Bernadett Kalmar

Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motoneurons of the spinal cord and motor cortex die, resulting in progressive paralysis. This condition has no cure and results in eventual death, usually within 1–5 years of diagnosis. Although the specific etiology of ALS is unknown, 20% of familial cases of the disease carry mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1). Transgenic mice overexpressing human mutant SOD1 have a phenotype and pathology that are very similar to that seen in human ALS patients. Here we show that treatment with arimoclomol, a coinducer of heat shock proteins (HSPs), significantly delays disease progression in mice expressing a SOD1 mutant in which glycine is substituted with alanine at position 93 (SOD1G93A). Arimoclomol-treated SOD1G93A mice show marked improvement in hind limb muscle function and motoneuron survival in the later stages of the disease, resulting in a 22% increase in lifespan. Pharmacological activation of the heat shock response may therefore be a successful therapeutic approach to treating ALS, and possibly other neurodegenerative diseases.

Induction of heat shock proteins for protection against oxidative stress

Heat shock proteins (Hsps) have been studied for many years and there is now a large body of evidence that demonstrates the role of Hsp upregulation in tissue and cell protection in a wide variety of stress conditions. Oxidative stress is known to be involved in a number of pathological conditions, including neurodegeneration, cardiovascular disease and stroke, and even plays a role in natural aging. In this review we summarize the current understanding of the role of Hsps and the heat shock response (HSR) in these pathological conditions and discuss the therapeutic potential of an Hsp therapy for these disorders. However, although an Hsp based therapy appears to be a promising approach for the treatment of diseases that involve oxidative damage, there are some significant hurdles that must be overcome before this approach can be successful. For example, to be effective an Hsp based therapy will need to ensure that the upregulation of Hsps occurs in the right place (i.e. be cell specific), at the right time and to a level and specificity that ensures that all the important binding partners, namely the co-chaperones, are also present at the appropriate levels. It is therefore unlikely that strategies that involve genetic modifications that result in overexpression of specific Hsps will achieve such sophisticated and coordinated effects. Similarly, it is likely that some pharmaceutical inducers of Hsps may be too generic to achieve the desired specific effects on Hsp expression, or may simply fail to reach their target cells due to delivery problems. However, if these difficulties can be overcome, it is clear that an effective Hsp based therapy would be of great benefit to the wide range of depilating conditions in which oxidative stress plays a critical role.

Late stage treatment with arimoclomol delays disease progression and prevents protein aggregation in the SOD1G93A mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motoneuron degeneration, resulting in muscle paralysis and death, typically within 1–5 years of diagnosis. Although the pathogenesis of ALS remains unclear, there is evidence for the involvement of proteasome dysfunction and heat shock proteins in the disease. We have previously shown that treatment with a co‐inducer of the heat shock response called arimoclomol is effective in the SODG93A mouse model of ALS, delaying disease progression and extending the lifespan of SODG93A mice ( Kieran et al. 2004 ). However, this previous study only examined the effects arimoclomol when treatment was initiated in pre‐ or early symptomatic stages of the disease. Clearly, to be of benefit to the majority of ALS patients, any therapy must be effective after symptom onset. In order to establish whether post‐symptomatic treatment with arimoclomol is effective, in this study we carried out a systematic assessment of different treatment regimes in SODG93A mice. Treatment with arimoclomol from early (75 days) or late (90 days) symptomatic stages significantly improved muscle function. Treatment from 75 days also significantly increased the lifespan of SODG93A mice, although treatment from 90 days has no significant effect on lifespan. The mechanism of action of arimoclomol involves potentiation of the heat shock response, and treatment with arimoclomol increased Hsp70 expression. Interestingly, this up‐regulation in Hsp70 was accompanied by a decrease in the number of ubiquitin‐positive aggregates in the spinal cord of treated SODG93A mice, suggesting that arimoclomol directly effects protein aggregation and degradation.

