A dopamine receptor antagonist L-745,870 suppresses microglia activation in spinal cord and mitigates the progression in ALS model mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1^H46R); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1^H46R transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.     

Macrophage colony stimulating factor (M-CSF) exacerbates ALS disease in a mouse model through altered responses of microglia expressing mutant superoxide dismutase

Macrophage colony stimulating factor (M-CSF) is a cytokine that regulates the survival, proliferation and maturation of microglial cells. Administration of M-CSF can promote neuronal survival in various models of central nervous system (CNS) injury. Here, in an attempt to induce a neuroprotective microglial cell phenotype and enhance motor neuron survival, mutant SOD1^G37R transgenic mice were treated, weekly, with M-CSF starting at onset of disease. Unexpectedly, M-CSF accelerated disease progression in SOD1^G37R mouse model of ALS. The shortened survival of M-CSF-treated animals was associated with diminished muscle innervation and enhanced adoption of a macrophage-like phenotype by microglial cells characterised by the upregulation of pro-inflammatory cytokines TNF-α and IL-1β and of the phagocytic marker CD68.     

ROCK inhibition improves axonal regeneration in a preclinical model of amyotrophic lateral sclerosis

Alteration of the RhoA/ROCK (Rho kinase) pathway has been shown to be neuroprotective in SOD1^G93A mice, the most commonly used animal model of ALS. Since previous studies indicate that, apart from neuroprotection, ROCK inhibitor Y-27632 can also accelerate regeneration of motor axons, we here assessed the regenerative capability of axons in SOD1^G93A mice with and without treatment with Y-27632. Regeneration of axons was examined after sciatic nerve crush in pre- and symptomatic SOD1^G93A mice. Proregenerative effects of Y-27632 were studied during the disease course in the SOD1^G93A mouse model. In symptomatic SOD1^G93A mice, axonal regeneration was markedly reduced compared to presymptomatic SOD1^G93A mice and wild types. Treatment with Y-27632 improved functional and morphological measures of motor axons after sciatic crush in all tested conditions. Y-27632 treatment did not increase the lifespan of symptomatic SOD1^G93A mice, but did improve axonal (re)innervation of neuromuscular junctions. Our study provides proof of concept that axonal regeneration of motor neurons harboring SOD1^G93A is impaired, but amenable for pharmacological interventions aiming to accelerate axonal regeneration. Given the lack of treatments for ALS, approaches to improve axonal regeneration, including by inhibiting ROCK, should be further explored.     

Inhibition of Rho‐kinase by Fasudil protects dopamine neurons and attenuates inflammatory response in an intranasal lipopolysaccharide‐mediated Parkinson's model

Microglia activation and inflammatory factors in brain microenvironment are associated with degeneration of neurons in the substantia nigra (SN) of Parkinson's disease (PD) patients and various PD models. There is increasing evidence that the Rho/ROCK (Rho kinase) signalling pathway may play a critical role in the inflammatory response, and ROCK inhibitor has been reported to have neuroprotective effects. In this study, we examined the neuroprotective potential and possible mechanism of ROCK inhibitor Fasudil in an intranasal lipopolysaccharide (LPS)‐induced PD model. ROCK was activated with LPS stimulation and inhibited by Fasudil treatment in this PD model. Behavioural tests demonstrated a clear improvement in motor performance after Fasudil treatment. Furthermore, Fasudil resulted in a significant attenuation of dopamine cell loss, α‐synuclein accumulation and inflammatory response with the reversion of inflammatory M1 to anti‐inflammatory M2 microglia, decreased NF‐кB activation, and IL‐12 and TNF‐α generation in the SN and olfactory bulb in this model. This study establishes a role for Fasudil in protecting against LPS‐mediated dopamine degeneration and provides a therapeutic strategy for the treatment of PD.     

The mitochondrial permeability transition pore in motor neurons: Involvement in the pathobiology of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause–effect relationships between mSOD1 and mitochondriopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.     

