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 (p75(NTR)) 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 p75(NTR) 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 p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) 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 p75(NTR) 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.     

Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival

Fibroblast growth factor-1 (FGF-1) is highly expressed in motor neurons and can be released in response to sublethal cell injury. Because FGF-1 potently activates astroglia and exerts a direct neuroprotection after spinal cord injury or axotomy, we examined whether it regulated the expression of inducible and cytoprotective heme oxygenase-1 (HO-1) enzyme in astrocytes. FGF-1 induced the expression of HO-1 in cultured rat spinal cord astrocytes, which was dependent on FGF receptor activation and prevented by cycloheximide. FGF-1 also induced Nrf2 mRNA and protein levels and prompted its nuclear translocation. HO-1 induction was abolished by transfection of astrocytes with a dominant-negative mutant Nrf2, indicating that FGF-1 regulates HO-1 expression through Nrf2. FGF-1 also modified the expression of other antioxidant genes regulated by Nrf2. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the amyotrophic lateral sclerosis-linked SOD1 G93A mutation. Overexpression of Nrf2 in astrocytes increased survival of co-cultured embryonic motor neurons and prevented motor neuron apoptosis mediated by nerve growth factor through p75 neurotrophin receptor. Taken together, these results emphasize the key role of astrocytes in determining motor neuron fate in amyotrophic lateral sclerosis.     

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1^G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 ^G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 ^G93A-dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.     

Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex

The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.     

Peroxynitrite transforms nerve growth factor into an apoptotic factor for motor neurons

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.     

Antisense peptide nucleic acid-mediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice

Re-expression of the death-signalling p75 neurotrophin receptor (p75NTR) is associated with injury and neurodegeneration in the adult nervous system. The induction of p75NTR expression in mature degenerating spinal motor neurons of humans and transgenic mice with amyotrophic lateral sclerosis (ALS) suggests a role of p75NTR in the progression of motor neuron disease (MND). In this study, we designed, synthesized and evaluated novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential gene knockdown therapeutic strategy for ALS. An 11-mer antisense PNA directed at the initiation codon, but not downstream gene sequences, dose-dependently inhibited p75NTR expression and death-signalling by nerve growth factor (NGF) in Schwann cell cultures. Antisense phosphorothioate oligonucleotide (PS-ODN) sequences used for comparison failed to confer such inhibitory activity. Systemic intraperitoneal administration of this antisense PNA to mutant superoxide dismutase 1 (SOD1G93A) transgenic mice significantly delayed locomotor impairment and mortality compared with mice injected with nonsense or scrambled PNA sequences. Reductions in p75NTR expression and subsequent caspase-3 activation in spinal cords were consistent with increased survival in antisense PNA-treated mice. The uptake of fluorescent-labelled antisense PNA in the nervous system of transgenic mice was also confirmed. This study suggests that p75NTR may be a promising antisense target in the treatment of ALS.     

FGF22 signaling regulates synapse formation during post‐injury remodeling of the spinal cord

The remodeling of axonal circuits after injury requires the formation of new synaptic contacts to enable functional recovery. Which molecular signals initiate such axonal and synaptic reorganisation in the adult central nervous system is currently unknown. Here, we identify FGF22 as a key regulator of circuit remodeling in the injured spinal cord. We show that FGF22 is produced by spinal relay neurons, while its main receptors FGFR1 and FGFR2 are expressed by cortical projection neurons. FGF22 deficiency or the targeted deletion of FGFR1 and FGFR2 in the hindlimb motor cortex limits the formation of new synapses between corticospinal collaterals and relay neurons, delays their molecular maturation, and impedes functional recovery in a mouse model of spinal cord injury. These results establish FGF22 as a synaptogenic mediator in the adult nervous system and a crucial regulator of synapse formation and maturation during post‐injury remodeling in the spinal cord.     

