport to execute the autophagic degradation of damaged cargo. Specific GTPases control macroautophagy (mitophagy), microautophagy, and chaperone-mediated autophagy (CMA). By inference, local GTP levels would control autophagy, if not in excess. Additional levels of control are imposed by the redox state of the cell, including thioredoxin involvement. Throughout this review, we emphasize the age-related changes that could contribute to deficits in GTP and AD. We conclude with prospects for boosting GTP levels and reversing age-related oxidative redox shift to restore autophagy. Therefore, GTP levels could regulate the numerous GTPases involved in endocytosis, autophagy, and vesicular trafficking. In aging, metabolic adaptation to a sedentary lifestyle could impair mitochondrial function generating less GTP and redox energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation.

NR1D1 downregulation in astrocytes induces a phenotype that is detrimental to cocultured motor neurons.

Nuclear receptor subfamily 1 group D member 1 (NR1D1, also known as Rev-erbα) is a nuclear transcription factor that is part of the molecular clock encoding circadian rhythms and may link daily rhythms with metabolism and inflammation. NR1D1, unlike most nuclear receptors, lacks a ligand-dependent activation function domain 2 and is a constitutive transcriptional repressor. Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Approximately 10%-20% of familial ALS is caused by a toxic gain-of-function induced by mutations of the Cu/Zn superoxide dismutase (SOD1). Dysregulated clock and clock-controlled gene expression occur in multiple tissues from mutant hSOD1-linked ALS mouse models. Here we explore NR1D1 dysregulation in the spinal cord of ALS mouse models and its consequences on astrocyte-motor neuron interaction. NR1D1 protein and mRNA expression are significantly downregulated in the spinal cord of symptomatic mice expressing mutant hSOD1, while no changes were observed in age-matched animals overexpressing wild-type hSOD1. In addition, NR1D1 downregulation in primary astrocyte cultures induces a pro-inflammatory phenotype and decreases the survival of cocultured motor neurons. NR1D1 orchestrates the cross talk between physiological pathways identified to be disrupted in ALS (e.g., metabolism, inflammation, redox homeostasis, and circadian rhythms) and we observed that downregulation of NR1D1 alters astrocyte-motor neuron interaction. Our results suggest that NR1D1 could be a potential therapeutic target to prevent astrocyte-mediated motor neuron toxicity in ALS.

Altered expression of clock and clock-controlled genes in a hSOD1-linked amyotrophic lateral sclerosis mouse model.

Most physiological processes in mammals are subjected to daily oscillations that are governed by a circadian system. The circadian rhythm orchestrates metabolic pathways in a time-dependent manner and loss of circadian timekeeping has been associated with cellular and system-wide alterations in metabolism, redox homeostasis, and inflammation. Here, we investigated the expression of clock and clock-controlled genes in multiple tissues (suprachiasmatic nucleus, spinal cord, gastrocnemius muscle, and liver) from mutant hSOD1-linked amyotrophic lateral sclerosis (ALS) mouse models. We identified tissue-specific changes in the relative expression, as well as altered daily expression patterns, of clock genes, sirtuins (Sirt1, Sirt3, and Sirt6), metabolic enzymes (Pfkfb3, Cpt1, and Nampt), and redox regulators (Nrf2, G6pd, and Pgd). In addition, astrocytes transdifferentiated from induced pluripotent stem cells from SOD1-linked and FUS RNA binding protein-linked ALS patients also displayed altered expression of clock genes. Overall, our results raise the possibility of disrupted cross-talk between the suprachiasmatic nucleus and peripheral tissues in hSOD1G93A mice, preventing proper peripheral clock regulation and synchronization. Since these changes were observed in symptomatic mice, it remains unclear whether this dysregulation directly drives or it is a consequence of the degenerative process. However, because metabolism and redox homeostasis are intimately entangled with circadian rhythms, our data suggest that altered expression of clock genes may contribute to metabolic and redox impairment in ALS. Since circadian dyssynchrony can be rescued, these results provide the groundwork for potential disease-modifying interventions.

FABP7 upregulation induces a neurotoxic phenotype in astrocytes.