Protective and Toxic Neuroinflammation in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disorder characterized by loss of motor neurons, resulting in paralysis and death. Multiple mechanisms of motor neuron injury have been implicated based upon the more than 20 different genetic causes of familial ALS. These inherited mutations compromise diverse motor neuron pathways leading to cell-autonomous injury. In the ALS transgenic mouse models, however, motor neurons do not die alone. Cell death is noncell-autonomous dependent upon a well orchestrated dialogue between motor neurons and surrounding glia and adaptive immune cells. The pathogenesis of ALS consists of 2 stages: an early neuroprotective stage and a later neurotoxic stage. During early phases of disease progression, the immune system is protective with glia and T cells, especially M2 macrophages/microglia, and T helper 2 cells and regulatory T cells, providing anti-inflammatory factors that sustain motor neuron viability. As the disease progresses and motor neuron injury accelerates, a second rapidly progressing phase develops, characterized by M1 macrophages/microglia, and proinflammatory T cells. In rapidly progressing ALS patients, as in transgenic mice, neuroprotective regulatory T cells are significantly decreased and neurotoxicity predominates. Our own therapeutic efforts are focused on modulating these neuroinflammatory pathways. This review will focus on the cellular players involved in neuroinflammation in ALS and current therapeutic strategies to enhance neuroprotection and suppress neurotoxicity with the goal of arresting the progressive and devastating nature of ALS.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0329-3) contains supplementary material, which is available to authorized users.Key Words: ALS, amyotrophic lateral sclerosis, neuroinflammation, inflammation, neurodegenerative disease     

Characterization of thoracic motor and sensory neurons and spinal nerve roots in canine degenerative myelopathy,a potential disease model of amyotrophic lateral sclerosis

Canine degenerative myelopathy (DM) is a progressive, adult‐onset, multisystem degenerative disease with many features in common with amyotrophic lateral sclerosis (ALS). As with some forms of ALS, DM is associated with mutations in superoxide dismutase 1 (SOD1). Clinical signs include general proprioceptive ataxia and spastic upper motor neuron paresis in pelvic limbs, which progress to flaccid tetraplegia and dysphagia. The purpose of this study was to characterize DM as a potential disease model for ALS. We previously reported that intercostal muscle atrophy develops in dogs with advanced‐stage DM. To determine whether other components of the thoracic motor unit (MU) also demonstrated morphological changes consistent with dysfunction, histopathologic and morphometric analyses were conducted on thoracic spinal motor neurons (MNs) and dorsal root ganglia (DRG) and in motor and sensory nerve root axons from DM‐affected boxers and Pembroke Welsh corgis (PWCs). No alterations in MNs or motor root axons were observed in either breed. However, advanced‐stage PWCs exhibited significant losses of sensory root axons, and numerous DRG sensory neurons displayed evidence of degeneration. These results indicate that intercostal muscle atrophy in DM is not preceded by physical loss of the motor neurons innervating these muscles, nor of their axons. Axonal loss in thoracic sensory roots and sensory neuron death suggest that sensory involvement may play an important role in DM disease progression. Further analysis of the mechanisms responsible for these morphological findings would aid in the development of therapeutic intervention for DM and some forms of ALS. © 2013 Wiley Periodicals, Inc.     

Fasudil,a Rho kinase inhibitor,drives mobilization of adult neural stem cells after hypoxia/reoxygenation injury in mice

Rho kinase (ROCK) is important in fundamental processes of cell proliferation and survival. Blockade of ROCK promotes stem cell survival in vitro and axonal regeneration in vivo, exhibiting therapeutic potential such as spinal cord injuries and stroke. Here, we used the model of hypoxia/reoxygenation (H/R) injury to explore the possibility whether Fasudil, a ROCK inhibitor in clinical application for subarachnoid hemorrhage and stroke, mobilizes adult neural stem cells in vivo. Most interestingly, Fasudil triggers neurogenesis especially in the subventricular zone after H/R. The increase of Brdu+ cholinergic neurons was observed in striatum and forebrain cortex of Fasudil-treated mice after 30 days. Further observation demonstrates that both levels of granulocyte colony-stimulating factor (G-CSF) and astrocytes expressing G-CSF were elevated in mice treated with Fasudil, as compared to mice injected with saline. In vitro H/R model of cultured astrocytes, Fasudil promoted astrocytes to produce G-CSF in a dose-dependent manner. In addition, antibody neutralization and receptor blocking of the G-CSF pathway clearly demonstrate that Fasudil-induced neurogenesis was mediated partially through astrocyte-derived G-CSF. Our results indicate that Fasudil might represent a promising therapeutic perspective by mobilizating endogenous adult neural stem cells in the CNS.     

