The pathogenesis of ALS likely involves disturbed intercellular communications between motor neurons and other cell types, as suggested by experiments using chimeric mice expressing mutated SOD-1. We investigated whether FGF-1 can contribute to induce sustained astrocyte activation characteristic of ALS. FGF-1 is highly expressed in motor neurons. It can be released from cells by oxidative stress, which might occur from SOD-1 aberrant function. Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that it can activate spinal cord astrocytes in a manner that decreased the survival of p75-expressing motor neurons in co-cultures. FGF-1 effects can be reproduced by treatment with exogenous peroxynitrite, which is consistent with the observation that FGF-1 induces oxidative stress and increases nitrotyrosine formation in cultured astrocytes. Activated astrocytes induce apoptosis of embryonic motor neuron by a mechanism involving nitric oxide (NO) and secretion of nerve growth factor (NGF). Both mediators were required to trigger p75-dependent apoptosis in pure motor neuron cultures. Since p75NTR is re-expressed only by affected motor neurons in ALS, it is proposed that astrocytic NGF may serve to eliminate damaged neurons. More recently, we found that FGF-1 also induced Nrf2 and caused prompted its nuclear translocation in spinal cord astrocytes. Nrf2 is a redox-sensitive transcription factor that regulates several ARE-containing genes, including HO-1 and enzymes involved in glutathion synthesis. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the ALS-linked SOD1 G93A mutation. Partial induction of ARE/Nrf2 pathway by FGF-1 cannot completely counteract p75NTR-dependent apoptosis. However further increase of antioxidant support by overexpression of Nrf2 in astrocytes resulted in increased survival of cocultured embryonic motor neurons and prevented p75-dependent motor neuron apoptosis mediated by p75 neurotrophin receptor. As expected, over expression of Nrf2 in astrocytes resulted in increased survival of cocultured embryonic motor neurons and prevented motor neuron apoptosis mediated by p75 neurotrophin receptor. Although partial induction of ARE/Nrf2 pathway by FGF-1 cannot completely counteract p75NTR-dependent apoptosis, further increase of antioxidant support by astrocytes following Nrf2 overexpression can prevent motor neuron apoptosis mediated by NGF. We hypothesizes that SOD-1 mutations linked to ALS sensitize astrocytes to FGF-induced oxidative damage and mitochondria dysfunction, leading to a catastrophic decrease in the neurotrophic and metabolic support for to motor neurons.
EXPERIMENTAL THERAPEUTICS IN TRANSGENIC MOUSE MODELS OF AMYOTROPHIC LATERAL SCLEROSIS
M. Flint Beal
Cornell University, New York, United States
A major goal in amyotrophic lateral sclerosis (ALS) is to develop new therapeutic agents. These efforts have focused on a number of different therapeutic approaches. We have studied whether COX-2 inhibitors with or without creatine could improve survival. The combination of Vioxx or Celebrex with creatine improves survival by as much as 31%. More recently we have studied whether agents which can block TNF-á which is induced in ALS motor neurons, have therapeutic effects. We found that treatment with thalidomide and lonalidomide attenuated weight loss, enhanced motor performance, decreased motor neuron cell death and increased life span significantly in G93A SOD1 mice. The thalidomide treated G93A mice showed a reduction in TNF-á and Fas-L immunoreactivity in lumbar spinal cord sections. We also recently in collaboration with Dr. John Crow have tested a manganese porhyphin catalytic anti-oxidant. This is a compound known as AEOL-10150. It is a manganese porhyphin mimetic. When administered after the onset of symptoms in the ALS mice it improves survival by as much as 3 fold. This was measured as the survival interval. We have also carried out some studies with the histone deacetylase inhibitor phenyl butyrate. Our initial results suggest that this does indeed have some efficacy in treating the G93A SOD1 transgenic mouse model of ALS. A major concern however is whether agents which are effective in this model will prove to be beneficial in patients. Unfortunately creatine and Celebrex both of which showed an efficacy in the transgenic mice have proved unsuccessful in patients. We have more recently done further looking at mitochondrial dysfunction in the G93A mice. We have found that the mutant SOD1 is localized to mitochondria where it forms aggregates and may impair mitochondrial function. Several recent experiments in transgenic mice appear to establish that copper can have no role in the toxicity of SOD in ALS, implying that aggregation of mutant SODs must account for the toxic gain of function in ALS. The principal hypothesis behind our work postulates that copper-containing, zinc-deficient SOD is toxic to motor neurons by catalyzing the formation of peroxynitrite from nitric oxide, oxygen and intracellular reductants. In primary cultures of motor neurons, we have shown that the loss of zinc from copper, zinc superoxide dismutase causes both wild-type and ALS-mutant SODs to be toxic to motor neurons via a nitric oxide-dependent mechanism. The ALS mutant SODs have a decreased affinity for zinc, making them slightly more prone to becoming zinc-deficient. The most damning evidence to date that appears to falsify the zinc-deficient hypothesis comes from the recent demonstration that an ALS-mutant SOD with all four ligands for copper mutated still develops motor neuron disease when highly expressed in transgenic mice. However, we have found that this particular Quad mutant form of SOD can bind copper in its zinc-binding site. While the Quad SOD no longer scavenges superoxide, it does act like other zinc-deficient SODs to generate peroxynitrite from oxygen, ascorbate and nitric oxide. Several elegant biochemical studies have demonstrated that the loss of copper and zinc from SOD allows the conserved cysteine bridge C57-146 to become more easily reduced, which causes SOD dimers to dissociate and then to polymerize into aggregates. However, there is surprisingly little evidence to show that aggregation of SOD actually kills cells and several studies have found no correlation of SOD aggregation with cell death in mice or in culture cells. One striking example is the inability of anyone to develop a mouse line developing motor neuron disease using the A4V SOD mutation, even though this SOD mutation causes a particularly aggressive form of ALS in humans and expresses large amounts of SOD aggregates in the transgenic mice. We found that mutating a cysteine at position 111 prevents much of the SOD aggregation and makes zinc-deficient SOD far more toxic to motor neurons in culture. We have also developed a A4V/C111S mouse that develops motor neuron disease in 13 months. Activation of mitogen activated protein kinases (MAPK) including p38 (p38MAPK) and JNK represents an important step in the pathways leading to neuron cell death in response to a variety of extracellular stimuli including excitotoxicity, oxidative stress and inflammatory processes. These stimuli have been implicated in the mechanisms of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Thus, we evaluated the involvement of MAPK in the progressive motor neuron degeneration in transgenic mice overexpressing SOD1G93A that, at certain level, recapitulate the clinical and neuropathological features of the human ALS. We showed a remarkable activation of p38MAPK in the spinal motor neurons of presymptomatic mice which was associated with the accumulation of phosphorylated neurofilaments in the perikarya and proximal axons, a pathological feature typical of ALS (Tortarolo et al. 2004). Interestingly, alpha p38MAPK was demonstrated to phosphorylate the side-arm domains of middle and heavy neurofilament (NF) chains leading to their aberrant phosphorylation (Ackerley et al. 2004). In line with this, we also showed that phosphorylated p38MAPK was a component of intracellular skein-like inclusions found in motor neurons of sporadic ALS (Bendotti et al.2004 ) indicating that activation of this kinase contribute to the pathological changes of motor neurons. We did not observe activation of JNK in the motor neurons of SOD1G93A mice at any stage of the disease, in line with previous observations in human ALS, suggesting a selective involvement of p38MAPK in the pathogenesis of ALS (Veglianese et al. 2004). We evaluated the cascade upstream the activation of p38MAPK and have found that also MKK3/6, MKK4 and ASK1 were rapidly activated in the motor neurons of SOD1G93A mice at the presymptomatic stage of the disease and this effect was related to an upregulation of TNF receptors, both 1 and 2, but not to FAS or IL1? receptors increase, indicating a selective pathway in the motor neuron death. Activation of p38MAPK was not restricted to motor neurons but also occurred in reactive glial cells, both astrocytes and microglia, with the progression of the disease. Since activated p38MAPK in glial cells is known to induce the release of pro-inflammatory factors and nitric oxide we suggest that this event may contribute to create a toxic microenvironment determining a self-propagating process of cell damage. Interestingly, minocycline, an antibiotics which delayed the progression of disease in SOD1mutant mice also inhibited the activation of p38MAPK in microglia (Kriz et al. 2002 and Van den Bosh,2002). Activated p38MAPK in reactive microglia of SOD1G93A spinal cord at the symptomatic stage was found in association with upregulation of TNF receptors, however other kinases upstream to p38 were not activated indicating an alternative mechanism of TNF-p38 linked pathway. As the disease progressed, phosphorylated p38MAPK also accumulated in hypertrophic astrocytes around degenerating motor neurons however this effect was not