Nicotinamide Adenine Dinucleotide Precursor Supplementation Modulates Neurite Complexity and Survival in Motor Neurons from Amyotrophic Lateral Sclerosis Models.

Aims: Increasing nicotinamide adenine dinucleotide (NAD+) availability has been proposed as a therapeutic approach to prevent neurodegeneration in amyotrophic lateral sclerosis (ALS). Accordingly, NAD+ precursor supplementation appears to exert neuroprotective effects in ALS patients and mouse models. The mechanisms mediating neuroprotection remain uncertain but could involve changes in multiple cell types. We investigated a potential direct effect of the NAD+ precursor nicotinamide mononucleotide (NMN) on the health of cultured induced pluripotent stem cell (iPSC)-derived human motor neurons and in motor neurons isolated from two ALS mouse models, that is, mice overexpressing wild-type transactive response DNA binding protein-43 (TDP-43) or the ALS-linked human superoxide dismutase 1 with the G93A mutation (hSOD1G93A). Results: NMN treatment increased the complexity of neuronal processes in motor neurons isolated from both mouse models and in iPSC-derived human motor neurons. In addition, NMN prevented neuronal death induced by trophic factor deprivation. In mouse and human motor neurons expressing ALS-linked mutant superoxide dismutase 1, NMN induced an increase in glutathione levels, but this effect was not observed in nontransgenic or TDP-43 overexpressing motor neurons. In contrast, NMN treatment normalized the TDP-43 cytoplasmic mislocalization induced by its overexpression. Innovation: NMN can directly act on motor neurons to increase the growth and complexity of neuronal processes and prevent the death induced by trophic factor deprivation. Conclusion: Our results support a direct beneficial effect of NAD+ precursor supplementation on the maintenance of the neuritic arbor in motor neurons. Importantly, this was observed in motor neurons isolated from two different ALS models, with and without involvement of TDP-43 pathology, supporting its therapeutic potential in sporadic and familial ALS.

FABP7 drives an inflammatory response in human astrocytes and is upregulated in Alzheimer's disease.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is characterized by the accumulation of intracellular neurofibrillary tangles, extracellular amyloid plaques, and neuroinflammation. In partnership with microglial cells, astrocytes are key players in the regulation of neuroinflammation. Fatty acid binding protein 7 (FABP7) belongs to a family of conserved proteins that regulate lipid metabolism, energy homeostasis, and inflammation. FABP7 expression is largely restricted to astrocytes and radial glia-like cells in the adult central nervous system. We observed that treatment of primary hippocampal astrocyte cultures with amyloid ß fragment 25-35 (Aß25-35) induces FABP7 upregulation. In addition, FABP7 expression is upregulated in the brain of APP/PS1 mice, a widely used AD mouse model. Co-immunostaining with specific astrocyte markers revealed increased FABP7 expression in astrocytes. Moreover, astrocytes surrounding amyloid plaques displayed increased FABP7 staining when compared to non-plaque-associated astrocytes. A similar result was obtained in the brain of AD patients. Whole transcriptome RNA sequencing analysis of human astrocytes differentiated from induced pluripotent stem cells (i-astrocytes) overexpressing FABP7 identified 500 transcripts with at least a 2-fold change in expression. Gene Ontology enrichment analysis identified (i) positive regulation of cytokine production and (ii) inflammatory response as the top two statistically significant overrepresented biological processes. We confirmed that wild-type FABP7 overexpression induces an NF-κB-driven inflammatory response in human i-astrocytes. On the other hand, the expression of a ligand-binding impaired mutant FABP7 did not induce NF-κB activation. Together, our results suggest that the upregulation of FABP7 in astrocytes could contribute to the neuroinflammation observed in AD.

Nicotinamide Adenine Dinucleotide (NAD+)-Dependent Signaling in Neurological Disorders.

Significance: Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling processes, which couples the enzymatic degradation of NAD+ to posttranslational modifications of proteins or the production of second messengers. Cellular NAD+ levels are dynamically controlled by synthesis and degradation, and dysregulation of this balance has been associated with acute and chronic neuronal dysfunction. Recent Advances: A decline in NAD+ has been observed during normal aging and since aging is the primary risk factor for many neurological disorders, NAD+ metabolism has become a promising therapeutic target and prolific research field in recent years. Critical Issues: In many neurological disorders, either as a primary feature or as consequence of the pathological process, neuronal damage is accompanied by dysregulated mitochondrial homeostasis, oxidative stress, or metabolic reprogramming. Modulating NAD+ availability appears to have a protective effect against such changes observed in acute neuronal damage and age-related neurological disorders. Such beneficial effects could be, at least in part, due to the activation of NAD+-dependent signaling processes. Future Directions: While in many instances the protective effect has been ascribed to the activation of sirtuins, approaches that directly test the role of sirtuins or that target the NAD+ pool in a cell-type-specific manner may be able to provide further mechanistic insight. Likewise, these approaches may afford greater efficacy to strategies aimed at harnessing the therapeutic potential of NAD+-dependent signaling in neurological disorders. Antioxid. Redox Signal. 39, 1150-1166.