Profilin 1 knockdown prevents ischemic brain damage by promoting M2 microglial polarization associated with the RhoA/ROCK pathway

Microglial polarization to the anti-inflammatory M2 phenotype is essential in resolving neuroinflammation, making it a promising therapeutic strategy for stroke intervention. The actin cytoskeleton is known to be important for the physiological functions of microglia, including migration and phagocytosis. Profilin 1 (PFN1), an actin-binding protein, is involved in the dynamic transformation and reorganization of actin. However, the role of PFN1 in microglial polarization and ischemia/reperfusion injury is unclear. The role of PFN1 on microglial polarization was examined in vitro in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGDR) and in vivo in male mice after transient middle cerebral artery occlusion (MCAO). Knockdown of PFN1 inhibited M1 microglial polarization and promoted M2 microglia polarization 48 hr after OGDR stimulation in BV2 cells and 7 days after MCAO-induced injury in male mice. RhoA/ROCK pathway was involved in the regulation of PFN1 during microglial polarization. Knockdown of PFN1 also significantly attenuated brain infarcts and edema, improved cerebral blood flow and neurological deficits in MCAO-injured mice. Inhibition of PFN1 effectively protected the brain against ischemia/reperfusion injuries by promoting M2 microglial polarization in vitro and in vivo.     

Motor neuron–immune interactions: the vicious circle of ALS

Because microglial cells, the resident macrophages of the CNS, react to any lesion of the nervous system, they have for long been regarded as potential players in the pathogenesis of several neurodegenerative disorders including amyotrophic lateral sclerosis, the most common motor neuron disease in the adult. In recent years, this microglial reaction to motor neuron injury, in particular, and the innate immune response, in general, has been implicated in the progression of the disease, in mouse models of ALS. The mechanisms by which microglial cells influence motor neuron death in ALS are still largely unknown. Microglial activation increases over the course of the disease and is associated with an alteration in the production of toxic factors and also neurotrophic factors. Adding to the microglial/macrophage response to motor neuron degeneration, the adaptive immune system can likewise influence the disease process. Exploring these motor neuron–immune interactions could lead to a better understanding in the physiopathology of ALS to find new pathways to slow down motor neuron degeneration.     

Epothilone D accelerates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Aims

Degeneration of the distal neuromuscular circuitry is a hallmark pathology of Amyotrophic Lateral Sclerosis (ALS). The potential for microtubule dysfunction to be a critical pathophysiological mechanism in the destruction of this circuitry is increasingly being appreciated. Stabilization of microtubules to improve neuronal integrity and pathology has been shown to be a particularly favourable approach in other neurodegenerative diseases. We present evidence here that treatment with the microtubule‐targeting compound Epothilone D (EpoD) both positively and negatively affects the spinal neuromuscular circuitry in the SOD1^G93A mouse model of ALS.

Methods

SOD1^G93A mice were treated every 5 days with 2 mg/kg EpoD. Evaluation of motor behaviour, neurological phenotype and survival was completed, with age‐dependent histological characterization also conducted, using the thy1‐YFP mouse. Motor neuron degeneration, axonal integrity, neuromuscular junction (NMJ) health and gliosis were also assessed.

Results

EpoD treatment prevented loss of the spinal motor neuron soma, and distal axon degeneration, early in the disease course. This, however, was not associated with protection of the NMJ synapse and did not improve motor phenotype or clinical progression. EpoD administration was also found to be neurotoxic at later disease stages. This was evidenced by accelerated motor neuron cell body loss, increasing gliosis, and was associated with detrimental outcomes to motor behaviour, clinical assessment and survival.

Conclusions

The results suggest that EpoD accelerates disease progression in the SOD1^G93A mouse model of ALS, and highlights that the pathophysiological involvement of microtubules in ALS is an evolving and underappreciated phenomenon.     

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1^G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1^G93A mice microglia do not express NLRP3, and SOD1^G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1^G93A mice. We show that both aggregated and soluble SOD1^G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1^G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43^Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1^G93A-mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.     