Interactions between beta-neuregulin and neurotrophins in motor neuron apoptosis

Neuregulins play a major role in the formation and stabilization of neuromuscular junctions, and are produced by both motor neurons and muscle. Although the effects and mechanism of neuregulins on skeletal muscle (e.g. regulation of acetylcholine receptor expression) have been studied extensively, the effects of neuregulins on motor neurons remain unknown. We report that neuregulin-1beta (NRGbeta1) inhibited apoptosis of rat motor neurons for up to 7 days in culture by a phosphatidylinositol 3 kinase-dependent pathway and synergistically enhanced motor neuron survival promoted by glial-derived neurotrophic factor (GDNF). However, binding of neurotrophins, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), to the p75 neurotrophin receptor (p75NTR) abolished the neuregulin anti-apoptotic effect on motor neurons. Inhibitors of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase prevented motor neuron death caused by co-incubation of NRGbeta1 and BDNF or NGF, as well as by trophic factor deprivation. Motor neuron apoptosis resulting from both trophic factor deprivation and exposure to NRGbeta1 plus neurotrophins required the induction of neuronal nitric oxide synthase and peroxynitrite formation. Because motor neurons express both p75NTR and neuregulin erbB receptors during the period of embryonic programmed cell death, motor neuron survival may be the result of complex interactions between trophic and death factors, which may be the same molecules acting in different combinations.     

The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.     

The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects

Basic fibroblast growth factor (FGF-2) promotes survival and/or neurite outgrowth from a variety of neurons in cell culture and regenerative processes in vivo. FGFs exert their effects by activating cell surface receptor tyrosine kinases. FGF receptor (FGFR) inhibitors have not been characterized on neuronal cell behaviors to date. In the present study, we show that the FGFR1 inhibitor PD 173074 potently and selectively antagonized the neurotrophic and neurotropic actions of FGF-2. Nanomolar concentrations of PD 173074 prevented FGF-2, but not insulin-like growth factor-1, support of cerebellar granule neuron survival under conditions of serum/K(+) deprivation; another FGF-2 inhibitor, SU 5402, was effective only at a 1,000-fold greater concentration. Neither PD 173074 nor SU 5402, at 100 times their IC(50) values, interfered with the survival of dorsal root ganglion neurons promoted by nerve growth factor, ciliary neurotrophic factor, or glial cell line-derived neurotrophic factor. PD 173074 and SU 5402 displayed 1,000-fold differential IC(50) values for inhibition of FGF-2-stimulated neurite outgrowth in PC12 cells and in granule neurons, and FGF-2-induced mitogen-activated protein kinase (p44/42) phosphorylation. The two inhibitors failed to disturb downstream signalling stimuli of FGF-2. PD 173074 represents a valuable tool for dissecting the role of FGF-2 in normal and pathological nervous system function without compromising the actions of other neurotrophic factors.     

Induction of motor neuron apoptosis by free 3-nitro-L-tyrosine

Peroxynitrite-dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein-linked 3-nitro-l-tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS-linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into alpha-tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into alpha-tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co-culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine-induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.     

Oxidative Stress and Autophagic Alteration in Brainstem of SOD1-G93A Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease involving both upper and lower motor neurons. The mechanism of motor neuron degeneration is still unknown. Although many studies have been performed on spinal motor neurons, few have been reported on brainstem and its motor nuclei. The aim of this study was to investigate oxidative stress and autophagic changes in the brainstem and representative motor nuclei of superoxide dismutase 1 (SOD1)-G93A mouse model of ALS. The expression levels of cluster of differentiation molecule 11b (CD11b), glial fibrillary acidic protein, glutamate–cysteine ligase catalytic subunit, heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, voltage-dependent anion-selective channel protein 1, Sequestosome 1/p62 (p62), microtubule-associated protein 1 light chain 3B (LC3), and SOD1 proteins in brainstem were examined by Western blot analysis. Immunohistochemistry and immunofluorescence were performed to identify the cellular localization of SOD1, p62, and LC3B, respectively. The results showed that there were progressive asctrocytic proliferation and microglial activation, induction of antioxidant proteins, and increased p62 and LC3II expression in brainstem of SOD1-G93A mice. Additionally, SOD1 and p62 accumulated in hypoglossal, facial, and red nuclei, but not in oculomotor nucleus. Furthermore, electron microscope showed increased autophagic vacuoles in affected brainstem motor nuclei. Our results indicate that brainstem share similar gliosis, oxidative stress, and autophagic changes as the spinal cord in SOD1-G93A mice. Thus, SOD1 accumulation in astrocytes and neurons, oxidative stress, and altered autophagy are involved in motor neuron degeneration in the brainstem, similar to the motor neurons in spinal cord. Therefore, therapeutic trials in the SOD1G93A mice need to target the brainstem in addition to the spinal cord.     