Fatty acid binding proteins (FABPs) are key regulators of lipid metabolism, energy homeostasis, and inflammation. They participate in fatty acid metabolism by regulating their uptake, transport, and availability of ligands to nuclear receptors. In the adult brain, FABP7 is especially abundant in astrocytes that are rich in cytoplasmic granules originated from damaged mitochondria. Mitochondrial dysfunction and oxidative stress have been implicated in the neurodegenerative process observed in amyotrophic lateral sclerosis (ALS), either as a primary cause or as a secondary component of the pathogenic process. Here we investigated the expression of FABP7 in animal models of human superoxide dismutase 1 (hSOD1)-linked ALS. In the spinal cord of symptomatic mutant hSOD1-expressing mice, FABP7 is upregulated in gray matter astrocytes. Using a coculture model, we examined the effect of increased FABP7 expression in astrocyte-motor neuron interaction. Our data show that FABP7 overexpression directly promotes an NF-κB-driven pro-inflammatory response in nontransgenic astrocytes that ultimately is detrimental for motor neuron survival. Addition of trophic factors, capable of supporting motor neuron survival in pure cultures, did not prevent motor neuron loss in cocultures with FABP7 overexpressing astrocytes. In addition, astrocyte cultures obtained from symptomatic hSOD1-expressing mice display upregulated FABP7 expression. Silencing endogenous FABP7 in these cultures decreases the expression of inflammatory markers and their toxicity toward cocultured motor neurons. Our results identify a key role of FABP7 in the regulation of the inflammatory response in astrocytes and identify FABP7 as a potential therapeutic target to prevent astrocyte-mediated motor neuron toxicity in ALS.

Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons. Astrocytes from diverse ALS models induce motor neuron death in co-culture. Enhancing NAD+ availability, or increasing the expression of the NAD+-dependent deacylases SIRT3 and SIRT6, abrogates their neurotoxicity in cell culture models. To determine the effect of increasing NAD+ availability in ALS mouse models we used two strategies, ablation of a NAD+-consuming enzyme (CD38) and supplementation with a bioavailable NAD+ precursor (nicotinamide riboside, NR). Deletion of CD38 had no effect in the survival of two hSOD1-linked ALS mouse models. On the other hand, NR-supplementation delayed motor neuron degeneration, decreased markers of neuroinflammation in the spinal cord, appeared to modify muscle metabolism and modestly increased the survival of hSOD1G93A mice. In addition, we found altered expression of enzymes involved in NAD+ synthesis (NAMPT and NMNAT2) and decreased SIRT6 expression in the spinal cord of ALS patients, suggesting deficits of this neuroprotective pathway in the human pathology. Our data denotes the therapeutic potential of increasing NAD+ levels in ALS. Moreover, the results indicate that the approach used to enhance NAD+ levels critically defines the biological outcome in ALS models, suggesting that boosting NAD+ levels with the use of bioavailable precursors would be the preferred therapeutic strategy for ALS.

Enhanced SIRT6 activity abrogates the neurotoxic phenotype of astrocytes expressing ALS-linked mutant SOD1.

Sirtuins (SIRTs) are NAD+-dependent deacylases that play a key role in transcription, DNA repair, metabolism, and oxidative stress resistance. Increasing NAD+ availability regulates endogenous SIRT activity, leading to increased resistance to oxidative stress and decreased mitochondrial reactive oxygen production in multiple cell types and disease models. This protection, at least in part, depends on the activation of antioxidant mitochondrial proteins. We now show that increasing total NAD+ content in astrocytes leads to the activation of the transcription factor nuclear factor, erythroid-derived 2, like 2 (Nfe2l2 or Nrf2) and up-regulation of the antioxidant proteins heme oxygenase 1 (HO-1) and sulfiredoxin 1 (SRXN1). Nrf2 activation also occurs as a result of SIRT6 overexpression. Mutations in Cu-Zn superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS). Astrocytes isolated from mutant human SOD1-overexpressing mice induce motor neuron death in coculture. Treatment with nicotinamide mononucleotide or nicotinamide riboside increases total NAD+ content in ALS astrocytes and abrogates their toxicity toward cocultured motor neurons. The observed neuroprotection depends on SIRT6 expression in astrocytes. Moreover, overexpression of SIRT6 in astrocytes by itself abrogates the neurotoxic phenotype of ALS astrocytes. Our results identify SIRT6 as a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.-Harlan, B. A., Pehar, M., Killoy, K. M., Vargas, M. R. Enhanced SIRT6 activity abrogates the neurotoxic phenotype of astrocytes expressing ALS-linked mutant SOD1.

Opposing actions of CRF-R1 and CB1 receptors on VTA-GABAergic plasticity following chronic exposure to ethanol.

