Marcelo R. Vargas

Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson's disease: Critical role for the astrocyte

Oxidative stress has been implicated in the etiology of Parkinsons disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. It is known that under conditions of oxidative stress, the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. To investigate the role of Nrf2 in the process of MPTP-induced toxicity, mice expressing the human placental alkaline phosphatase (hPAP) gene driven by a promoter containing a core ARE sequence (ARE-hPAP) were used. ARE-hPAP mice were injected (30 mg/kg) once per day for 5 days and killed 7 days after the last MPTP injection. In response to this design, ARE-dependent gene expression was decreased in striatum whereas it was increased in substantia nigra. The same MPTP protocol was applied in Nrf2+/+ and Nrf2−/− mice; Nrf2 deficiency increases MPTP sensitivity. Furthermore, we evaluated the potential for astrocytic Nrf2 overexpression to protect from MPTP toxicity. Transgenic mice with Nrf2 under control of the astrocyte-specific promoter for the glial fribillary acidic protein (GFAP-Nrf2) on both a Nrf2+/+ and Nrf2−/− background were administered MPTP. In the latter case, only the astrocytes expressed Nrf2. Independent of background, MPTP-mediated toxicity was abolished in GFAP-Nrf2 mice. These striking results indicate that Nrf2 expression restricted to astrocytes is sufficient to protect against MPTP and astrocytic modulation of the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing neuronal death in PD.

The Nrf2–ARE Pathway

Transcriptional activation of protective genes is mediated by a cis‐acting element called the antioxidant responsive element (ARE). The transcription factor Nrf2 (NF–E2‐related factor 2) binds to the ARE. Activation of this pathway protects cells from oxidative stress‐induced cell death. Increased oxidative stress is associated with neuronal cell death during the pathogenesis of multiple chronic neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, Huntingtons disease, and amyotrophic lateral sclerosis. We hypothesize that Nrf2–ARE activation is a novel neuroprotective pathway that confers resistance to a variety of oxidative, stress‐related, neurodegenerative insults. In recent studies, primary neuronal cultures treated with chemical activators of the Nrf2–ARE pathway displayed significantly greater resistance to oxidative stress‐induced neurotoxicity. Similar cultures generated from ARE–hPAP reporter mice demonstrated selective activation of the Nrf2–ARE pathway in astrocytes, suggesting that Nrf2 activation in astrocytes somehow confers resistance to naive neurons. Further, in chemical models of neurodegeneration, Nrf2 knockout mice are significantly more sensitive to mitochondrial complex I and II inhibitors. Combining these observations with the results implying that the astrocyte is central to Nrf2–ARE‐mediated neuroprotection, we transplanted Nrf2‐overexpressing astrocytes into the mouse striatum prior to lesioning with malonate. This procedure led to dramatic protection against malonate‐induced neurotoxicity. Translating this to other chemical and genetic models of neurodegeneration will be discussed.

Nrf2 Activation in Astrocytes Protects against Neurodegeneration in Mouse Models of Familial Amyotrophic Lateral Sclerosis

Activation of the transcription factor Nrf2 in astrocytes coordinates the upregulation of antioxidant defenses and confers protection to neighboring neurons. 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. Non-neuronal cells, including astrocytes, shape motor neuron survival in ALS and are a potential target to prevent motor neuron degeneration. The protective effect of Nrf2 activation in astrocytes has never been examined in a chronic model of neurodegeneration. We generated transgenic mice over-expressing Nrf2 selectively in astrocytes using the glial fibrillary acidic protein (GFAP) promoter. The toxicity of astrocytes expressing ALS-linked mutant hSOD1 to cocultured motor neurons was reversed by Nrf2 over-expression. Motor neuron protection depended on increased glutathione secretion from astrocytes. This protective effect was also observed by crossing the GFAP-Nrf2 mice with two ALS-mouse models. Over-expression of Nrf2 in astrocytes significantly delayed onset and extended survival. These findings demonstrate that Nrf2 activation in astrocytes is a viable therapeutic target to prevent chronic neurodegeneration.

Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis.

Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS‐mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75NTR) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75NTR was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75NTR +/+ mouse embryos but had no effect in p75NTR –/– knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice‐stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75NTR blocking‐antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75‐expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.