Patient‐derived olfactory mucosa for study of the non‐neuronal contribution to amyotrophic lateral sclerosis pathology

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease which currently has no cure. Research using rodent ALS models transgenic for mutant superoxide dismutase 1 (SOD1) has implicated that glial–neuronal interactions play a major role in the destruction of motor neurons, but the generality of this mechanism is not clear as SOD1 mutations only account for less than 2% of all ALS cases. Recently, this hypothesis was backed up by observation of similar effects using astrocytes derived from post‐mortem spinal cord tissue of ALS patients which did not carry SOD1 mutations. However, such necropsy samples may not be easy to obtain and may not always yield viable cell cultures. Here, we have analysed olfactory mucosa (OM) cells, which can be easily isolated from living ALS patients. Disease‐specific changes observed when ALS OM cells were co‐cultured with human spinal cord neurons included decreased neuronal viability, aberrant neuronal morphology and altered glial inflammatory responses. Our results show the potential of OM cells as new cell models for ALS.     

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1^G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1^G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.     

Induction of cyclooxygenase-2 in reactive glial cells by the CD40 pathway: relevance to amyotrophic lateral sclerosis

An inflammatory process in association with reactive gliosis has been suggested to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). One of the key findings is a marked increase in the level of cyclooxygenase-2 (COX-2), a therapeutic target of ALS. We investigated the expression of CD40 in the spinal cord of a transgenic mouse model of ALS (G93A mice), and its relevance to COX-2 upregulation. CD40 was predominantly expressed in neurons in normal spinal cord and upregulated in reactive glial cells in spinal cord injury. In the spinal cord of G93A mice, the expression of CD40 was increased in both reactive microglia and astrocytes, where COX-2 was especially increased. The level of COX-2 was upregulated in microglia and astrocytes by CD40 stimulation in vitro. CD40 stimulation in primary spinal cord cultures caused motor neuron loss that was protected by selective COX-2 inhibitor. These results suggest that CD40, which is upregulated in reactive glial cells in ALS, participates in motor neuron loss via induction of COX-2.     

The p75 neurotrophin receptor: effects on neuron survival in vitro and interaction with death domain-containing adaptor proteins

The p75 neurotrophin receptor (p75NTR) is a death domain (DD) containing receptor of the TNF/FAS(APO-1) family. p75NTR has recently been shown to mediate apoptosis in certain types of neurons as well as in oligodendrocytes. The molecular mechanisms by which p75NTR stimulates apoptosis are still unknown. Here, we have tested whether overexpression of p75NTR could modulate survival of sympathetic neurons cultured in the presence or absence of NGF. Moreover, using the yeast two-hybrid system, we tested whether p75NTR intracellular domain was able to dimerize or interact with known DD-containing proteins including FADD, RIP, RAIDD and TRADD. We found that over-expression of p75NTR had no effect on the survival of sympathetic neurons cultured in the presence of NGF but instead delayed neuronal death following NGF deprivation. These results strongly support the finding that p75NTR is not involved in the apoptosis process induced by NGF deprivation in sympathetic neurons. We also foun d that the intracellular domain of p75NTR failed to associate either with itself or with other known DD-containing proteins. This suggests that the mechanisms by which p75NTR triggers apoptosis in certain cell types are different from those used by other receptors of the TNF/FAS family.     

Nogo-A and Nogo-66 receptor in amyotrophic lateral sclerosis

Nogo/reticulon (RTN)-4 has been strongly implicated as a disease marker for the motor neuron disease amyotrophic lateral sclerosis (ALS). Nogo isoforms, including Nogo-A, are ectopically expressed in the skeletal muscle of ALS mouse models and patients and their levels correlate with the disease severity. The notion of a direct involvement of Nogo-A in ALS aetiology is supported by the findings that Nogo-A deletion in mice reduces muscle denervation and prolongs survival, whereas overexpression of Nogo-A destabilizes motor nerve terminals and promotes denervation. Another intriguing, and somewhat paradoxical, recent finding revealed that binding of the Nogo-66 receptor (NgR) by either agonistic or antagonistic Nogo-66-derived peptides protects against p75 neurotrophin receptor (p75(NTR))-dependent motor neuron death. Ligand binding by NgR could result in subsequent engagement of p75(NTR), and this association could preclude pro-apoptotic signalling by the latter. Understanding the intricate interplay among Nogo isoforms, NgR and p75(NTR) in ALS disease progression may provide important, therapeutically exploitable information.