Elizabeth C. Lopes

Impaired Extracellular Secretion of Mutant Superoxide Dismutase 1 Associates with Neurotoxicity in Familial Amyotrophic Lateral Sclerosis

Mutations in the intracellular metalloenzyme superoxide dismutase 1 (SOD1) are linked to neurotoxicity in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism. Golgi fragmentation and endoplasmic reticulum stress are early hallmarks of spinal motor neuron pathology in transgenic mice overexpressing mutant SOD1, suggesting that dysfunction of the neuronal secretory pathway may contribute to ALS pathogenesis. We therefore proposed that mutant SOD1 directly engages and modulates the secretory pathway based on recent evidence of SOD1 secretion in diverse human cell lines. Here, we demonstrate that a fraction of active endogenous SOD1 is secreted by NSC-34 motor neuron-like cells via a brefeldin-A (BFA)-sensitive pathway. Expression of enhanced green fluorescent protein-tagged mutant human SOD1 (hSOD1-EGFP) in NSC-34 cells induced frequent cytoplasmic inclusions and protein insolubility that correlated with toxicity. In contrast, transfection of non-neuronal COS-7 cells resulted in mutant hSOD1-EGFP cytoplasmic inclusions, oligomerization, and fragmentation without detectable toxicity. Importantly, impaired secretion of hSOD1-EGFP was common to all 10 SOD1 mutants tested relative to wild-type protein in NSC-34 cells. Treatment with BFA inhibited hSOD1-EGFP secretion with pronounced BFA-induced toxicity in mutant cells. Extracellular targeting of mutant hSOD1-EGFP via SOD3 signal peptide fusion attenuated cytoplasmic inclusion formation and toxicity. The effect of elevated extracellular SOD1 was then evaluated in a transgenic rat model of ALS. Chronic intraspinal infusion of exogenous wild-type hSOD1 significantly delayed disease progression and endpoint in transgenic SOD1G93A rats. Collectively, these results suggest novel extracellular roles for SOD1 in ALS and support a causal relationship between mutant SOD1 secretion and intraneuronal toxicity.

A potential role for the p75 low-affinity neurotrophin receptor in spinal motor neuron degeneration in murine and human amyotrophic lateral sclerosis.

INTRODUCTION: The p75 neurotrophin receptor has been recognized as a death-signalling molecule under certain circumstances. Its role in motor neuron degeneration in amyotrophic lateral sclerosis (ALS) was analysed in SOD1-G93A transgenic mice and in spinal cords from human amyotrophic lateral sclerosis. METHOD: The precise loss of motor neurons in SOD1-G93A transgenic mice from birth to adulthood was established using the unbiased fractionator/optical dissector neuronal counting technique. RESULTS: This study showed an early trend in the loss of lumbar motor neurons in SOD1-G93A mice, beginning at birth and progressing to a massive 80% reduction by 4 months of age, when the disease is severe. This study also found that the p75 neurotrophin receptor was expressed in lumbar motor neurons in symptomatic SOD1-G93A mice and in motor neurons in the cervical spinal cords of patients with ALS. CONCLUSIONS: The murine and human ALS data suggest that the p75 neurotrophin receptor may play a death-signalling role in the pathogenesis of motor neuron degeneration. The precise mechanism by which this receptor drives the apoptotic process, both in murine SOD1-G93A motor neuron degeneration and in human amyotrophic lateral sclerosis, remains to be determined.

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

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

Magnetic resonance imaging reveals neuronal degeneration in the brainstem of the superoxide dismutase 1G93A G1H transgenic mouse model of amyotrophic lateral sclerosis

Magnetic resonance imaging (MRI) is becoming the preferred neuroimaging modality for the diagnosis of human amyotrophic lateral sclerosis (ALS). A useful animal model of ALS is the superoxide dismutase 1G93A G1H transgenic mouse, which shows many of the clinico‐pathological features of the human condition. We have employed a 4.7‐Tesla MRI instrument to determine whether a noninvasive imaging approach can reveal pathological changes in the nervous system of this animal model. Our T2‐weighted MRI revealed consistent changes in brain and brainstem of these mice. Hyperintensities, indicative of neuropathology, were observed in several areas including the nucleus ambiguus, facial nucleus, trigeminal motor nucleus, rostroventrolateral reticular nucleus, lateral paragigantocellular nucleus and the substantia nigra. Histology analysis including neuronal counts of the imaged brains confirmed the T2‐weighted MRI findings. Enlarged ventricles and hypointense striations, indicative of global atrophy, were also observed in the brain and cerebellum. This atrophy was confirmed by fresh brain weight data. The extensive global degeneration involving multiple structures suggests a multisystem disease that is similar to human ALS.