Combination of early constraint-induced movement therapy and fasudil enhances motor recovery after ischemic stroke in rats

Purpose: Constraint-induced movement therapy (CIMT) is a promising technique for the recovery of upper extremity movement in chronic stroke patients. However, the effectiveness of its use in acute ischemia has not been confirmed. Myelin-associated inhibitors, which have upregulated functions in tissues affected by acute focal infarction, limit axonal regeneration via activation of the Rho–Rho-associated protein kinase (ROCK) pathway. The present study examined whether early CIMT combined with the ROCK inhibitor fasudil promotes motor recovery after acute ischemic stroke. Materials and Methods: Rats were trained to perform the skilled-reach test and then subjected to middle cerebral artery occlusion (MCAO), producing a stroke affecting the preferred forelimb. Rats were assigned to one of four groups (N = 6/group): (nontreated) Control, CIMT, Fasudil, or CIMT+fasudil. CIMT and/or intraperitoneal infusion of fasudil were initiated 1 day postMCAO. Skilled reach and foot fault test data were collected once before and repeatedly over 4 weeks after the operation. Infarct volumes were calculated. Results: All four groups showed similar forelimb impairment before treatment. The performance of CIMT alone group was similar to that of controls on both tests. Fasudil alone facilitated recovery in the foot-fault test, but not in the skilled-reach test. Rats in the CIMT+fasudil group demonstrated enhanced recovery in both tests, including better performance over time than the Fasudil group on the foot-fault test. Infarct size did not differ significantly between the groups. Conclusions: Early CIMT promotes motor recovery after acute ischemic stroke when it is administered with fasudil pharmacotherapy, but not without it.     

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1^G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1^G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1^G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00953-z.Key Words: ALS, NF-κB-p65, microglia, BANA.     

Excitability properties of mouse motor axons in the mutant SOD1G93A 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 SOD1^G93A 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 SOD1^G93A 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     

Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS

Transgenic mice that highly over-express a mutated human CuZn superoxide dismutase (SOD1) gene [gly⁹³→ala; TgN(SOD1-G93A)G1H line] found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease that is characterized by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of motor activity. The mutant Cu,Zn SOD exhibits essentially normal SOD activity but also generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. Consequently, lipid and protein oxidative damage to the spinal motor neurons occurs and is associated with disease onset and progression. In the present study, we investigated the time course of microglial (major histocompatibility-II antigen immunoreactivity) and astrocytic (glial fibrillary acidic protein immunoreactivity) activation in relation to the course of motor neuron disease in the TgN(SOD1-G93A)G1H FALS mice. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but pre-disease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to non-transgenic littermates, the TgN(SOD1-G93A)G1H mice showed significantly increased numbers of activated astrocytes (P < 0.01) at 100 days of age in both the cervical and lumbar spinal cord regions. However, at 120 days of age, the activation lost statistical significance. In contrast, microglial activation was significantly increased several-fold at both 100 and 120 days. We hypothesize that astrocytic activation may exert a trophic influence on the motor neurons that is insufficiently maintained late in the course of the disease. On the other hand, the sustained, intense microglial activation may conceivably contribute to the oxidative stress and damage involved in the disease process. If true, then agents which inhibit microglia may help to limit disease progression. GLIA 23:249–256, 1998. © 1998 Wiley-Liss, Inc.     

Overexpression of human wild-type FUS causes progressive motor neuron degeneration in an age- and dose-dependent fashion

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are relentlessly progressive neurodegenerative disorders with overlapping clinical, genetic and pathological features. Cytoplasmic inclusions of fused in sarcoma (FUS) are the hallmark of several forms of FTLD and ALS patients with mutations in the FUS gene. FUS is a multifunctional, predominantly nuclear, DNA and RNA binding protein. Here, we report that transgenic mice overexpressing wild-type human FUS develop an aggressive phenotype with an early onset tremor followed by progressive hind limb paralysis and death by 12 weeks in homozygous animals. Large motor neurons were lost from the spinal cord accompanied by neurophysiological evidence of denervation and focal muscle atrophy. Surviving motor neurons in the spinal cord had greatly increased cytoplasmic expression of FUS, with globular and skein-like FUS-positive and ubiquitin-negative inclusions associated with astroglial and microglial reactivity. Cytoplasmic FUS inclusions were also detected in the brain of transgenic mice without apparent neuronal loss and little astroglial or microglial activation. Hemizygous FUS overexpressing mice showed no evidence of a motor phenotype or pathology. These findings recapitulate several pathological features seen in human ALS and FTLD patients, and suggest that overexpression of wild-type FUS in vulnerable neurons may be one of the root causes of disease. Furthermore, these mice will provide a new model to study disease mechanism, and test therapies.     

