S[+] Apomorphine is a CNS penetrating activator of the Nrf2-ARE pathway with activity in mouse and patient fibroblast models of amyotrophic lateral sclerosis.

Compelling evidence indicates that oxidative stress contributes to motor neuron injury in amyotrophic lateral sclerosis (ALS), but antioxidant therapies have not yet achieved therapeutic benefit in the clinic. The nuclear erythroid 2-related-factor 2 (Nrf2) transcription factor is a key regulator of an important neuroprotective response by driving the expression of multiple cytoprotective genes via its interaction with the antioxidant response element (ARE). Dysregulation of the Nrf2-ARE system has been identified in ALS models and human disease. Taking the Nrf2-ARE pathway as an attractive therapeutic target for neuroprotection in ALS, we aimed to identify CNS penetrating, small molecule activators of Nrf2-mediated transcription in a library of 2000 drugs and natural products. Compounds were screened extensively for Nrf2 activation, and antioxidant and neuroprotective properties in vitro. S[+]-Apomorphine, a receptor-inactive enantiomer of the clinically approved dopamine-receptor agonist (R[-]-apomorphine), was identified as a nontoxic Nrf2 activating molecule. In vivo S[+]-apomorphine demonstrated CNS penetrance, Nrf2 induction, and significant attenuation of motor dysfunction in the SOD1(G93A) transgenic mouse model of ALS. S[+]-apomorphine also reduced pathological oxidative stress and improved survival following an oxidative insult in fibroblasts from ALS patients. This molecule emerges as a promising candidate for evaluation as a potential neuroprotective agent in ALS patients in the clinic.

A comparison of in vitro properties of resting SOD1 transgenic microglia reveals evidence of reduced neuroprotective function.

BACKGROUND: Overexpression of mutant copper/zinc superoxide dismutase (SOD1) in rodents has provided useful models for studying the pathogenesis of amyotrophic lateral sclerosis (ALS). Microglia have been shown to contribute to ALS disease progression in these models, although the mechanism of this contribution remains to be elucidated. Here, we present the first evidence of the effects of overexpression of mutant (TG G93A) and wild type (TG WT) human SOD1 transgenes on a set of functional properties of microglia relevant to ALS progression, including expression of integrin ß-1, spreading and migration, phagocytosis of apoptotic neuronal cell debris, and intracellular calcium changes in response to an inflammatory stimulus. RESULTS: TG SOD1 G93A but not TG SOD1 WT microglia had lower expression levels of the cell adhesion molecule subunit integrin ß-1 than their NTG control cells [NTG (G93A) and NTG (WT), respectively, 92.8 ± 2.8% on TG G93A, 92.0 ± 6.6% on TG WT, 100.0 ± 1.6% on NTG (G93A), and 100.0 ± 2.7% on NTG (WT) cells], resulting in decreased spreading ability, with no effect on ability to migrate. Both TG G93A and TG WT microglia had reduced capacity to phagocytose apoptotic neuronal cell debris (13.0 ± 1.3% for TG G93A, 16.5 ± 1.9% for TG WT, 28.6 ± 1.8% for NTG (G93A), and 26.9 ± 2.8% for NTG (WT) cells). Extracellular stimulation of microglia with ATP resulted in smaller increase in intracellular free calcium in TG G93A and TG WT microglia relative to NTG controls (0.28 ± 0.02 µM for TG G93A, 0.24 ± 0.03 µM for TG WT, 0.39 ± 0.03 µM for NTG (G93A), and 0.37 ± 0.05 µM for NTG (WT) microglia). CONCLUSIONS: These findings indicate that, under resting conditions, microglia from mutant SOD1 transgenic mice have a reduced capacity to elicit physiological responses following tissue disturbances and that higher levels of stimulatory signals, and/or prolonged stimulation may be necessary to initiate these responses. Overall, resting mutant SOD1-overexpressing microglia may have reduced capacity to function as sensors of disturbed tissue/cellular homeostasis in the CNS and thus have reduced neuroprotective function.

Contrasting effects of cerebrospinal fluid from motor neuron disease patients on the survival of primary motor neurons cultured with or without glia.