The distal hereditary motor neuropathies

The distal hereditary motor neuropathies (dHMN) comprise a heterogenous group of diseases that share the common feature of a length-dependent predominantly motor neuropathy. Many forms of dHMN have minor sensory abnormalities and/or a significant upper-motor-neuron component, and there is often an overlap with the axonal forms of Charcot–Marie–Tooth disease (CMT2) and with juvenile forms of amyotrophic lateral sclerosis and hereditary spastic paraplegia. Eleven causative genes and four loci have been identified with autosomal dominant, recessive and X-linked patterns of inheritance. Despite advances in the identification of novel gene mutations, 80% of patients with dHMN have a mutation in an as-yet undiscovered gene. The causative genes have implicated proteins with diverse functions such as protein misfolding (HSPB1, HSPB8, BSCL2), RNA metabolism (IGHMBP2, SETX, GARS), axonal transport (HSPB1, DYNC1H1, DCTN1) and cation-channel dysfunction (ATP7A and TRPV4) in motor-nerve disease. This review will summarise the clinical features of the different subtypes of dHMN to help focus genetic testing for the practising clinician. It will also review the neuroscience that underpins our current understanding of how these mutations lead to a motor-specific neuropathy and highlight potential therapeutic strategies. An understanding of the functional consequences of gene mutations will become increasingly important with the advent of next-generation sequencing and the need to determine the pathogenicity of large amounts of individual genetic data.

The role of heat shock proteins in Amyotrophic Lateral Sclerosis: The therapeutic potential of Arimoclomol☆

Arimoclomol is a hydroxylamine derivative, a group of compounds which have unique properties as co-inducers of heat shock protein expression, but only under conditions of cellular stress. Arimoclomol has been found to be neuroprotective in a number of neurodegenerative disease models, including Amyotrophic Lateral Sclerosis (ALS), and in mutant Superoxide Dismutase 1 (SOD1) mice that model ALS, Arimoclomol rescues motor neurons, improves neuromuscular function and extends lifespan. The therapeutic potential of Arimoclomol is currently under investigation in a Phase II clinical trial for ALS patients with SOD1 mutations. In this review we summarize the evidence for the neuroprotective effects of enhanced heat shock protein expression by Arimoclomol and other inducers of the Heat Shock Response. ALS is a complex, multifactorial disease affecting a number of cell types and intracellular pathways. Cells and pathways affected by ALS pathology and which may be targeted by a heat shock protein-based therapy are also discussed in this review. For example, protein aggregation is a characteristic pathological feature of neurodegenerative diseases including ALS. Enhanced heat shock protein expression not only affects protein aggregation directly, but can also lead to more effective clearance of protein aggregates via the unfolded protein response, the proteasome-ubiquitin system or by autophagy. However, compounds such as Arimoclomol have effects beyond targeting protein mis-handling and can also affect additional pathological mechanisms such as oxidative stress. Therefore, by targeting multiple pathological mechanisms, compounds such as Arimoclomol may be particularly effective in the development of a disease-modifying therapy for ALS and other neurodegenerative disorders.

Upregulation of heat shock proteins rescues motoneurones from axotomy-induced cell death in neonatal rats.

Heat shock proteins (hsps) are induced in a variety of cells following periods of stress, where they promote cell survival. In this study, we examined the effect of upregulating hsp expression by treatment with BRX-220, a co-inducer of hsps, on the survival of injured motoneurones. Following sciatic nerve crush at birth, rat pups were treated daily with BRX-220. The expression of hsp70 and hsp90, motoneurone survival, and muscle function was examined at various intervals later and the number of functional motor units was assessed by in vivo isometric tension recordings. Fourteen days after injury, significantly more motoneurones survived in the BRX-220-treated group (39 +/- 2.8%) compared to the saline-treated group (21 +/- 1.7%). Moreover, in the BRX-220-treated group no further loss of motoneurones occurred, so that at 10 weeks 42 +/- 2.1% of motoneurones survived compared to 15 +/- 0.6% in the untreated group. There were also more functional motor units in the hindlimb muscles of BRX-220-treated animals. In addition, treatment with BRX-220 resulted in a significant increase in the expression of hsp70 and hsp90 in glia and neurones. Thus, treatment with BRX-220, a co-inducer of hsps, protects motoneurones from axotomy-induced cell death.