Synaptic loss in the proximal axon of anterior horn neurons in motor neuron disease

This report deals with an ultrastructural investigation of the synapses of the proximal axons of normal-appearing anterior horn neurons of 7 patients with amyotrophic lateral sclerosis (ALS) and 4 patients with motor neuron disease who had no upper motor neuron and corticospinal tract involvement (lower motor neuron disease, LMND). Specimens from 12 age-matched individuals who died of non-neurological diseases served as controls. Proximal axons directly emanating from the normal-appearing neurons were examined: 42 axons were from ALS patients, 43 from LMND patients and 87 from controls. Our results show that the number of synapses on axon hillocks, as well as the lengths of the synaptic contact and of the active zone were reduced in both groups of patients (P<0.0001), but no significant differences were seen between patients and controls with respect to the synaptic parameters of initial axon segments. There was no overall difference between ALS and LMND patients. These findings suggest that the electrophysiological functions pertaining to integration of electrical inputs into the axon and information transduction on the axon may be greatly impaired in the early stages of motor neuron diseases, and that the observed synaptic alterations may be pathological events, likely to be due to anterior horn neuron degeneration.     

Motor unit function during evolution of proximal axonal swellings.

beta,beta'-Iminodipropionitrile (IDPN) impairs axonal transport of neurofilaments; their accumulation leads to the formation of proximal swellings in motor axons. Similar proximal swellings are a feature of some cases of motor neuron disease such as amyotrophic lateral sclerosis (ALS). Motor units in IDPN-treated animals were assessed to determine their relative susceptibilities to impaired function and whether the functional changes resulting from proximal axonal swellings share certain electromyographic features with ALS. Intrinsic properties of medial gastrocnemius motoneurones (MN) and contractile responses of their motor units were examined during the evolution of proximal axonal swellings in cats administered IDPN (50 mg/kg once weekly) for 7, 14 or 35 days. While conduction velocities were significantly decreased in all motor unit types by 35 days, the conduction slowing was greater in fast fatigable (types FF and FI) motor units than in fatigue resistant (types FR and S) motor units. Normal correlations between axonal conduction velocity and MN input resistance (Rin) and the inverse relationship between Rin and rheobase were lost with progression of the neuropathy. Twitch and maximum tetanic tension developed by fast-fatigable motor units declined early in the neuropathy, whereas fatigue-resistant units did not show similar changes until later stages of the intoxication. In some motor units, irregular and abnormal tetanic tensions were elicited by repetitive MN discharge. At 14 and 35 days, a novel, intermediate motor unit response classified as slow and fatigable (SF) was observed. Conduction block, characterized by repetitive MN firing without a corresponding contractile response, was observed in some type FF and S units by 35 days. Morphometric analysis of muscle fiber types showed significant atrophy, particularly in the type I fibers at 14-35 days; the atrophy reversed following cessation of IDPN administration. The influence of proximal axonal swellings on motor unit function in IDPN neuropathy is discussed in terms of reported electrophysiological alterations in motoneurone disease.     

Minocycline in amyotrophic lateral sclerosis: a pilot study

Abstract Recent studies indicate that minocycline exerts neuroprotective effects in vitro and in vivo, and suggest that the drug may represent a novel therapeutic approach to amyotrophic lateral sclerosis (ALS). In this study we investigated the safety of combined treatment with minocycline and riluzole in ALS. Twenty ALS patients were randomised into two groups and administered either riluzole (50 mg b.i.d.) or riluzole and minocycline (100 mg i.d.) for 6 months. Disease progression was measured by means of the ALS-Functional Rating Scale score at monthly intervals. Respiratory function was measured at the beginning of the study and repeated after 3 and 6 months of treatment. Combined treatment with minocycline and riluzole was not followed by significant side effects. This pilot study shows that minocycline and riluzole can be taken safely together. Further trials are needed to assess efficacy of such treatment.