associated with an upregulation of TNF receptors neither with activation of MKK3,4 and 6 or ASK1 suggesting that different pathways are involved in the cellular signalling among different cell types. In conclusion, these data suggest that a specific intracellular signalling pathway involving the TNFalpha receptors and the downstream p38MAPK kinase cascade participates to the early events leading to motor neuron death in SOD1 mutant mice. TNF receptors may also activate p38MAPK in reactive microglial cells, through an alternative cascade, and this may contribute to the propagation of damage to adjacent cells. Thus, pharmacological or other strategies able to modulate the activity of this pathway may provide therapeutic benefit to ALS patients. Although amyotrophic lateral sclerosis (ALS) was described more than 130 years ago, the mechanism underlying death of motor neurons in this common adult motor neuron disease has remained a mystery. With the discovery of mutations in one gene [Cu/Zn superoxide dismutase (SOD1)] as a primary cause of some forms of ALS, model systems have been developed which have helped us begin to understand the disease and test potential new compounds for therapy. Several other genes have been implicated as risk factors including neurofilaments, cytoplasmic dynein and its processivity factor dynactin, and vascular endothelial growth factor (VEGF) as contributors to, or causes of, motor neuron diseases. The recent discovery of mutations in genes linked to the more rare motor neuron diseases will provide new clues as to why the motor neuron is particularily vulnerable in the disease. Furthermore, the application of novel approaches to therapy using Ribonucleic Acid inteference (RNAi) technology to down-regulate mutant protein and the realization that motor neuorns although central in the disease process are not the only cell type involved mark exciting progress in the field of ALS. This overview focuses on some of the recent advances made in understanding the disease and how this may eventually lead to more effective therapies for ALS. Amyotrophic Lateral Sclerosis (ALS) is a severe disease characterized by neurogenic amyotrophy and degeneration of upper and lower motorneurons. Although ALS patients usually experience reductions in fat free mass (FFM), hypermetabolism has been reported. The diagnosis ALS is made clinically on the basis of neurological examination and history. Most important is the demonstration of the involvement of both upper and lower motor neurons and the absence of signs pointing to lesions in other neuronal systems. Lab tests are mainly carried out to exclude diseases which can mimic ALS. The most helpful technical test is still EMG which can demonstrate disseminated denervation in muscles appearing clinically normal. Currently no specific biomarkers of ALS are available. Neurophysiological and imaging techniques serve as surrogate markers. Amyotrophic lateral sclerosis (ALS) is a motor neuron disease of unknown origin. About 2% of ALS cases are linked to mutations in the gene encoding copper/zinc superoxide dismutase (SOD1). Expression of mutant forms of SOD1 (SOD1m) in transgenic mice leads to motor neuron death and an ALS-like phenotype. Strikingly, motor neuron death in SOD1m mice is not cell autonomous which indicates that the fate of motor neurons is dictated by its environment rather than by its genotype. This suggests that ALS is a more systemic pathology than previously expected. In addition, several studies, including ours, demonstrated that SOD1m expression triggered mitochondrial dysfunction not only in primarily affected tissues such as spinal cord but also in peripheral cells such as skeletal muscle or lymphocytes. Indeed, we recently showed that SOD1m transgenic mice are affected by a generalized defect in energy homeostasis primarily due to a skeletal muscle hypermetabolism. Most importantly, we showed that compensating this energy defect by increasing energy intake of SOD1m mice offered neuroprotection and increased survival of these mice. We are currently studying the mechanisms of this protection. In particular, we showed that SOD1m mice are hypolipidemic due to an increased clearance of triglycerides-rich lipoproteins. The implications of these findings as well as their potential relevance towards human ALS will be discussed. Both protein-linked and free nitrotyrosine are present in the spinal cord of patients and animal models of ALS. Nitrotyrosine immunoreactivity is also increased in cultured motor neurons undergoing apoptosis induced by trophic factor deprivation. Peptides containing tyrosine residues prevented tyrosine nitration and apoptosis induced by trophic factor deprivation, but not if the tyrosine is replaced by phenylalanine or proline. Tyrosine has no effect on the survival of motor neurons cultured with or without trophic factors. However, free nitrotyrosine, at pathological relevant concentrations, induced motor neuron apoptosis in the presence of trophic factors. Nitrotyrosine-induced apoptosis was not prevented by inhibition of nitric oxide production or by the superoxide and peroxynitrite scavengers MnTBAP and FeTCPP. Incubation of proliferating cultures of myoblasts with nitrotyrosine inhibited cell growth and differentiation, while stimulating cell death. In contrast, incubation of differentiated myotubes with nitrotyrosine did not affect cell viability. The dialyzed conditioned media from myotube cultures sustained motor neuron survival in the absence of pure trophic factors. However, the dialyzed conditioned media from nitrotyrosine-treated myotubes induced motor neuron death, even in the presence of a combination of trophic factors. Free nitrotyrosine was incorporated into the skeletal muscle ?-tubulin of newborn rats. The possible role of muscle in the pathogenesis of ALS was tested by middle intensity exercise in G93A transgenic mice. Exercise accelerated the onset of disease symptoms and death. We hypothesized that the kynurenine pathway (KP) and more particularly the excitotoxin quinolinic acid (QUIN) are involved in the neuropathogenesis of amyotrophic lateral sclerosis (ALS). Prominent microgliosis is a pathological hallmark in patients with advanced disease. Activated microglia produce QUIN in neurotoxic concentrations. We previously showed that chronic exposure to QUIN at low concentrations (350 nM) causes ultra-structural changes in cultured human neurons. Using gas chromatography / mass spectrometry (GC/MS), QUIN and picolinic acid (PIC), another KP metabolite, concentrations have been quantified in matching serum and CSF samples from patients with ALS. We found that both QUIN and PIC are strongly elevated in serum and to a lesser extent in CSF. These data imply that the KP may potentially play a role in motor neuron death in ALS.
The biological basis of preferential motor neuron degeneration in amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) remains incompletely understood, and effective therapies to prevent the lethal consequences of this disorder are not yet available. Since 1993, more than 100 mutant variants of the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) have been identified in familial ALS. Many studies have sought to distinguish abnormal properties shared by these proteins that may contribute to age-dependent motor neuron loss in ALS. SOD1 is an abundant 32-kDa homodimeric enzyme that catalyzes the dismutation of superoxide radicals (O2-·) to O2 and H2O2. Transgenic rodent models that express mutant SOD1s have also provided tools to investigate the pathophysiology of mutant SOD1-mediated ALS and to test new therapies. Evidence to date implicates a gain of toxicity by mutant SOD1s. Possible mechanisms of toxicity include aberrant oxidative chemistry, formation of noxious aggregates or oligomers, saturation of clearance mechanisms or chaperone functions, impairment of glutamate re-uptake, dysfunction of mitochondria, altered neurofilaments or axonal transport, and triggering of inflammatory or apoptotic cascades. Complex networks of cellular interactions and age-associated changes may link mutant SOD1s and other stresses to motor neuron death in ALS. Our laboratory and collaborators have compared biophysical and biochemical properties of wild type and mutant SOD1 proteins to discern features shared by the pathogenic variants. Crystal structures obtained from metallated “wild type like” (WTL) SOD1 mutants, which retain the ability to bind copper and zinc and exhibit normal specific activity, indicate a native-like structure with only subtle changes to the backbone fold. In contrast, a group of “metal binding region” (MBR) SOD1 mutants that are deficient in copper and zinc exhibit thermal destabilization and disorder of the zinc binding and electrostatic channel loops near the metal binding sites. Furthermore, WTL SOD1 mutants differ from wild type SOD1 in their susceptibility to reduction of the intrasubunit disulfide bond between Cys-57 and Cys-146 at physiological pH and temperature. This bond anchors the disulfide loop to the SOD1 â-barrel and helps to maintain the dimeric configuration of the protein. Cleavage of the disulfide linkage renders the WTL mutants vulnerable to metal loss. This may be a key factor in vivo that perturbs relatively well-folded WTL mutants to resemble the destabilized and locally misfolded MBR mutant species. Consistent with this, we have observed that both purified MBR and WTL mutants and corresponding mutant species in SOD1 transgenic mouse spinal cord lysates exhibit abnormally increased hydrophobic surface properties. SOD1 proteins with disordered loops or increased monomerization are expected to be more susceptible to self-association or aberrant interactions with other cellular constituents. How these interactions of partially unfolded SOD1 may relate to toxicity at the cellular and systems levels remains to be determined.