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.

Effects of RAGE inhibition on the progression of the disease in hSOD1G93A ALS mice.

Astrocytes play a key role in the progression of amyotrophic lateral sclerosis (ALS) by actively inducing the degeneration of motor neurons. Motor neurons isolated from receptor for advanced glycation end products (RAGE)-knockout mice are resistant to the neurotoxic signal derived from ALS-astrocytes. Here, we confirmed that in a co-culture model, the neuronal death induced by astrocytes over-expressing the ALS-linked mutant hSOD1G93A is prevented by the addition of the RAGE inhibitors FPS-ZM1 or RAP. These inhibitors also prevented the motor neuron death induced by spinal cord extracts from symptomatic hSOD1G93A mice. To evaluate the relevance of this neurotoxic mechanism in ALS pathology, we assessed the therapeutic potential of FPS-ZM1 in hSOD1G93A mice. FPS-ZM1 treatment significantly improved hind-limb grip strength in hSOD1G93A mice during the progression of the disease, reduced the expression of atrophy markers in the gastrocnemius muscle, improved the survival of large motor neurons, and reduced gliosis in the ventral horn of the spinal cord. However, we did not observe a statistically significant effect of the drug in symptoms onset nor in the survival of hSOD1G93A mice. Maintenance of hind-limb grip strength was also observed in hSOD1G93A mice with RAGE haploinsufficiency [hSOD1G93A ;RAGE(+/-)], further supporting the beneficial effect of RAGE inhibition on muscle function. However, no benefits were observed after complete RAGE ablation. Moreover, genetic RAGE ablation significantly shortened the median survival of hSOD1G93A mice. These results indicate that the advance of new therapies targeting RAGE in ALS demands a better understanding of its physiological role in a cell type/tissue-specific context.

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.

Redox Biology in Neurological Function, Dysfunction, and Aging.

Reduction oxidation (redox) reactions are central to life and when altered, they can promote disease progression. In the brain, redox homeostasis is recognized to be involved in all aspects of central nervous system (CNS) development, function, aging, and disease. Recent studies have uncovered the diverse nature by which redox reactions and homeostasis contribute to brain physiology, and when dysregulated to pathological consequences. Redox reactions go beyond what is commonly described as oxidative stress and involve redox mechanisms linked to signaling and metabolism. In contrast to the nonspecific nature of oxidative damage, redox signaling involves specific oxidation/reduction reactions that regulate a myriad of neurological processes such as neurotransmission, homeostasis, and degeneration. This Forum is focused on the role of redox metabolism and signaling in the brain. Six review articles from leading scientists in the field that appraise the role of redox metabolism and signaling in different aspects of brain biology including neurodevelopment, neurotransmission, aging, neuroinflammation, neurodegeneration, and neurotoxicity are included. An original research article exemplifying these concepts uncovers a novel link between oxidative modifications, redox signaling, and neurodegeneration. This Forum highlights the recent advances in the field and we hope it encourages future research aimed to understand the mechanisms by which redox metabolism and signaling regulate CNS physiology and pathophysiology. Antioxid. Redox Signal. 28, 1583-1586.

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.

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.

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.

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.

Electrophilic nitro-fatty acids prevent astrocyte-mediated toxicity to motor neurons in a cell model of familial amyotrophic lateral sclerosis via nuclear factor erythroid 2-related factor activation.

Nitro-fatty acids (NO2-FA) are electrophilic signaling mediators formed in tissues during inflammation, which are able to induce pleiotropic cytoprotective and antioxidant pathways including up regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) responsive genes. Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons associated to an inflammatory process that usually aggravates the disease progression. In ALS animal models, the activation of the transcription factor Nrf2 in astrocytes confers protection to neighboring neurons. It is currently unknown whether NO2-FA can exert protective activity in ALS through Nrf2 activation. Herein we demonstrate that nitro-arachidonic acid (NO2-AA) or nitro-oleic acid (NO2-OA) administrated to astrocytes expressing the ALS-linked hSOD1(G93A) induce antioxidant phase II enzyme expression through Nrf2 activation concomitant with increasing intracellular glutathione levels. Furthermore, treatment of hSOD1(G93A)-expressing astrocytes with NO2-FA prevented their toxicity to motor neurons. Transfection of siRNA targeted to Nrf2 mRNA supported the involvement of Nrf2 activation in NO2-FA-mediated protective effects. Our results show for the first time that NO2-FA induce a potent Nrf2-dependent antioxidant response in astrocytes capable of preventing motor neurons death in a culture model of ALS.

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.

Mitochondria-targeted catalase reverts the neurotoxicity of hSOD1G9³A astrocytes without extending the survival of ALS-linked mutant hSOD1 mice.

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1G93A astrocyte cultures. It also reverted the toxicity of hSOD1G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.

Absence of Nrf2 or its selective overexpression in neurons and muscle does not affect survival in ALS-linked mutant hSOD1 mouse models.

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant 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 and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1(G93A) mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.

Temporal patterns of tyrosine nitration in embryo heart development.

Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development.