Antisense peptide nucleic acid targeting GluR3 delays disease onset and progression in the SOD1 G93A mouse model of familial ALS

Glutamate excitotoxicity is strongly implicated as a major contributing factor in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Excitotoxicity results from elevated intracellular calcium ion (Ca2+) levels, which in turn recruit cell death signaling pathways. Recent evidence suggests that α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor subunit (GluR) stoichiometry is a dominant factor leading to excess Ca2+ loading in neurodegeneration. In particular, the Ca2+ permeable glutamate receptor subunit 3 (GluR3) has been implicated in several neurologic conditions such as bipolar disorder and epilepsy. Recent proteomic analysis within our group on the copper zinc superoxide dismutase (SOD1)G93A transgenic mouse model of familial ALS (FALS) reveals a potentially deleterious upregulation of GluR3 in spinal cord compared to that in wild‐type littermates. Based on this finding we designed a 12mer antisense peptide nucleic acid (PNA) directed against GluR3. This sequence significantly reduced levels of GluR3 protein and protected neuroblastoma × spinal cord (NSC‐34) cells against death induced by the AMPA receptor‐specific agonist (S)‐5‐fluorowillardiine. We subsequently treated SOD1G93A mice thrice weekly with intraperitoneal injections of the antisense PNA (2.5 mg/kg) commencing at postnatal day 50. Mice treated with the antisense sequence had significantly extended survival compared to mice injected with a nonsense sequence. Western blot analysis, however, did not reveal a significant reduction in GluR3 protein levels in whole extracts of the lumbar spinal cord. These results suggest that interference with the GluR3 component of the AMPA receptor assembly may be a novel strategy for controlling excitotoxic destruction of motor neurons and may lead to new therapeutic opportunities for the treatment of human ALS.

Properties of slow- and fast-twitch muscle fibres in a mouse model of amyotrophic lateral sclerosis

This investigation was undertaken to determine if there are altered histological, pathological and contractile properties in presymptomatic or endstage diseased muscle fibres from representative slow-twitch and fast-twitch muscles of SOD1 G93A mice in comparison to wildtype mice. In presymptomatic SOD1 G93A mice, there was no detectable peripheral dysfunction, providing evidence that muscle pathology is secondary to motor neuronal dysfunction. At disease endstage however, single muscle fibre contractile analysis demonstrated that fast-twitch muscle fibres and neuromuscular junctions are preferentially affected by amyotrophic lateral sclerosis-induced denervation, being unable to produce the same levels of force when activated by calcium as muscle fibres from their age-matched controls. The levels of transgenic SOD1 expression, aggregation state and activity were also examined in these muscles but there no was no preference for muscle fibre type. Hence, there is no simple correlation between SOD1 protein expression/activity, and muscle fibre type vulnerability in SOD1 G93A mice.

Neuromuscular accumulation of mutant superoxide dismutase 1 aggregates in a transgenic mouse model of familial amyotrophic lateral sclerosis

Superoxide dismutase 1 (SOD1) aggregates are a histological and biochemical correlate of disease progression in neural tissues from mutant SOD1-linked forms of familial amyotrophic lateral sclerosis (FALS). In the present study, we assayed the monomeric and high molecular weight mutant SOD1 content of nervous, muscle and visceral tissues from transgenic SOD1(G93A) mice using immunoblotting and zymograms. A progressive age-dependent increase in mutant SOD1 level, aggregation and stabilisation by cross-species heterodimers was determined in lumbar spinal cord, sciatic nerve and gastrocnemius muscle. Such biochemical abnormalities were not present in cervical spinal cord, brainstem and diaphragm muscle, nor common to endogenous mouse SOD1. Mutant dismutase activity in general did not increase correspondingly with accumulating protein at later ages. These results suggest that peripheral targets such as hindlimb skeletal muscle and nerve accumulate mutant SOD1 aggregates and may therefore be susceptible to mutant SOD1-mediated toxicity, in addition to lower and upper motor neurons of the central nervous system in transgenic FALS mice.