Neuroinflammation is the concept that innate immune reactions occur in the central nervous system, via glial cell biology, thereby contributing significantly to neurodegeneration. One decade ago the neuroinflammation hypothesis was controversial and widely disputed; now, it has gained credence as a common feature of many (perhaps all) age-related neurodegenerative conditions. Research into neuroinflammation in ALS has lagged somewhat behind similar research in other neurodisease areas. Nonetheless significant progress has been made due largely to the accessibility of robust animal models. Exposure to environmental neurotoxicants may play a role in the etiology of ALS. Existing evidence most clearly suggests a role for heavy metals, particularly lead, but results are inconsistent. Genetic susceptibility may also be important. We conducted a case-control study of ALS in New England, USA, from 1993 to 1996 in order to characterize the relationships of ALS to lead exposure and to genetic polymorphisms affecting neurologic function or lead metabolism. Cases (N=109) were recruited at two hospitals in Boston, MA; ALS diagnosis based on El Escorial criteria was verified by a neurologist. Population controls (N=256) identified by random digit dialing were frequency matched to cases by age, sex, and region of residence within New England. We used a structured interview to collect information on occupational, residential, and recreational exposure to lead as well as information on demographics, lifestyle, and medical history. We measured blood and bone lead levels, the latter using noninvasive K x-ray fluorescence, and we collected whole blood as a source of DNA for studies of genetic susceptibility. Laboratory measures were available for most cases and a subset (N=41) of controls. We explored the relationship of ALS to several lifestyle factors. Cigarette smoking was associated with increased ALS risk [odds ratio (OR) 1.7, 95% confidence interval (CI) 1.0-2.8], but alcohol use had no relationship to ALS. Family history of ALS was associated with increased risk of ALS. We examined dietary intake of calcium, magnesium, and antioxidants. Overall, most dietary factors were not related to ALS risk, although modestly protective associations were suggested for magnesium (OR 0.7) and lycopene (OR 0.5). In analyses of the relationship of ALS to lead exposure, we found that risk of ALS was associated with an increase in self-reported occupational exposure to lead (OR 1.9, 95% CI 1.1-3.3), with a dose-response for lifetime days of lead exposure. In contrast, ALS was not related to residential or recreational exposure to lead. Risk of ALS was also associated with elevations in both blood and bone lead levels: ORs (95% CIs) were 1.9 (1.4-2.6) for each ug/dl increase in blood lead, 3.6 (0.6-21) for each unit increase in log-transformed patella lead, and 2.3 (0.4-15) for each unit increase in log-transformed tibia lead. These results extend previous reports based entirely on interview data, showing for the first time an association of ALS with lead biomarkers, and suggest a potential role for lead exposure in the etiology of ALS. We also explored the role of genetic susceptibility to lead exposure. We evaluated the relationship of ALS to polymorphisms in the genes for delta-aminolevulinic acid dehydratase (ALAD) and the vitamin D receptor (VDR), which have both been implicated in susceptibility to lead toxicity. The ALAD 2 allele was associated with decreased lead levels in both patella and tibia, although not in blood, and with an increase in ALS risk (OR 1.9, 95% CI 0.6-6.3). In contrast, the VDR B allele was not associated with lead levels or ALS risk. These novel findings suggest that genetic susceptibility conferred by polymorphisms in ALAD may affect ALS risk, possibly through a mechanism related to changes in lead toxicokinetics. We evaluated the relationship of ALS to vascular endothelial growth factor (VEGF), an angiogenic growth factor that mediates responses to hypoxia. We confirmed a previous report that risk of ALS is associated with polymorphisms in the VEGF promoter region that determine two specific haplotypes (OR 3.0, 95% CI 0.7-13). These findings suggest that the mechanism of ALS pathogenesis may involve impaired response to hypoxia, consistent with previous evidence that oxidative stress is involved in ALS pathophysiology. In summary, we found that risk of ALS was associated with occupational exposure to lead and increased blood and bone lead levels. ALS was also associated with cigarette smoking but not with alcohol use. Polymorphisms in ALAD and the VEGF promoter were associated with ALS. Thus ALS likely has a multifactorial etiology involving both environmental exposures and genetic susceptibility. A causal approach to treatment of motor neuron diseases has historically always met with scepticism. However, the discovery of mutations in the SOD1 gene back in 1993 which is causal for a subgroup of patients and the subsequent development of an animal model has paved the way to numerous experimental therapeutic approaches. In contrast, inspite of large investments the approaches to causal treatments in humans were often disappointing, recent examples are the German high-dose vitamine E trial, the Dutch creatin trial, and the European pentoxyphylline, Ono 2506 and TCH-346 trial. S-nitrosylation refers to the binding of a NO group to a thiol (usually a cysteine) on a peptide or protein. S-nitrosylated peptides serve as reservoirs of nitric oxide (NO) in cells and transfer their NO groups to protein thiols in transnitrosation reactions. S-nitrosylation of critical cysteine residues on proteins regulates protein function and cell signaling. We investigated whether disruption of protein S-nitrosylation contributes to amyotrophic lateral sclerosis (ALS) pathogenesis. It has previously been shown that WT SOD1 catalyzes the reductive decomposition of S-nitrosylated peptides to yield NO and the disulfide. We hypothesized that SOD1 mutants catabolize S-nitrosylated peptides faster than WT SOD1 due increased access of S-nitrosylated peptides to the active site copper of some SOD1 mutants. An aberrant increase in the catabolism of peptide S-nitrosothiols (SNOs) may contribute to motor neuron death by disrupting cell signaling pathways regulated by S-nitrosylation. To test this hypothesis, we first demonstrated that SOD1 mutants decompose peptide SNOs significantly faster than WT SOD1 in cell free systems. We then demonstrated that both peptide and protein SNO levels are decreased in cells expressing mutant as compared to WT SOD1. We have identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as one of the proteins that is denitrosylated in cells expressing SOD1 mutants. GAPDH, a protein originally thought to play a role only in glycolysis, has recently been found to have multiple additional functions including the regulation of transcription and apoptosis. In cells expressing SOD1 mutants, not only the extent of S-nitrosylation, but also the subcellular localization of GAPDH is altered. Finally our data suggest that SNO catabolism is increased in the spinal cords of transgenic mice expressing copper-binding SOD1 mutants as compared to WT SOD1. These findings raise the possibility that increased catabolism of SNOs is a novel toxic gain-of-function of SOD1 mutants that contributes to ALS pathogenesis. Amyotrophic lateral sclerosis (ALS) is the most popular motor neuron disease linked to fatal outcome within 3-5 years. The precise pathogenetic mechanism underlying motoneuronal death in ALS remains to be elucidated. There are currently no effective therapies for ALS. Since 1990, a series of clinical trials have been conducted in ALS in many countries, involving a large number of patients. They provided a huge number of crucial information, even if they are less than expected due to the restrictive policy of transfer of databases by most pharmaceutical companies. A central working hypothesis of our laboratory is that neurological diseases, including amyotrophic lateral sclerosis (ALS) result from a failure in compensatory mechanisms to cell stress. Accordingly, our team has focused its efforts on identifying transcription factors that mediate adaptive responses to oxidative stress. These efforts have resulted in the identification of a signaling pathway leading to the activation of the transcriptional activator, hypoxia inducible factor-1 (HIF-1) as a mediator of protective responses in neurons. Specifically, we have correlated the protective effects of iron chelators such as deferoxamine and mimosine with their ability to stabilize HIF-1 protein levels; to induce activity of a canonical hypoxia response element driven luciferase reporter; and to increase the expression of known HIF target genes such as vascular endothelial growth factor (VEGF), erythropoietin (Epo), and glycolytic enzymes. Additional experiments have determined that iron chelators activate HIF-1 by inhibiting a class of enzymes known as the iron and 2-oxoglutarate dependent prolyl 4-hydroxylases. Indeed, we have used low molecular weight as well as peptide inhibitors of the HIF prolyl 4-hydroxylases to show that inhibition of this class of dioxygenases is sufficient to protect neurons from oxidative stress in vitro and permanent focal ischemia in vivo. Finally, we have used an FDA-approved library to identify novel HIF prolyl 4-hydroxylase inhibitors and have identified several clinically approved compounds that possess the ability to upregulate VEGF and Epo in the nervous system. As VEGF appears to be necessary and sufficient to enhance motor neuron viability, small molecule activators of HIF-1 are well positioned to act as therapeutics in ALS.
Impairment and loss of the glutamate transporter GLT1 (a.k.a. EAAT2) has been reported in both sporadic and familial cases of amyotrophic lateral sclerosis (ALS) as well as in rodent models of the disease. Caspase-3 (cp-3) is pathologically activated in transgenic SOD1 mice model of ALS and its activation has been described both in neurons and astrocytes in the spinal cord. GLT1 is one of five high affinity glutamate transporters and it is responsible for the reuptake of more than 90% of the released glutamate. GLT1 has a predominant glial localization and has one cp-3 putative site in its cytoplasmic, C-terminal domain (-DTID-S). As motor neurons depend on GLT1 uptake in astrocytes to avoid excitotoxicity, it is possible that in ALS GLT1 becomes a substrate for cp-3 cleavage and that the resulting impairment of the transporter leads to excitotoxic damage of the motor neurons. In the present study, we investigated whether the glutamate transporter GLT1 could be a substrate for activated cp-3. We found that GLT1 is cleaved in vitro by active cp-3 and that the cleavage occurs at the consensus site at the aspartate residue in position 505. Other caspases such as caspase-7/8 or 6 do not cleave GLT1. GLT1 cleavage appears to be selective as the other mayor glial glutamate transporter GLAST is insensitive to cp-3 activation. Functionally, cp-3 activation causes a time-dependent impairment of GLT1. Xenopus oocytes expressing human GLT1 (EAAT2) and injected with the active form of cp-3 showed a progressive inhibition of GLT1-mediated uptake current and uptake of substrate (~60% within 30 min), paralleled by a loss of GLT1 immunoreactivity. Cp-3 cleavage of GLT1 occurs also in ALS SOD1-G93A mice and SOD1-H46R rats. In these animal models of ALS, the time-course of appearance of the cp-3-derived GLT1 fragments paralleled the time-course of cp-3 activation during the disease progression. In conclusion, we have demonstrated that cp-3 cleaves and inactivated the glutamate transporter GLT1. This event occurs in vivo in SOD1 transgenic animal models of ALS, and likely contributes to the excitotoxic damage to motor neurons in ALS.