Dopamine neurons in the ventral tegmental area (VTA) influence learned behaviors and neuropsychiatric diseases including addiction. The stress peptide corticotrophin-releasing factor (CRF) contributes to relapse to drug and alcohol seeking following withdrawal, although the cellular actions are poorly understood. In this study, we show that presynaptic CRF type 1 receptors (CRF-R1) potentiate GABA release onto mouse VTA dopamine neurons via a PKC-Ca2+ signaling mechanism. In naive animals, activation of CRF-R1 by bath application of CRF or ethanol enhanced GABAA inhibitory postsynaptic currents (IPSCs). Following 3 days of withdrawal from four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure, spontaneous IPSC frequency was enhanced while CRF and ethanol potentiation of IPSCs was intact. However, withdrawal for 3 weeks or more was associated with reduced spontaneous IPSC frequency and diminished CRF and ethanol responses. Long-term withdrawal was also accompanied by decreased sensitivity to the CB1 receptor agonist WIN55212 as well as greatly enhanced sensitivity to the CB1 antagonist AM251. Inclusion of BAPTA in the internal recording solution restored the responsiveness to CRF or ethanol and reduced the potentiating actions of AM251. Together, these data suggest that GABAA inhibition of VTA dopamine neurons is regulated by presynaptic actions of CRF and endocannabinoids and that long-term withdrawal from CIE treatment enhances endocannabinoid-mediated inhibition, thereby suppressing CRF facilitation of GABA release. Such findings have implications for understanding the impact of chronic alcohol on stress-related, dopamine-mediated alcohol-seeking behaviors.

Decreased glutathione levels cause overt motor neuron degeneration in hSOD1WT over-expressing mice.

Mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. Several lines of evidence have shown that SOD1 mutations cause ALS through a gain of a toxic function that remains to be fully characterized. A significant share of our understanding of the mechanisms underlying the neurodegenerative process in ALS comes from the study of rodents over-expressing ALS-linked mutant hSOD1. These mutant hSOD1 models develop an ALS-like phenotype. On the other hand, hemizygous mice over-expressing wild-type hSOD1 at moderate levels (hSOD1WT, originally described as line N1029) do not develop paralysis or shortened life-span. To investigate if a decrease in antioxidant defenses could lead to the development of an ALS-like phenotype in hSOD1WT mice, we used knockout mice for the glutamate-cysteine ligase modifier subunit [GCLM(-/-)]. GCLM(-/-) mice are viable and fertile but display a 70-80% reduction in total glutathione levels. GCLM(-/-)/hSOD1WT mice developed overt motor symptoms (e.g. tremor, loss of extension reflex in hind-limbs, decreased grip strength and paralysis) characteristic of mice models over-expressing ALS-linked mutant hSOD1. In addition, GCLM(-/-)/hSOD1WT animals displayed shortened life span. An accelerated decrease in the number of large neurons in the ventral horn of the spinal cord and degeneration of spinal root axons was observed in symptomatic GCLM(-/-)/hSOD1WT mice when compared to age-matched GCLM(+/+)/hSOD1WT mice. Our results show that under conditions of chronic decrease in glutathione, moderate over-expression of wild-type SOD1 leads to overt motor neuron degeneration, which is similar to that induced by ALS-linked mutant hSOD1 over-expression.

Nitration and Glycation Turn Mature NGF into a Toxic Factor for Motor Neurons: A Role for p75NTR and RAGE Signaling in ALS.

INTRODUCTION: Glycating stress can occur together with oxidative stress during neurodegeneration and contribute to the pathogenic mechanism. Nerve growth factor (NGF) accumulates in several neurodegenerative diseases. Besides promoting survival, NGF can paradoxically induce cell death by signaling through the p75 neurotrophin receptor (p75NTR). The ability of NGF to induce cell death is increased by nitration of its tyrosine residues under conditions associated with increased peroxynitrite formation. AIMS: Here we investigated whether glycation also changes the ability of NGF to induce cell death and assessed the ability of post-translational modified NGF to signal through the receptor for advanced glycation end products (RAGEs). We also explored the potential role of RAGE-p75NTR interaction in the motor neuron death occurring in amyotrophic lateral sclerosis (ALS) models. RESULTS: Glycation promoted NGF oligomerization and ultimately allowed the modified neurotrophin to signal through RAGE and p75NTR to induce motor neuron death at low physiological concentrations. A similar mechanism was observed for nitrated NGF. We provide evidence for the interaction of RAGE with p75NTR at the cell surface. Moreover, we observed that post-translational modified NGF was present in the spinal cord of an ALS mouse model. In addition, NGF signaling through RAGE and p75NTR was involved in astrocyte-mediated motor neuron toxicity, a pathogenic feature of ALS. INNOVATION: Oxidative modifications occurring under stress conditions can enhance the ability of mature NGF to induce neuronal death at physiologically relevant concentrations, and RAGE is a new p75NTR coreceptor contributing to this pathway. CONCLUSION: Our results indicate that NGF-RAGE/p75NTR signaling may be a therapeutic target in ALS. Antioxid. Redox Signal. 28, 1587-1602.