Increased glutathione biosynthesis by Nrf2 activation in astrocytes prevents p75NTR‐dependent motor neuron apoptosis

Astrocytes may modulate the survival of motor neurons in amyotrophic lateral sclerosis (ALS). We have previously shown that fibroblast growth factor‐1 (FGF‐1) activates astrocytes to increase secretion of nerve growth factor (NGF). NGF in turn induces apoptosis in co‐cultured motor neurons expressing the p75 neurotrophin receptor (p75NTR) by a mechanism involving nitric oxide (NO) and peroxynitrite formation. We show here that FGF‐1 increased the expression of inducible nitric oxide synthase and NO production in astrocytes, making adjacent motor neurons vulnerable to NGF‐induced apoptosis. Spinal cord astrocytes isolated from transgenic SOD1G93A rats displayed increased NO production and spontaneously induced apoptosis of co‐cultured motor neurons. FGF‐1 also activates the redox‐sensitive transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2) in astrocytes. Because Nrf2 increases glutathione (GSH) biosynthesis, we investigated the role of GSH production by astrocytes on p75NTR‐dependent motor neuron apoptosis. The combined treatment of astrocytes with FGF‐1 and t‐butylhydroquinone (tBHQ) increased GSH production and secretion, preventing motor neuron apoptosis. Moreover, Nrf2 activation in SOD1G93A astrocytes abolished their apoptotic activity. The protection exerted by increased Nrf2 activity was overcome by adding the NO donor DETA‐NONOate to the co‐cultures or by inhibiting GSH synthesis and release from astrocytes. These results suggest that activation of Nrf2 in astrocytes can reduce NO‐dependent toxicity to motor neurons by increasing GSH biosynthesis.

Mitochondrial Superoxide Production and Nuclear Factor Erythroid 2-Related Factor 2 Activation in p75 Neurotrophin Receptor-Induced Motor Neuron Apoptosis

Nerve growth factor (NGF) can induce apoptosis by signaling through the p75 neurotrophin receptor (p75NTR) in several nerve cell populations. Cultured embryonic motor neurons expressing p75NTR are not vulnerable to NGF unless they are exposed to an exogenous flux of nitric oxide (•NO). In the present study, we show that p75NTR-mediated apoptosis in motor neurons involved neutral sphingomyelinase activation, increased mitochondrial superoxide production, and cytochrome c release to the cytosol. The mitochondria-targeted antioxidants mitoQ and mitoCP prevented neuronal loss, further evidencing the role of mitochondria in NGF-induced apoptosis. In motor neurons overexpressing the amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1G93A (SOD1G93A) mutation, NGF induced apoptosis even in the absence of an external source of •NO. The increased susceptibility of SOD1G93A motor neurons to NGF was associated to decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and downregulation of the enzymes involved in glutathione biosynthesis. In agreement, depletion of glutathione in nontransgenic motor neurons reproduced the effect of SOD1G93A expression, increasing their sensitivity to NGF. In contrast, rising antioxidant defenses by Nrf2 activation prevented NGF-induced apoptosis. Together, our data indicate that p75NTR-mediated motor neuron apoptosis involves ceramide-dependent increased mitochondrial superoxide production. This apoptotic pathway is facilitated by the expression of ALS-linked SOD1 mutations and critically modulated by Nrf2 activity.

Astrogliosis in Amyotrophic Lateral Sclerosis: Role and Therapeutic Potential of Astrocytes

SummaryAmyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by the progressive loss of motor neurons. Although the molecular mechanism underlying motor neuron degeneration remains unknown; non-neuronal cells (including astrocytes) shape motor neuron survival in ALS. Astrocytes closely interact with neurons to provide an optimized environment for neuronal function and respond to all forms of injury in a typical manner known as reactive astrogliosis. A strong reactive astrogliosis surrounds degenerating motor neurons in ALS patients and ALS-animal models. Although reactive astrogliosis in ALS is probably both primary and secondary to motor neuron degeneration; astrocytes are not passive observers and they can influence motor neuron fate. Due to the important functions that astrocytes perform in the central nervous system; it is of key importance to understand how these functions are altered when astrocytes become reactive in ALS. Here; we review the current evidences supporting a potential toxic role of astrocytes and their viability as therapeutic targets to alter motor neuron degeneration in ALS.