In VEGF transgenic mice, the hypoxia response element in the VEGF promoter was deleted. This resulted in reduced VEGF expression in the spinal cord and caused adult-onset, progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis (ALS). The mechanism through which these reduced VEGF levels caused selective motor neuron death is unknown. We therefore examined the direct effect of VEGF on cultured motor neurons and found VEGF to have a direct neurotrophic effect on motor neurons in vitro. Survival and vulnerability to excitotoxicity of motor neurons from VEGF mice was similar to that of motor neurons from non-transgenic littermates. The VEGF concentration in the spinal cord of mutant SOD1G93A mice was not different from that in SOD1WT overexpressing mice. Upregulation of VEGF in the spinal cord by housing mutant SOD1G93A mice in hypoxic conditions did not affect their life span, while crossbreeding the VEGF mice with SOD1G93A mice resulted in a shorter life span of the double transgenics. Moreover, intracerebroventricular delivery of recombinant VEGF in SOD1G93A rats delayed onset of paralysis by 17 days, improved motor performance and prolonged survival by 22 days. This VEGF treatment was particularly effective in rats with forelimb onset. These results show that VEGF is a neurotrophic factor for motor neurons, that shortage of this neurotrophic factor may contribute to the selective motor neuron death observed in humans and animals and that supplementation with VEGF could have therapeutic potential.
RNA interference (RNAi) can achieve gene silencing in a wide variety of eukaryotes. Early work suggested that RNAi can silence genes with single nucleotide specificity. In many neurodegenerative diseases, neuronal degeneration develops in heterozygous individuals. In these cases, while the mutant allele expresses toxic proteins and causes cell death, the wild type allele expresses the normal protein that performs essential functions. Therefore, the ideal treatment for these diseases is selectively silencing the mutant while maintaining the wild type gene expression. To understand the rules for designing allele-specific silencing, we carried out a systematic investigation using human Cu, Zn superoxide dismutase (SOD1) gene as a model. We tested all possible mismatches between the siRNA and its target. We also tested the optimal position where placing of the mismatch generates the most specific siRNA. Our results indicate that only a subset of mismatches, when placed at certain positions of the small interfering RNA (siRNA), generate effective allele-specific siRNAs.
Human recombinant CuZnSOD (hrCuZnSOD) is inactivated by peroxynitrite in a dose-dependent way. The concentrations of peroxynitrite that decreased the activity by 50% (IC-50) were 35 and 100 µM at 1 and 8 µM hrCuZnSOD respectively, at pH 7.4, 37 ºC. Peroxynitrite reacted with hrCuZnSOD with a second order rate constant of (9.4 ± 1.0) x 103 M-1 s-1 per monomer at pH 7.5, 37 ºC. EPR experiments suggest that histidinyl radical formation is involved in hrCuZnSOD inactivation. Exposure to heavy metals, including lead (Pb) has been proposed as a risk factor for ALS. However, the pathogenic effect of Pb in ALS remains controversial. We investigated the effects of low-level Pb exposure in transgenic mice expressing the G93A SOD-1 mutation. Mice were exposed to 200 mg/l or 600 mg/l Pb acetate in drinking water from PND20 until death. Unexpectedly, Pb exposure caused an increase in survival by an average of 2 weeks with both doses, when compared to transgenic littermates treated with vehicle only (sodium acetate). To determine whether Pb directly affected neuronal survival, we investigated its effects on purified embryonic motor neurons cultures. Addition of 10µM concentration of Pb to the culture medium caused 25% motor neuron death. Since astrocytes represent the major storage of Pb in the CNS, we then explored whether Pb influenced the ability of astrocytes to support the survival of motor neurons maintained in the absence of trophic factors. Exposure of astrocytes to 10 µM Pb for 24h increased the trophic activity of conditioned medium for motor neurons by 20-25% (with respect to sodium acetate), suggesting that Pb stimulates the production of diffusible trophic factors. Preliminary results show that Pb potently increased the expression of the antioxidant enzyme heme-oxygenase-1 and the trophic factor VEGF in astrocyte monolayers. Chronic low-level Pb exposure in drinking water also increased the levels of VEGF mRNA in the spinal cords of G93A SOD-1 mice treated with Pb. These data suggest that the appropriate stimulation of astrocytes can delay the progression of motor neuron degeneration in ALS, in spite of Pb neurotoxicity. A toxic “gain-of-function” of mutant Cu–Zn superoxide dismutase 1 (SOD1) has been involved in the pathogenesis in familial amyotrophic lateral sclerosis (ALS). Expression of mutant forms of the human SOD1 gene in mice and rats causes a degeneration of motor neurons and astrocyte activation. Previous studies have showed a decrease in mitochondrial respiration in brain and spinal cord from G93A-SOD1 mice, which could be linked to oxidative damage. In transgenic early symptomatic rats expressing the G93A SOD-1 mutation, we found a reduction in oxygen consumption by mitochondria purifed from the spinal cord. We also found a high degree of protein oxidative damage in the spinal cord as evidenced by immunolabeling for DMPO-protein adducts by specific antibodies, following systemic injections of DMPO. Astrocytes in the anterior horn of early symptomatic G93A rats were also labeled after DMPO injections, suggesting the occurrence of oxidative stress and a potential compromise of mitochondria. Mitochondria from cultured brain astrocytes isolated from SOD/G93A transgenic rats showed a marked decreased of oxygen consumption. Addition of DMPO partially restored mitochondria respiration. Human SOD-1 expressed by the transgen was accumulated in purified astrocytic mitochondria, suggesting a functional link between SOD-1 mutation and decreased mitochondrial activity. Finally, we found that G93A transgenic astrocytes did not support motor neuron survival in cocultures in the absence of trophic factors, as compared to normal astrocytes. These data suggest that mitochondrial dysfunction is an early event in astrocytes expressing mutated SOD-1. The metabolic consequences of such mitochondrial dysfunction may underlie the deficitary neurotrophic support of transgenic astrocytes. In an otherwise tightly coupled electron transport chain, approximately 1-3% of mitochondrial oxygen consumed is incompletely reduced and leads to the production of reactive oxygen species (ros). While not “harvested” for oxidative phosphorylation, these “leaky” electrons can quickly interact with molecular oxygen to form superoxide anion, the predominant ros in mitochondria. Mtdna lacks protective histones and has minimal repair mechanisms rendering mitochondria particularly vulnerable to oxidative damage. Superoxide has been implicated in various diseases including hypertension, atherosclerosis, diabetes, parkinson's, and amyotrophic lateral sclerosis. A variety of mechanisms, including excitotoxicity, oxidation-mediated damage, neurofilament aggregation and autoimmunity had been suggested to explain the pathogenesis of ALS. Observations made in animals models and ALS patients support the involvement of autoimmune mechanisms in sporadic ALS pathogenesis. The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily and may exert pro-apoptotic actions when activated by its ligands including nerve growth factor (NGF). In adult motor neurons, p75NTR expression is linked to neuronal injury or disease and its expression has been implicated in motor neuron death observed in Amyotrophic Lateral Sclerosis (ALS). Pure embryonic motor neuron cultures were not sensitive to exogenous NGF (100 ng/ml), in spite of the high level of p75NTRexpression. However, when co-cultured on the top of astrocyte monolayers motor neurons became vulnerable to exogenous NGF. The effect of astrocytes was reproduced in pure motor neuron cultures by the production of low steady state concentrations of nitric oxide (<50nM) by NOC-18. NOC-18 did not affect neuronal survival but rendered motor neurons vulnerable to NGF-induced apoptosis. Motor neuron loss induced by NGF involved JNK pathway, NADPH oxidase activation and was prevented by superoxide and peroxynitrite scavengers, suggesting that the execution of motor neuron apoptosis requires endogenous peroxynitrite formation. Moreover, motor neurons expressing the G93A SOD1 mutation were sensitive to NGF induced apoptosis without the addition of nitric oxide. These results suggest that endogenous production of nitric oxide and peroxynitrite contribute to motor neuron degeneration in ALS by facilitating apoptosis induced by NGF/p75NTR. Amyotrophic lateral sclerosis (ALS) is a motor neuron disease of unknown origin. About 2% of ALS cases are linked to mutations in the gene encoding copper/zinc superoxide dismutase (SOD1). Expression of mutant forms of SOD1 (SOD1m) in transgenic mice leads to motor neuron death and an ALS-like phenotype. Using an unbiased subtractive suppressive hybridization screen, we have identified a clone encoding the neurite outgrowth inhibitor Nogo that is specifically up-regulated in the lumbar