The effect of treatment with BRX-220, a co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury

In this study, we examined the effect BRX-220, a co-inducer of heat shock proteins, in injury-induced peripheral neuropathy. Following sciatic nerve injury in adult rats and treatment with BRX-220, the following features of the sensory system were studied: (a) expression of calcitonin gene-related peptide (CGRP); (b) binding of isolectin B4 (IB4) in dorsal root ganglia (DRG) and spinal cord; (c) stimulation-evoked release of substance P (SP) in an in vitro spinal cord preparation and (d) nociceptive responses of partially denervated rats. BRX-220 partially reverses axotomy-induced changes in the sensory system. In vehicle-treated rats there is a decrease in IB4 binding and CGRP expression in injured neurones, while in BRX-220-treated rats these markers were better preserved. Thus, 7.0 +/- 0.6% of injured DRG neurones bound IB4 in vehicle-treated rats compared to 14.4 +/- 0.9% in BRX-220-treated animals. Similarly, 4.5 +/- 0.5% of DRG neurones expressed CGRP in the vehicle-treated group, whereas 9.0 +/- 0.3% were positive in the BRX-220-treated group. BRX-220 also partially restored SP release from spinal cord sections to electrical stimulation of primary sensory neurones. Behavioural tests carried out on partially denervated animals showed that BRX-220 treatment did not prevent the emergence of mechanical or thermal hyperalgesia. However, oral treatment for 4 weeks lead to reduced pain-related behaviour suggesting either slowly developing analgesic actions or enhancement of recovery processes. Thus, the morphological improvement seen in sensory neurone markers was accompanied by restored functional activity. Therefore, treatment with BRX-220 promotes restoration of morphological and functional properties in the sensory system following peripheral nerve injury.

A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis.

SUMMARY Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in the death of motor neurons in the brain and spinal cord. The disorder generally strikes in mid-life, relentlessly leading to paralysis and death, typically 3–5 years after diagnosis. No effective treatments are available. Up to 10% of ALS is familial, usually autosomal dominant. Several causative genes are known and, of these, mutant superoxide dismutase 1 (SOD1) is by far the most frequently found, accounting for up to 20% of familial ALS. A range of human mutant SOD1 transgenic mouse strains has been produced, and these largely successfully model the human disease. Of these, the most widely used is the SOD1 mouse, which expresses a human SOD1 transgene with a causative G93A mutation. This mouse model is excellent for many purposes but carries up to 25 copies of the transgene and produces a great excess of SOD1 protein, which might affect our interpretation of disease processes. A variant of this strain carries a deletion of the transgene array such that the copy number is dropped to eight to ten mutant SOD1 genes. This ‘deleted’ ‘low-copy’ mouse undergoes a slower course of disease, over many months. Here we have carried out a comprehensive analysis of phenotype, including nerve and muscle physiology and histology, to add to our knowledge of this ‘deleted’ strain and give baseline data for future studies. We find differences in phenotype that arise from genetic background and sex, and we quantify the loss of nerve and muscle function over time. The slowly progressive pathology observed in this mouse strain could provide us with a more appropriate model for studying early-stage pathological processes in ALS and aid the development of therapies for early-stage treatments.

Activation of the heat shock response in a primary cellular model of motoneuron neurodegeneration-evidence for neuroprotective and neurotoxic effects

Pharmacological up-regulation of heat shock proteins (hsps) rescues motoneurons from cell death in a mouse model of amyotrophic lateral sclerosis. However, the relationship between increased hsp expression and neuronal survival is not straightforward. Here we examined the effects of two pharmacological agents that induce the heat shock response via activation of HSF-1, on stressed primary motoneurons in culture. Although both arimoclomol and celastrol induced the expression of Hsp70, their effects on primary motoneurons in culture were significantly different. Whereas arimoclomol had survival-promoting effects, rescuing motoneurons from staurosporin and H2O2 induced apoptosis, celastrol not only failed to protect stressed motoneurons from apoptosis under same experimental conditions, but was neurotoxic and induced neuronal death. Immunostaining of celastrol-treated cultures for hsp70 and activated caspase-3 revealed that celastrol treatment activates both the heat shock response and the apoptotic cell death cascade. These results indicate that not all agents that activate the heat shock response will necessarily be neuroprotective.

Excitability properties of mouse motor axons in the mutant SOD1(G93A) model of amyotrophic lateral sclerosis.

Non‐invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1G93A mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild‐type mice were compared before onset of signs and during disease progression (4–19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1G93A axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model. Muscle Nerve, 2010