Nicotinamide Adenine Dinucleotide Metabolism and Neurodegeneration.

SIGNIFICANCE: Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling processes, which involve the cleavage of NAD+ coupled to posttranslational modifications of proteins or the production of second messengers. Either as a primary cause or as a secondary component of the pathogenic process, mitochondrial dysfunction and oxidative stress are prominent features of several neurodegenerative diseases. Activation of NAD+-dependent signaling pathways has a major effect in the capacity of the cell to modulate mitochondrial function and counteract the deleterious effects of increased oxidative stress. Recent Advances: Progress in the understanding of the biological functions and compartmentalization of NAD+-synthesizing and NAD+-consuming enzymes have led to the emergence of NAD+ metabolism as a major therapeutic target for age-related diseases. CRITICAL ISSUES: Three distinct families of enzymes consume NAD+ as substrate: poly(ADP-ribose) polymerases (PARPs), ADP-ribosyl cyclases (CD38/CD157) and sirtuins. Two main strategies to increase NAD+ availability have arisen. These strategies are based on the utilization of NAD+ intermediates/precursors or the inhibition of the NAD+-consuming enzymes, PARPs and CD38. An increase in endogenous sirtuin activity seems to mediate the protective effect that enhancing NAD+ availability confers in several models of neurodegeneration and age-related diseases. FUTURE DIRECTIONS: A growing body of evidence suggests the beneficial role of enhancing NAD+ availability in models of neurodegeneration. The challenge ahead is to establish the value and safety of the long-term use of these strategies for the treatment of neurodegenerative diseases. Antioxid. Redox Signal. 28, 1652-1668.

Role and Therapeutic Potential of Astrocytes in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. The molecular mechanism underlying the progressive degeneration of motor neuron remains uncertain but involves a non-cell autonomous process. In acute injury or degenerative diseases astrocytes adopt a reactive phenotype known as astrogliosis. Astrogliosis is a complex remodeling of astrocyte biology and most likely represents a continuum of potential phenotypes that affect neuronal function and survival in an injury-specific manner. In ALS patients, reactive astrocytes surround both upper and lower degenerating motor neurons and play a key role in the pathology. It has become clear that astrocytes play a major role in ALS pathology. Through loss of normal function or acquired new characteristics, astrocytes are able to influence motor neuron fate and the progression of the disease. The use of different cell culture models indicates that ALS-astrocytes are able to induce motor neuron death by secreting a soluble factor(s). Here, we discuss several pathogenic mechanisms that have been proposed to explain astrocyte-mediated motor neuron death in ALS. In addition, examples of strategies that revert astrocyte-mediated motor neuron toxicity are reviewed to illustrate the therapeutic potential of astrocytes in ALS. Due to the central role played by astrocytes in ALS pathology, therapies aimed at modulating astrocyte biology may contribute to the development of integral therapeutic approaches to halt ALS progression.

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.

Changes in Protein Expression and Lysine Acetylation Induced by Decreased Glutathione Levels in Astrocytes.

Astrocytes and neurons form a highly specialized functional unit, and the loss or gain of astrocytic functions can influence the initiation and progression of different neurodegenerative diseases. Neurons depend on the antioxidant protection provided by neighboring astrocytes. Glutathione (γ-l-glutamyl-l-cysteinyl-glycine) is a major component of the antioxidant system that defends cells against the toxic effects of reactive oxygen/nitrogen species. A decline in glutathione levels has been observed in aging and neurodegenerative diseases, and it aggravates the pathology in an amyotrophic lateral sclerosis-mouse model. Using a SILAC-based quantitative proteomic approach, we analyzed changes in global protein expression and lysine acetylation in primary astrocyte cultures obtained from wild-type mice or those deficient in the glutamate-cysteine ligase modifier subunit (GCLM). GCLM knockout astrocytes display an ∼80% reduction in total glutathione levels. We identified potential molecular targets and novel sites of acetylation that are affected by the chronic decrease in glutathione levels and observed a response mediated by Nrf2 activation. In addition, sequence analysis of peptides displaying increased acetylation in GCLM knockout astrocytes revealed an enrichment of cysteine residues in the vicinity of the acetylation site, which suggests potential crosstalk between lysine-acetylation and cysteine modification. Regulation of several metabolic and antioxidant pathways was observed at the level of protein expression and lysine acetylation, revealing a coordinated response involving transcriptional and posttranslational regulation.