spinal cord of asymptomatic G86R transgenic mice. This result was confirmed by Northern-blot. In spinal cord of 90 days old G86R mice NogoA mRNA levels are significantly increased as compared to the wild-type mice. In order to identify the cell type(s) over-expressing NogoA in the spinal cord, we have performed immunohistochemistry using specific Nogo-A antibody. In G86R transgenic mice, in addition to a basal immunoreactivity in glial cells also found in wild-type mice, Nogo-A is strongly induced in a subset of motor neurons. This result has been confirmed to spinal cord of ALS patients. Furthermore, using a double labelling, we have shown that in Nogo-A positive motor neurons nogo receptor (NgR) immunoreactivity was decreased. In contrast, in motoneurons immunoreactive to NgR, Nogo-A staining was faint or absent. Distal axonopathy of motor fibers is an early event associated with motor neuron degeneration in ALS. Mitochondria dysfunction and aggregation of mutated SOD-1 have been recently shown to contribute to ALS pathogenesis. We have studied the subcellular distribution of mitochondria and the protein SOD-1 in motor axons of rats expressing the G93A SOD-1 mutation. Immunohistochemistry was performed on axonal wholemounts micro-dissected form ventral or dorsal roots of asymptomatic (65-days old) transgenic or non-transgenic rats. Mitochondria and SOD-1 protein displayed a homogenous distribution along motor and sensory fibers in non-transgenic rats. No obvious accumulation mitochondrial clusters was observed. In constrat, motor axons from G93A rats showed accumulation of mitochondria in discrete clusters at regular intervals located at the axonal cortex. Remarkably, most of human G93A SOD-1 expressed by the transgen was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of the mutated enzyme to axonal mitochondria as previously described. The mitochondrial clusters displayed increased immunoreactivity for nitrotyrosine. Although mutated SOD-1 was highly expressed in sensory axons of G93A rats, we observed a low degree of colocalization with mitochondria and the occurrence of only few clusters in these fibers. The cytoskeleton was not overtly altered in the SOD-1/mitochondrial clusters. These results further support the contribution of axonopathy as an early event in motor neuron degeneration and suggest a pathogenic role of the interaction of mutated SOD-1 with mitochondria. Nuclear factor erythroid 2-related factor 2 (Nrf2) through the antioxidant response element (ARE) is known to coordinate the up-regulation of cytoprotective genes involved in combating oxidative stress. Genes that are regulated by this mechanism include antioxidant enzyme heme oxygenase-1 and enzymes related to the biosynthesis and utilization of glutathione. Because oxidative stress plays a key role in motor neuron loss observed in ALS, we investigated the status of Nrf2 pathway in rats expressing the ALS-linked SOD1 G93A mutation. Nrf2 and HO-1 levels were increased at the onset of the disease and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of SOD1 G93A rats. These observations implicate the initiation of an endogenous protective response in SOD1 G93A rats, which eventually is overwhelmed by apoptotic mechanisms leading to paralysis and death. Since ARE-mediated gene induction is mainly restricted to astrocyte cell populations, we investigate whether astrocytes with increased Nrf2 activity could rescue co-cultured motor neurons from NGF/p75NTR-dependent apoptosis. Astrocytes transiently transfected with Nrf2 expression vector or treated with tert-butylhydroquinone (tBHQ) prevented p75NTR-mediated motor neuron death. neuronal survival and reduced p75NTR-dependent apoptosis. Therefore, upregulation of ARE/Nrf2 pathway in astrocytes may serve as a therapeutic approach in ALS. The amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurological disease, characterized for the degeneration of upper and lower motor neurons, which invariably has a fatal outcome.
SOD AGGREGATION AS BEING PROTECTIVE IN ALS
Joseph Beckman1, Alvaro Estévez
2 and Luis Barbeito3
1 Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, USA;
2 Department of Physiology & Biophysics, University of Alabama at Birmingham, Birmingham, USA;
3 Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable
SIGNALLING BY MITOGEN ACTIVATED PROTEIN KINASES PLAY A ROLE IN THE MOTOR NEURON DEGENERATION
Bendotti C., Lo Coco D., Tortarolo M., Veglianese P.
Dept. Neuroscience, Institute for Pharmacological Research ”Mario Negri”, Milano, Italy
Acknowledgments: This study was supported by Telethon, Italy (GP0222Y01), Fondazione Monzino and MND Association, UK. The financial support of the Istituto Italiano di Cultura di Montevideo to the meeting attendance of C.B. is greatly acknowledged.
CURRENT HIGHLIGHTS IN AMYOTROPHIC LATERAL SCLEROSIS: FROM DISEASE MECHANISMS TO THERAPIES
Lucie I. Bruijn
The ALS Association, 4338 Lavender Drive, Palm Harbor FL34685
HYPERMETABOLISM IN ALS CORRELATIONS WITH CLINICAL PARAMETERS
Desport J.C, Preux P.M, Torny F, Lacoste M, Couratier P
The objective was to analyse in a longitudinal study the metabolic level of 44 ALS patients and correlations with clinical parameters.
We measured the resting energy expenditure (REE) of 44 patients (sex ratio M/F: 0.69, mean age: 61.5 ± 10.1 years) at T1 (mean delay since first symptoms 587.2 ± 485.6 days) and T2 (mean interval T1-T2: 332.8 ± 272 days) and investigated the factors correlated with metabolic level. Nutritional evaluation included bioelectrical impedance analysis (with phase angle), indirect calorimetry and calculation of the body mass index. Neurologic assessment included an evaluation of tempheral and central neurologic deficit. Forced vital capacity was measured.
We confirm that ALS patients were hypermetabolic at T1 and T2 by an average of 12-13% more than in a reference healtly population. FFM and age were significantly associated with REE. During evolution, we noted a significant decrease in FVC, weight, BMI and phase angle. 56.8% of patients were hypermetabolic at T1 and 47.7% at T2. Hypermetabolism remained stable with time. Hypermetabolism at T1 did not influence the varation of clinical parameters at T2.
ALS patients are hypermetabolic. Hypermetabolism remained stable with time and was not influenced by variation in neurological and respiratory parameters.
BIOMARKERS AND SURROGATE MARKERS OF AMYOTROPHIC LATERAL SCLEROSIS (ALS)
Reinhard Dengler
Department of Neurology of the Medical School Hannover and Center of Systemic Neurosciences Hannover, Germany
There is an intensive search for specific biomarkers of ALS. A very promising approach uses proteomic studies, especially analysis of the proteins in the cerebrospinal fluid (CSF). Methodological descriptions have already been published, but no report on a disease-specific protein. Mass spectrometry is mostly used in conjunction with cleaving of proteins to smaller polypeptides. The SELDI technique which is a special mass spectrometry particularly appropriate to identify low molecular proteins is also frequently used. More effort is required by the 2-D-gel electrophoresis which may be appropriate to demonstrate also larger proteins. A combination of both mass spectrometry and 2-D-gel electrophoresis could be the best way. The expectation is justified that proteomics may find a place in the diagnosis of ALS within the next years. Demonstration of pro-inflammatory cytokines in the CSF such as monocyte-chemoattractant-protein-1 (MCP1) in ALS is of theoretical interest, but appears unspecific.
Imaging techniques, especially modern MRI may improve early diagnosis by demonstrating upper motor neuron involvement preclinically. Diffusion tensor imaging (DTI) separates between normal and ALS in group comparisons and may be improved to provide reliable diagnostic findings in individual patients. Magnetization Transfer imaging (MTI) may also have a potential in the diagnosis of individual cases. Both techniques and also functional MRI could be valuable in monitoring the disease course, e.g. in clinical treatment trials. Voxel based morphometry can unspecifically demonstrate cortical atrophy in the motor region and beyond and its extension in the course of the disease.
Clinical neurophysiology provides markers of both upper motor neuron involvement and loss of lower motor neurons. The latter can be closely monitored by the EMG technique “motor unit number estimate” (MUNE) which provides a fairly good measure of surviving motor neurons. Simple standardized measurement of compound muscle action potentials in response to supramaximal electrical nerve stimulation may be almost equally informative. A very sensitive technique to detect early upper motor neuron involvement seems to be the TMS modification “triple stimulation technique” (TST). We recently found significant changes with this technique in patients with suspected ALS who clinically did not show upper motor neuron signs. It would be worthwhile to compare the MRI technique DTI with TST to find out which method is more sensitive.
AMYOTROPHIC LATERAL SCLEROSIS: FATTER IS BETTER
Luc Dupuis
INSERM U692, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg, France
NITROTYROSINE, MUSCLE AND MOTOR NEURON DEGENERATION
Alvaro G. Estévez
University of Alabama at Birmingham, Birmingham, Alabama
DOES SKELETAL MUSCLE CONTRIBUTE TO AMYOTROPHIC LATERAL SCLEROSIS?