Astrocyte-Specific Overexpression of Nrf2 Delays Motor Pathology and Synuclein Aggregation throughout the CNS in the Alpha-Synuclein Mutant (A53T) Mouse Model

Alpha synuclein (SYN) is a central player in the pathogenesis of sporadic and familial Parkinsons disease (PD). SYN aggregation and oxidative stress are associated and enhance each others toxicity. It is unknown whether the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a role against the toxicity of SYN. To examine this, mice selectively overexpressing Nrf2 in astrocytes (GFAP-Nrf2) were crossed with mice selectively expressing human mutant SYN (hSYNA53T) in neurons. Increased astrocytic Nrf2 delayed the onset and extended the life span of the hSYNA53T mice. This correlated with increased motor neuron survival, reduced oxidative stress, and attenuated gliosis in the spinal cord, as well as a dramatic decrease in total hSYNA53T and phosphorylated (Ser129) hSYNA53T in Triton-insoluble aggregates. Furthermore, Nrf2 in astrocytes delayed chaperone-mediated autophagy and macroautophagy dysfunction observed in the hSYNA53T mice. Our data suggest that Nrf2 in astrocytes provides neuroprotection against hSYNA53T-mediated toxicity by promoting the degradation of hSYNA53T through the autophagy-lysosome pathway in vivo. Thus, activation of the Nrf2 pathway in astrocytes is a potential target to develop therapeutic strategies for treating pathologic synucleinopathies including PD.

Complexity of Astrocyte-Motor Neuron Interactions in Amyotrophic Lateral Sclerosis

Neurons and surrounding glial cells compose a highly specialized functional unit. In amyotrophic lateral sclerosis (ALS) astrocytes interact with motor neurons in a complex manner to modulate neuronal survival. Experiments using chimeric mice expressing ALS-linked mutations to Cu,Zn superoxide dismutase (SOD-1) suggest a critical modulation exerted by neighboring non-neuronal cell types on disease phenotype. When perturbed by primary neuronal damage, e.g. expression of SOD-1 mutations, neurons can signal astrocytes to proliferate and become reactive. Fibroblast growth factor-1 (FGF-1) can be released by motor neurons in response to damage to induce astrocyte activation by signaling through the receptor FGFR1. FGF-1 stimulates nerve growth factor (NGF) expression and secretion, as well as activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor. Nrf2 leads to the expression of antioxidant and cytoprotective enzymes such as heme oxygenase-1 and a group of enzymes involved in glutathione metabolism that prevent motor neuron degeneration. However, prolonged stimulation with FGF-1 or SOD-mediated oxidative stress in astrocytes may disrupt the normal neuron-glia interactions and lead to progressive neuronal degeneration. The re-expression of p75 neurotrophin receptor and neuronal NOS in motor neurons in parallel with increased NGF secretion by reactive astrocytes may be a mechanism to eliminate critically damaged neurons. Consequently, astrocyte activation in ALS may have a complex pathogenic role.

Astrocyte-specific overexpression of Nrf2 protects striatal neurons from mitochondrial complex II inhibition.

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that is known to regulate a variety of cytoprotective genes through the antioxidant response element (ARE). This endogenous response is one of the major pathways by which cells are protected from xenobiotic or innate oxidative insults. Furthermore, in neural systems, astrocyte-specific activation of Nrf2 is known to protect neurons. In previous work, our laboratory found that Nrf2 protects from intrastriatal injections of the mitochondrial complex II inhibitor malonate. Here, we extend these results to show that multiple methods of astrocyte-specific Nrf2 overexpression provide protection from neurotoxicity in vivo. GFAP-Nrf2 transgenic mice are significantly more resistant to malonate lesioning. This outcome is associated with an increased basal resistance, but more so, an enhanced Nrf2 response to lesioning that attenuated the ensuing neurotoxicity. Furthermore, striatal transplantation of neuroprogenitor cells overexpressing Nrf2 that differentiate into astrocytes after grafting also significantly reduced malonate toxicity. Overall, these data establish that enhanced astrocytic Nrf2 response and Nrf2 preconditioning are both sufficient to protect from acute lesions from mitochondrial complex II inhibition.