Motor neuronal (MN) degeneration in motor neuron disease (MND) often starts focally before spreading to neighbouring MN populations, suggesting soluble factors may contribute to disease propagation. Whether cerebrospinal fluid (CSF) from MND patients contains such factors has been difficult to prove. We aimed to determine the effect of glia on the response of MNs to CSF from MND patients. Primary rat spinal MNs grown in mono-culture or cocultured with glia were exposed to CSF from patients (MND-CSF) or controls (Con-CSF) and survival measured by cell counting. In mono-culture both MND-CSF and Con-CSF reduced MN survival with MND-CSF reducing MN survival by less than Con-CSF. In coculture MN survival was unchanged by exposure to MND-CSF while exposure to Con-CSF improved MN survival. In separate experiments, murine MNs grown in mono-culture and stressed by growth factor withdrawal were partially rescued by the application of monocyte chemoattractant protein-1 (MCP-1), a trophic factor previously found to be elevated in MND-CSF. Our results suggest that MND-CSF may contain factors harmful to MNs as well as factors protective of MNs, the interplay of which is altered by the presence of glial cells. These preliminary results further emphasize the importance of MN environment to MN health.

Oxidative stress in ALS: key role in motor neuron injury and therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by death of motor neurons leading to muscle wasting, paralysis, and death, usually within 2-3 years of symptom onset. The causes of ALS are not completely understood, and the neurodegenerative processes involved in disease progression are diverse and complex. There is substantial evidence implicating oxidative stress as a central mechanism by which motor neuron death occurs, including elevated markers of oxidative damage in ALS patient spinal cord and cerebrospinal fluid and mutations in the antioxidant enzyme superoxide dismutase 1 (SOD1) causing approximately 20% of familial ALS cases. However, the precise mechanism(s) by which mutant SOD1 leads to motor neuron degeneration has not been defined with certainty, and the ultimate trigger for increased oxidative stress in non-SOD1 cases remains unclear. Although some antioxidants have shown potential beneficial effects in animal models, human clinical trials of antioxidant therapies have so far been disappointing. Here, the evidence implicating oxidative stress in ALS pathogenesis is reviewed, along with how oxidative damage triggers or exacerbates other neurodegenerative processes, and we review the trials of a variety of antioxidants as potential therapies for ALS.

Mutant SOD1 G93A microglia have an inflammatory phenotype and elevated production of MCP-1.

The inflammatory response in amyotrophic lateral sclerosis (ALS) is well documented but the underlying cellular mechanisms have not been fully elucidated. We report that microglia isolated from the mutant human superoxide dismutase 1 (SOD1) G93A transgenic mouse model of ALS have an increased response to the inflammatory stimulus, lipopolysaccharide. Cell surface area and F4/80 surface marker, both indicators of cell activation, are increased relative to transgenic wild-type human SOD1 microglia. Monocyte chemoattractant protein-1, known to be increased in ALS, is produced at three-fold higher levels by SOD1 G93A than by wild-type human SOD1 microglia, under activating conditions. This novel finding implicates ALS microglia as a source of the increased monocyte chemoattractant protein-1 levels detected in ALS patients and in the ALS mouse model.

An in vitro screening cascade to identify neuroprotective antioxidants in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen.

There is now compelling evidence of mitochondrial dysfunction in motor neuron disease (MND), but the molecular basis of these abnormalities is unknown. It is also unclear whether the observed mitochondrial dysfunction plays a central role in disease pathogenesis, and if so, whether its amelioration might present therapeutic opportunities. We adopted a candidate generation approach using proteomics to screen for changes in mitochondrial protein expression in a well-validated cell-culture model of superoxide dismutase 1 (SOD1) related familial MND (fMND). Changed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. Protein candidates included apoptotic regulators, anti-oxidants and components of the electron transport chain. Confirmatory Western blotting was performed, and validated protein expression changes were further investigated. Peroxiredoxin 3 (Prx3), a mitochondrial thioredoxin-dependent hydroperoxidase, is downregulated in the presence of mutant SOD1 in both our cell-culture model and in the spinal cord mitochondria of mutant SOD1 transgenic mice. We confirm the expression of Prx3 within the mitochondria of spinal motor neurons in mouse and humans by immunohistochemistry. Using quantitative real-time PCR (Q-PCR), we show that Prx3 is also downregulated in spinal motor neurons from patients with both sporadic (sMND) and SOD1-related fMND. In a disease characterized by oxidative stress, this represents a potentially important deficit in mitochondrial anti-oxidant defence. Recent evidence suggests that oxidative stress from aberrant copper chemistry may not play a major part in the pathogenesis of SOD1-related fMND. From the results of this study we propose disruption of mitochondrial anti-oxidant defence as an alternative mechanism whereby mutant SOD1 may generate oxidative stress within motor neurons. We further demonstrate that ebselen, an anti-oxidant drug already safely used in human studies and that acts as a Prx mimic, is able to ameliorate the toxicity of mutant SOD1 in our cell-culture model. We conclude by showing that ebselen is capable of inducing transcription of the anti-oxidant response element (ARE) and postulate that ebselen may act both by the transcriptional upregulation of anti-oxidant proteins, and directly as an anti-oxidant in its own right.

Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target.

The cause(s) of amyotrophic lateral sclerosis (ALS) is not fully understood in the vast majority of cases and the mechanisms involved in motor neuron degeneration are multi-factorial and complex. There is substantial evidence to support the hypothesis that oxidative stress is one mechanism by which motor neuron death occurs. This theory becomes more persuasive with the discovery that mutation of the anti-oxidant enzyme, superoxide dismutase 1 (SOD1), causes disease in a significant minority of cases. However, the precise mechanism(s) by which mutant SOD1 leads to motor neuron degeneration have not been defined with certainty, and trials of anti-oxidant therapies have been disappointing. Here, we review the evidence implicating oxidative stress in ALS pathogenesis, discuss how oxidative stress may affect and be affected by other proposed mechanisms of neurodegeneration, and review the trials of various anti-oxidants as potential therapies for ALS.

Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS.

Familial amyotrophic lateral sclerosis (FALS) is caused, in 20% of cases, by mutations in the Cu/Zn superoxide dismutase gene (SOD1). Although motor neuron injury occurs through a toxic gain of function, the precise mechanism(s) remains unclear. Using an established NSC34 cellular model for SOD1-associated FALS, we investigated the effects of mutant SOD1 specifically in cells modelling the vulnerable cell population, the motor neurons, without contamination from non-neuronal cells present in CNS. Using gene expression profiling, 268 transcripts were differentially expressed in the presence of mutant human G93A SOD1. Of these, 197 were decreased, demonstrating that the presence of mutant SOD1 leads to a marked degree of transcriptional repression. Amongst these were a group of antioxidant response element (ARE) genes encoding phase II detoxifying enzymes and antioxidant response proteins (so-called 'programmed cell life' genes), the expression of which is regulated by the transcription factor NRF2. We provide evidence that dysregulation of Nrf2 and the ARE, coupled with reduced pentose phosphate pathway activity and decreased generation of NADPH, represent significant and hitherto unrecognized components of the toxic gain of function of mutant SOD1. Other genes of interest significantly altered in the presence of mutant SOD1 include several previously implicated in neurodegeneration, as well as genes involved in protein degradation, the immune response, cell death/survival and the heat shock response. Preliminary studies on isolated motor neurons from SOD1-associated motor neuron disease cases suggest key genes are also differently expressed in the human disease.

S1P and LPA trigger Schwann cell actin changes and migration.

The processes by which a Schwann cell (SC) migrates towards, wraps around and, in some cases, myelinates an axon are incompletely understood. The complex morphological rearrangements involved in these events require fundamental changes in the actin cytoskeleton. Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are two modulators of the actin cytoskeleton, and receptors for these signalling lipids are expressed on SCs at the time of differentiation. Previous work has revealed a role for LPA in SC survival, morphology and differentiation, but the effects of S1P have received less attention. Here we show that S1P and LPA both cause major rearrangements to the actin cytoskeleton in primary rat SCs and the SCL4.1/F7 rat SC line. S1P and LPA caused formation of lamellipodia and a circular geodesic actin network. We also show that S1P and LPA increased cell migration. The small GTPases RhoA and Rac1 were both activated by S1P/LPA treatment, but the actin rearrangements were dependent on Rac1 and not RhoA. These effects of S1P/LPA could be mimicked by SCL4.1/F7 cell-conditioned medium, which was found to contain S1P. Reduction in cellular synthesis of S1P by adding the sphingosine kinase inhibitor dimethyl sphingosine during medium conditioning reduced the ability of conditioned medium to cause actin rearrangements. These results support a role for S1P as an autocrine signal regulating the actin cytoskeleton during Schwann cell development.