Jose-Luis Gonzalez de Aguilar
INSERM U-692, Université Louis Pasteur, Strasbourg, France
Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disease characterized by the selective loss of upper and lower motor neurons, and progressive muscle atrophy. Although many hypotheses have been put forward, the etiology of ALS is still poorly understood. Interestingly, recent studies postulated that motor neurons in ALS do not die because of an autonomous cell death program but other cells (particularly astrocytes and muscle fibers) may also contribute to the disease process. Our previous studies showed a characteristic expression pattern of the three major isoforms of the neurite outgrowth inhibitor Nogo (including Nogo-A, -B and –C) in skeletal muscles of ALS patients and SOD1(G86R) mice. In addition, we found that the increased levels of Nogo-A and Nogo-B, which had been barely detectable in muscles of control subjects, correlate significantly with the severity of motor impairment of ALS patients, as determined by the clinically validated ALS functional rating scale. Here we report that the transient expression of Nogo-A in skeletal muscle fibers is sufficient to injury the neuromuscular junction by inducing dismantlement of the post-synaptic structure and loss of pre-synaptic terminals. These findings strongly suggest that initial alterations in skeletal muscle, preceding the onset of disease symptoms, are able to trigger loss of neuromuscular junction integrity, axonal degeneration and muscle denervation, rather than motor neuron death. To investigate this question further, we used a high-density oligonucleotide microarray approach as a means to analyze gene expression on a large scale in skeletal muscle biopsies obtained from ALS patients. We also compared the skeletal muscle transcriptome of SOD1(G86R) mice with that of sciatic nerve-axotomized mice. The analysis of these databases is currently in progress.
KYNURENINE PATHWAY IN THE PATHOGENESIS OF AMYOTROPHIC LATERAL SCLEROSIS
Gilles J Guillemin1, George Smythe2, Vincent Meininger3 and Bruce J Brew1
1Centre for Immunology, Neuroimmnulogy Dept, St Vincent's Hospital, Sydney, NSW, Australia, 2010;
2Biomedical Mass Spectrometry Facility, University of NSW, Sydney, NSW 2052, Australia;
3Centre SLA Salpêtrière Fédération de Neurologie, l'Hôpital de la Pitié-Salpêtrière, 47 Boulevard de l'Hôpital, 75013 Paris, France.
MUTANT SOD1 INSTABILITY: IMPLICATIONS FOR TOXICITY IN ALS
Lawrence J. Hayward
Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Supported by NIH-NINDS NS44170, the ALS Association, and the Muscular Dystrophy Association
NEUROINFLAMMATION: A PRINCIPLE FOR RESEARCH AND THERAPY DEVELOPMENT IN ALS
Kenneth Hensley
Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma USA
Transgenic mice expressing familial als-associated, mutant g93a-sod1 experience reliable age-related neuroinflammation manifest by changes in astrocyte morphology, microglial proliferation, cytokine expression and the production of reactive oxygen species. In the g93a-sod1 mouse, both cytokine elaboration and oxidative stress begin in late pre-symptomatic stages of disease and accelerate thereafter. In addition to cytokine output, eicosanoid producing enzymes of both the cyclooxygenase (prostaglandin) and lipoxygenase (leukotriene) pathways are markedly induced in the g93a-sod1 mouse in an age-dependent fashion. Moreover primary astroglia cultured from g93a-sod1 neonates are in a “metastable” state that is exquisitely poised to hyper-express inflammatory mediators of both the cytokine and the eicosanoid types as well as remarkable excesses of reactive oxygen species.
The ultimate cause and the pathogenic relevance of glial inflammation remain unclear, in ALS and other neurodegenerative diseases. In mutant SOD1-associated ALS the genesis of neuroinflammation may relate to aberrant SOD1 accumulation in mitochondria, leading to perturbations in glial signal transduction pathways. If one makes a working assumption that neuroinflammation is generally detrimental in ALS, a series of rational strategies can be formulated for cell culture screens of bioactive agents that can be subsequently tested in the mouse model. Employing this strategy, a number of novel natural products have been identified that suppress glial activation in vitro. In particular, a constellation of small molecules that inhibit arachidonate metabolism through the lipoxygenase pathway, have shown in vitro and in vivo action including modest amelioration of symptoms in the G93A-SOD1 mouse. These findings begin to provide a basis for further research that could both identify novel molecular targets for pharmacotherapy and suggest new lead structures for development of safe neuro-active medicines.
PROTECTING MOTOR NEURONS FROM TOXIC INSULTS BY INHIBITING TRKB SIGNALING
Jelena Mojsilovic-Petrovic and Robert Kalb
Children's Hospital of Philadelphia, Philadelphia, PA 19104
The central pathologic event in ALS is the selective degeneration of motor neurons. One event that triggers cell death is the excessive activation of glutamate receptors (excitotoxicity). Cell death can also be triggered by expression of mutant proteins such as superoxide dysmutase (SOD1) or p150glued. In cultures of spinal cord, motor neurons will die if transiently exposed to an excitotoxic challenge or if they express mutant SOD1 or p150glued. In our tissue culture system these insults only kill motor neurons if Brain-derived neuronotrophic factor (BDNF) signaling via TrkB is intact. Inhibiting TrkB activation with tyrosine kinase antagonist is neuro-protective. In addition to activation by its ligand BDNF, TrkB can be trans-activated by stimulating G-protein coupled adenosine receptors. We found that inhibiting endogenous activation of adenosine receptors could reduce activation of TrkB. Application adenosine receptor antagonists also protected motor neurons from excitotoxic challenge or express mutant SOD1 or p150glued. Future work will examine the applicability of these findings to in vivo models of ALS.
Supported by the ALS Association and the NIH
LEAD EXPOSURE AS A RISK FACTOR FOR AMYOTROPHIC LATERAL SCLEROSIS
F Kamel, DM Umbach, H Hu, TL Munsat, JM Shefner, MP Longnecker, TA Lehman, PD Terry, JA Taylor, and DP Sandler
EXPERIMENTAL THERAPIES OF MOTOR NEURON DISEASES
Albert C. Ludolph
University of Ulm, Germany
How can we get to treatments more efficiently and fast ?
In conclusion, 125 years after Charcot therapeutics are in development and are a realistic goal for MND. However, enthusiasm must be accompanied by advances in methodology, both for experimental treatment in mice and men.
ALTERED S-NITROSYLATION IN ALS
Joan B. Mannick
University of Massachusetts, USA
TOXICITY BY MUTANT SOD1 IS ANTAGONIZED BY ALSIN, THE PRODUCT OF THE ALS2 GENE, AND ACTIVITY-DEPENDENT NEUROTROPHIC FACTOR
Masaaki Matsuoka and Ikuo Nishimoto
Department of Pharmacology and neuroscience, KEIO University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Several lines of evidence have supported the notion that proapoptotic property of familial ALS (FALS)-linked mutant Cu/Zn-superoxide dismutase I (SOD I) genes plays an important role in the pathogenesis of some of autosomal dominant FALS patients. In contrast, until recently, there had been no studies directly indicating the function of alsinLF, the product of the secondly identified FALS-linked ALS2 gene, linked to the onset of autosomal recessive motoneuron diseases.
Our recent studies have revealed that alsinLF is an anti-apoptotic protein that specifically protects motoneurons from toxicity by SOD1 mutants, but not toxicity by other neurodegenerative insults. We have further found that the alsinLF-mediated neuroprotective signal is transmitted to the Rac1/PI3 kinase/Akt3 anti-apoptotic axis.
Independently, we also found that activity-dependent neurotrophic factor (ADNF) protected neurons from death caused by FALS-linked SOD1 mutants though Ca2+/calmodulin-dependent protein kinase IV. Furthermore, we have showed that intracerebroventricularly administered ADNF improved motor performance of G93A-SOD1 transgenic mice, a widely used mouse model of ALS. Most recently, we have succeeded in developing more potent ADNF derivatives elongating survival of G93A-SOD1 transgenic mice. These new anti-ALS drug candidates are waiting for clinical trial.
It is thus anticipated that ADNF-derived agents will be combined with agents stimulating alsinLF-mediated motoneuronal protection in upcoming research looking for curative therapy of ALS.
CLINICAL TRIALS IN ALS: WHAT DID WE LEARN FROM RECENT TRIALS IN HUMANS
Vincent Meininger
ALS national referral and coordinating centre. Hôpital Salpétrière, 47 boulevard de l'Hôpital, 75013 Paris, France.
Prognostic factors. The most extensively documented are age, disease duration, diagnostic delay, site of onset and respiratory status. Based on a combination of these and other factors scoring systems have been developed which attempt to predict survival expectancy. These scores had various limitations, for example, some were derived from a relatively small sample of patients, whereas others were based on historical data, raising the question of the quality of the data collected. In contrast, data which have been collected during clinical trials are more secure due to a good quality of follow up. In studies which included a large range of patients allow predicting expected survival time for individual patients. In a large riluzole study we develop a simplified score which contains simple categorical values to make it easy to handle in daily clinical practice. Nine variables can be evaluated by examination of the patient, namely: age, disease duration, number of regions with atrophy, spasticity and fasciculations, and cough and swallowing and the distal muscle score which is easily performed using the standardised MRC procedure. Two non clinical variables, creatinine levels and slow vital capacity are routinely performed. This score should therefore be practical for daily use in the clinic and useful for planning disease management in ALS.
Preclinical studies. Recent results obtained in humans raise the question of the validity of both in vitro and in vivo mouse models to predict the efficacy of a compound in human patients. Discrepancies between transgenic mice (G93A) and ALS patients have been observed for most compounds, such vitamin E, gabapentine, topiramate, creatine, pentoxifylline. Only riluzole showed a positive effect in human and in mouse. The possible shortcomings are: the lack of study for a dose response relationship; the huge difference between the doses of drug used in animals and the dose usable in humans; the lack of study analyzing for possible interaction with other drugs as riluzole. It is also necessary to consider that we need to take into account that in animals treatment was initiated long before onset of muscle weakness, while ALS patients are well advanced into their disease course when enrolled in clinical trials.