Effect of p75 neurotrophin receptor antagonist on disease progression in transgenic amyotrophic lateral sclerosis mice

Neurotrophin level imbalances and altered p75 neurotrophin receptor (p75NTR) expression are implicated in spinal motor neuron degeneration in human and mouse models of amyotrophic lateral sclerosis (ALS). Recently, elevated reactive astrocyte‐derived nerve growth factor (NGF) was linked to p75NTR‐expressing motor neuron death in adult transgenic ALS mice. To test the role of NGF‐dependent p75NTR‐mediated signalling in ALS, we examined the effects of a cyclic decapeptide antagonist of p75NTR ligand binding by using neurotrophin‐stimulated cell death assays and transgenic ALS mice. Murine motor neuron‐like (NSC‐34) cell cultures expressed full‐length and truncated p75NTR, tyrosine receptor kinase B (TrkB), and the novel neurotrophin receptor homolog‐2 (NHR2) but were TrkA deficient. Accordingly, treatment of cells with NGF induced dose‐dependent cell death, which was significantly blocked by the cyclic decapeptide p75NTR antagonist. Application of brain‐derived neurotrophic factor, neurotrophin‐3, or neurotrophin‐4 to cultures increased cell proliferation, and such trophic effects were abolished by pretreatment with the tyrosine kinase inhibitor K‐252a. Systemic administration of a modified cyclic decapeptide p75NTR antagonist conjugated to the TAT4 cell permeabilization sequence to presymptomatic transgenic SOD1G93A mice affected neither disease onset nor disease progression, as determined by hindlimb locomotor, grip strength, and survival analyses. These studies suggest that disrupting NGF‐p75NTR interactions by using this approach is insufficient to alter the disease course in transgenic ALS mice. Thus, alternate ligand‐independent pathways of p75NTR activation or additional NGF receptor targets may contribute to motor neuron degeneration in ALS mice.

Behavioural and anatomical effects of systemically administered leukemia inhibitory factor in the SOD1G93A G1H mouse model of familial amyotrophic lateral sclerosis

We investigated the anatomical and behavioural effects of daily intraperitoneal injection of 25 microg/kg of LIF in the SOD1(G93A G1H) mouse model of familial ALS. We found some subtle beneficial behavioural changes in LIF treated mice. These included later onset of clinical disease in females as determined by clinical scoring; better grip strength in males; and delayed development of motor impairment in males as determined by the rotarod test. However, we found no significant rescue of motoneurons or prolongation of survival as a result of this systemic dose of LIF in these mice.

Loss of synaptophysin‐positive boutons on lumbar motor neurons innervating the medial gastrocnemius muscle of the SOD1G93A G1H transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a common form of motor neuron disease (MND) that involves both upper and lower nervous systems. In the SOD1G93A G1H transgenic mouse, a widely used animal model of human ALS, a significant pathology is linked to the degeneration of lower motor neurons in the lumbar spinal cord and brainstem. In the current study, the number of presynaptic boutons immunoreactive for synaptophysin was estimated on retrogradely labeled soma and proximal dendrites of α and γ motor neurons innervating the medial gastrocnemius muscle. No changes were detected on both soma and proximal dendrites at postnatal day 60 (P60) of α and γ motor neurons. By P90 and P120, however, α motor neuron soma had a reduction of 14 and 33% and a dendritic reduction of 19 and 36%, respectively. By P90 and P120, γ motor neuron soma had a reduction of 17 and 41% and a dendritic reduction of 19 and 35%, respectively. This study shows that levels of afferent innervation significantly decreased on surviving α and γ motor neurons that innervate the medial gastrocnemius muscle. This finding suggests that the loss of motor neurons and the decrease of synaptophysin in the remaining motor neurons could lead to functional motor deficits, which may contribute significantly to the progression of ALS/MND.