Function/survival. It is often proposed that function is closely related to survival leading recently to the assumption that functional measures can act as “surrogate markers” for survival. In most studies there are significant correlations between survival and the slopes of deterioration of functional variables. However, these correlations are not strong enough to suggest that these functional endpoints are able to explain the overall survival parameter. Analyses of most trials urge caution and suggest that we reconsider the wisdom of basing the design of phase 3 trials solely on functional measures. For example, in the riluzole trial, the drug had a significant effect on survival, but no effect on function. In the xaliproden trial, there was a positive effect on function but no effect on survival. In the CNTF and pentoxifylline trials there was a deleterious effect on survival and a lack of efficacy on muscle strength. A deleterious effect on function with no effect on survival was reported in a combined analysis of the two gabapentin trials. Similar conclusion about independence between survival and function has been recently demonstrated in animal models.
All these data strengthen the need for more collaborative works between basic scientists and clinicians in the design of preclinical and clinical trials.
THE WISDOM OF THE BODY: PROLYL HYDROXYLASE INHIBITION, HYPOXIA INDUCIBLE FACTOR-1 ACTIVATION AND ADAPTIVE RESPONSES TO OXIDATIVE STRESS AND HYPOXIA IN THE NERVOUS SYSTEM
Rajiv R. Ratan M.D., Ph.D., Ambreena Siddiq, Ph.D., JoAnn Gensert, Ph.D., Kyungsun Suh, Ph.D., Philipp Lange, M.D., Leila Aminova, Ph.D., Brett Langley, Ph.D. and Juan Chavez, Ph.D.
Burke/Cornell Medical Research Institute and Departments of Neurology and Neuroscience, Weill Medical College of Cornell University
CASPASE-3 MEDIATED PROCESSING OF THE GLUTAMATE TRANSPORTER EAAT2 IN ALS
Davide Trotti
Massachusetts General Hospital, Harvard Medical School, Charlestown – Boston, USA
VEGF AND MOTOR NEURON DEGENERATION
Ludo Van Den Bosch
Neurobiology, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium
RNAI THERAPY AGAINST MUTANT SOD1 THAT CAUSES ALS
Zuoshang Xu
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
For diseases that are caused by many point mutations (e.g. ALS that is caused by more than 100 mutations in the SOD1 gene), our results imply that all mutations cannot be silenced with allele specificity. To circumvent this problem, we have designed a replacement RNAi strategy. In this strategy, both mutants and the wild type SOD1 are inhibited by RNAi. The wild type SOD1 function is then replaced by a designed wild type SOD1 gene that is resistant to the RNAi. To demonstrate the concept of this strategy, we screened numerous short hairpin RNAs (shRNAs) and found two highly efficacious ones. Two replacement genes that resist RNAi mediated by each of these two shRNAs were then designed. We demonstrated that the shRNAs silenced the endogenous SOD1 gene and that the replacement gene can restore the levels of the SOD1 protein and its function in the presence of the shRNA. By placing the shRNA-synthesizing cassette and the replacement gene on the same viral vectors, this strategy can be used to treat all ALS cases that are caused by mutations in the SOD1 gene. We are currently testing this strategy in mutant SOD1 transgenic mice.
SOD INACTIVATION BY PEROXYNITRITE AND FLUXES OF SUPEROXIDE AND NITRIC OXIDE
Demicheli, V.§¶; Quijano, C.§ , Alvarez, B.¶ and Radi, R.§
§Depto. de Bioquímica, Facultad de Medicina and ¶Laboratorio de Enzimología, Facultad de Ciencias, UDELAR, Montevideo, Uruguay
In order to approximate this situation to a physiological condition, we studied the activity of hrCuZnSOD exposed to fluxes of superoxide and nitric oxide. Exposure of hrCuZnSOD to equimolar fluxes (10 µM/min) of nitric oxide and superoxide yielded a time-dependent inactivation, reaching a maximum of 70% of inactivation after 40 min of incubation. Then, we exposed hrCuZnSOD for 10 min to a constant flux of superoxide (10 µM/min) varying the rate of nitric oxide production from 5 to 30 µM/min. We observed that, the higher the flux of nitric oxide, the lower the activity of the enzyme. A maximum of 50% of inactivation was achieved with 30 µM/min nitric oxide and 10 µM/min superoxide. Similar profiles were obtained when exposing the enzyme to higher superoxide fluxes (30 µM/min) and varying nitric oxide fluxes from 15 to 90 µM/min, for shorter periods (3 min). Immunospintrapping experiments using DMPO as spin trap, showed that a protein radical intermediate is formed during the exposure of hrCuZnSOD to the fluxes.
We have also exposed hrMnSOD to equimolar fluxes (10 µM/min) of superoxide and nitric oxide, obtaining a maximum of ~40% of inactivation after 10 min of incubation. The loss of activity was associated with tyrosine nitration.
CuZnSOD and MnSOD inactivation by peroxynitrite leads to an increase of the rate of reaction of superoxide with nitric oxide, generating thus, more peroxynitrite. The increase in the formation of oxidants promoted by this positive feedback system could be relevant in the onset of several pathologies.
CHRONIC LOW-LEVEL LEAD EXPOSURE INCREASES SURVIVAL OF G93A SOD-1 TRANSGENIC MICE
Ana G. Barbeito#, Natalia Guelfi&, Marcelo R. Vargas&, Mariana Pehar&, Joseph Beckman¶, Luis Barbeito& , Patricia Cassina#
#Departamento de Histología, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay; &Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo 11600, Uruguay; ¶Linus Pauling Institute, Environmental Health Sciences Center, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA.
MITOCHONDRIAL DYSFUNCTION AND OXIDATIVE STRESS IN G93A SOD TRANSGENIC ASTROCYTES
Patricia Cassina#, Adriana Cassina¶, Mariana Pehar&, Raquel Castellanos#, Marcelo R. Vargas&, Joseph S. Beckman‡, Rafael Radi¶ and Luis Barbeito&
#Departamento de Histología, y ¶Centro de Investigaciones Biomédicas en Radicales Libres, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay. & Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo 11600, Uruguay.
‡ Linus Pauling Institute, Environmental Health Sciences Center, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA.
FLUOROGENIC DETECTION OF MITOCHONDRIAL SUPEROXIDE IN LIVE CELLS
Michael S. Janes1,2, Dani M. Hill 1, Caleb M. Cardon1, Kristine M. Robinson3, Julia R. Walls1, Wai-Yee Leung1, Joseph S. Beckman3,4, Michael J. Ignatius1
1Invitrogen-Molecular Probes Labeling and Detection Technologies, Eugene, Oregon;2Molecular and Cellular Biology Program, Oregon State University, Corvallis, Oregon; 3Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon; 4Linus Pauling Institute, Oregon State University, Corvallis, Oregon
We have developed a novel fluorogenic dye for the highly selective detection of mitochondrial superoxide in live cells. The assumption that mitochondria serve as the major intracellular source of ROS has been based largely on experiments with isolated mitochondria rather than with live cells. To establish selectivity for superoxide, we employed various cell-free systems to generate ROS and radical nitrogen species. The probe was readily oxidized by superoxide but not by other oxidative species. The oxidation product becomes highly fluorescent upon binding nucleic acids and oxidation of the probe was prevented by superoxide dismutase. The probe is permeable to live cells and is rapidly and selectively targeted to mitochondria. Once in the mitochondria, the compound is oxidized by superoxide and red fluorescence is easily visible as distinct reticula.
This reagent may enable researchers to delineate artifacts of isolated mitochondrial preparations from direct measurements of superoxide generated in the mitochondria of live cells. It may also provide a valuable tool in the discovery of agents that modulate oxidative stress in various pathologies.
SIGNALING PATHWAYS REGULATING SYNAPTIC POTENTIATION TRIGGERED BY ALS-IgG IN MOTOR NERVE TERMINALS
R.M. Pagani 1, R. Reisin 2 & O.D. Uchitel 1
1 IFIBYNE-CONICET, Fac. Ciencias Exactas y Naturales, UBA; 2 Servicio de Neurofisiología, Hospital Británico
IgG from ALS patients (ALS-IgG) trigger synaptic potentiation at the neuromuscular junction. We hypothesize that ALS-IgG-induced synaptic potentiation may be an early step of the motor neuronal degeneration. Thus, we have investigated the molecular target localization and signaling mechanisms of ALS-IgG-induced synaptic potentiation.
We observed that ALS-IgG interacts with a molecular target localized at the motor nerve terminals and that synaptic potentiation requires Ca2+ influx through Cav2.2 calcium channels and concomitant activation of a signaling pathway involving PLC, IP3 and ryanodine sensitive Ca2+ stores. These findings supports the hypothesize that immunological factors may play an important role in the disease process
CONTRIBUTION OF NITRIC OXIDE AND PEROXYNITRITE TO P75NTR- DEPENDED MOTOR NEURON APOPTOSIS
Mariana Pehar1; Patricia Cassina2; Marcelo R. Vargas1; Alvaro G. Estévez3; Joseph S. Beckman4 and Luis Barbeito1
1 Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable and 2 Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; 3 Department of Physiology & Biophysics, University of Alabama at Birmingham, Birmingham, USA; 4 Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, USA
NOGOA UPREGULATION IN ALS MOTOR NEURONS: INSIGHTS INTO A POTENTIAL FUNCTION IN MOTOR NEURON SURVIVAL
Frédérique Rene
INSERM U692, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg, France
These results suggest a relationship between Nogo-A overexpression and NgR down-regulation. The possible implication of this cross-talk in motor neurons death is currently under investigation in in vitro models.
(This work was supported by AFM)
OCCURRENCE OF MITOCHONDRIAL CLUSTERS ASSOCIATED WITH G93A SOD-1 PROTEIN IN MOTOR AXONS IN A TRANSGENIC MODEL OF AMYOTROPHIC LATERAL SCLEROSIS (ALS)
José Sotelo Silveira1,2, Sofía Horjales2, German Cota2, Martín Baraibar3, Julio Battistoni 3, Mario Señorale2, Mónica Marín2, Ricardo Ehrlich2, Luis Barbeito1
1Secc. Bioquímica, Facultad de Ciencias; 2 Depto. Neurobiología Celular y Molecular, IIBCE; 3 Lab. de Inmunotecnología, Cat. Inmunología Fac Ciencias-Fac. Química.
NRF2 ACTIVATION IN SPINAL CORD ASTROCYTES AND MOTOR NEURON SURVIVAL
Marcelo R. Vargas&, Mariana Pehar&, Patricia Cassina#, Laura Martínez-Palma#, John A. Thompson¶, Joseph S. Beckman‡,and Luis Barbeito&
& Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo 11600, Uruguay; #Departamento de Histología, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay; ¶ Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA and ‡ Linus Pauling Institute, Environmental Health Sciences Center, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA
EPIDEMIOLOGY OF ALS IN URUGUAY. INCIDENCE, PREVALENCE AND RISK FACTORS
Vázquez, C, Ketzoian, C, Legnani, C, Rega, I., Sánchez, N, Perna, To, Penela, M, Aguirrezábal, X, Fraquia, M, Velázquez, M, Orique, C, Medici, M.
Instituto de Neurología-Hospital de Clínicas, Facultad de Medicina-Universidad de la República, Montevideo, Uruguay
Epidemiological studies report the crude incidence of ALS to vary from 0,31 to 3,2 for 100,000 of the population year, and the prevalence from 2.6 to 6.4 per 100,000 of the population.
The potential risk factor for ALS remains controversial. Previous case-control studies found increased risk for ALS among rural populations and in occupations involving physical activity (athletics, rugby, soccer). Other risk factors include exposure to chemical solvents, electrical field, heavy metals and traumatism.
The purpose of this study was document the incidence and prevalence of ALS in the Republic Oriental del Uruguay and examine the potential risk factor for ALS.
We conducted a prospective, population based study of ALS for the 2-year period 2002 to 2003.
To ensure complete case ascertainment, multiple sources of information were used.
The El Escorial diagnostic criteria for ALS were applied to all cases enrolled.
Each patient was evaluated and regularly followed up during his or her illness.
Between January 1st, 2002, and December 31st, 2003, 143 patients were diagnosed with probable and definite ALS (126 definite, 17 probable) including 93 men and 50 women.(sex ratio 1,8)
The mean age at onset was 58,5 + 12 years (57,5 years for men, 60,5 years for women)
The mean time to reach diagnosis varied widely with a mean of 16.8 months.
The average annual incidence rate was 1,32 per 100.000 persons year.
On December 31st, 2002 the prevalence was 1,9 per 100.000 of the total population.
The incidence and prevalence found in Uruguay is comparable to reported to date in another regions of the world.
For the analysis of risk factors was conducted a case control study and Odds Ratio calculated for each factor. We found a statistically significant association for work place in rural areas and physical activity.
This is the first prospective population based study performed in Latin America that estimate the incidence, prevalence and risk factors of the ELA in a entire country with geographic and ethnic homogeneous characteristics.
List of Participants
Lucie Bruijn / The ALS Association, Palm Harbor, US /
lbruijn@snet.net
Caterina Bendotti / Mario Negri Institute, Milan, Italy / bendotti@marionegri.it
Robert Kalb / University of Pennsylvania, Philadelphia, US / kalb@email.chop.edu
Masaaki Matsuoka / Keio University, Tokyo, Japan / sakimatu@sc.itc.keio.ac.jp
Davide Trotti / Harvard Medical School, Boston, US / DTROTTI@PARTNERS.ORG
Gilles J. Guillemin / University of New South Wales, Sydney, Australia / g.guillemin@cfi.unsw.edu.au
Jean-Philippe Loeffler / Université Louis Pasteur, Strasbourg, France / loeffler@neurochem.u-strasbg.fr
Lawrence Hayward / University of Massachusetts, Worcester, US / Lawrence.Hayward@umassmed.edu
Joseph Beckman / Oregon State University, US / joe.beckman@oregonstate.edu
Joan B. Mannick / University of Massachusetts, US / joan.Mannick@umassmed.edu
Alvaro G. Estevez / University of Alabama at Birmingham, US / Estevez@PHYSIOLOGY.UAB.EDU
Rafael Radi / Facultad de Medicina, Uruguay / rradi@fmed.edu.uy
Reinhard Dengler / Medizinische Hochschule Hannover, Germany / dengler.reinhard@mh-hannover.de
Freya Kamel / NIEHS, North Carolina, USA / kamel@niehs.nih.gov
Ludo Van Den Bosch / University of Leuven, Belgium / Ludo.VandenBosch@med.kuleuven.ac.be
Luis Barbeito / IIBCE, Uruguay / lbarb@iibce.edu.uy
Kenneth Hensley / Oklahoma Medical Research Foundation, US / Kenneth-hensley@omrf.ouhsc.edu
José Luis González de Aguilar / ULP, Strasbourg, France / gonzalez@neurochem.u-strasbg.fr
Philippe Couratier / CHU Dupuytren, Limoges, France
/ philippe.couratier@unilim.fr
Luc Dupuis / INSERM U692, Faculté de Médecine, Strasbourg, France / ldupuis@neurochem.u-strasbg.fr
M. Flint Beal / Cornell University, NY, US / fbeal@med.cornell.edu
Vincent Meininger / Hôpital de la Salpetrière, Paris, France / vincent.meininger@psl.ap-hop-paris.fr
Zuoshang Xu / University of Massachusetts, Worcester, US / zuoshang.xu@umassmed.edu
Juan Chávez / Cornell University, NY, US / JCHAVEZ@burke.org
Albert Ludolph / University of Ulm, Germany / sekretariat.neurologie@rku.de
Mario Medici / Instituto de Neurología, Uruguay
Ana Gabriela Barbeito / Facultad de Medicina, Uruguay
Cristina Vázquez / Facultad de Medicina, Uruguay
Frédérique Rene / ULP, Strasbourg, France / f_rene@neurochem.u-strasbg.fr
Marcelo Vargas / IIBCE, Uruguay
Rafael Pagani / IFIBYNE-CONICET, Argentine / mpagani@fbmc.fcen.uba.ar
Mariana Pehar / IIBCE, Uruguay / mpehar@iibce.edu.uy
José Sotelo Silveira / IIBCE, Uruguay
Michael Janes / Invitrogen-Molecular Probes, Eugene, US / mike.janes@invitrogen.com
Patricia Cassina / Facultad de Medicina, Uruguay / pcassina@fmed.edu.uy
Beatriz Alvarez / Facultad de Ciencias, Uruguay / beatriz.alvarez@fcien.edu.uy
Homero Rubbo / Facultad de Medicina, Uruguay / hrubbo@fmed.edu.uy
Adriana Cassina / Facultad de Medicina, Uruguay / acassina@fmed.edu.uy
Francisco Morales / Facultad de Medicina, Uruguay / fmorales@fmed.edu.uy
Abayuba Perna / Instituto de Neurología, Uruguay
Pedro Alzari / Institut Pasteur, France
Martín Baraibar / Instituto de Higiene, Uruguay
Laura Martínez / Facultad de Medicina, Uruguay
Inés Pose / Facultad de Medicina, Uruguay / ipose@fmed.edu.uy
Livea Fujita Barbosa / Universidad de Sao Paulo, Brasil / livea@iq.usp.br
Juan Gil / Instituto de Neurología, Uruguay
Carlos Ketzoian / Instituto de Neurología, Uruguay
Ximena Aguirrezabal / Instituto de Neurología, Uruguay
Renato Verdugo / Universidad de Chile, Chile / renatoverdugo@vtr.net
Ricardo Maccioni / Universidad de Chile, Chile / isabelro@manquehue.net
Sofía Horjales / Facultad de Ciencias, Uruguay / shorjales@fcien.edu.uy
Roberto Sica / Hospital Ramos Mejía, Argentina
Alberto Dubrovsky / Hospital Francés, Argentina