Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.

Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.

Monocyte locomotion inhibitory factor produced by E. histolytica improves motor recovery and develops neuroprotection after traumatic injury to the spinal cord.

Monocyte locomotion inhibitory factor (MLIF) is a pentapeptide produced by Entamoeba histolytica that has a potent anti-inflammatory effect. Either MLIF or phosphate buffered saline (PBS) was administered directly onto the spinal cord (SC) immediately after injury. Motor recovery was evaluated. We also analyzed neuroprotection by quantifying the number of surviving ventral horn motor neurons and the persistence of rubrospinal tract neurons. To evaluate the mechanism through which MLIF improved the outcome of SC injury, we quantified the expression of inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10), and transforming growth factor- ß (TGF- ß ) genes at the site of injury. Finally, the levels of nitric oxide and of lipid peroxidation were also determined in peripheral blood. Results showed that MLIF improved the rate of motor recovery and this correlated with an increased survival of ventral horn and rubrospinal neurons. These beneficial effects were in turn associated with a reduction in iNOS gene products and a significant upregulation of IL-10 and TGF- ß expression. In the same way, MLIF reduced the concentration of nitric oxide and the levels of lipid peroxidation in systemic circulation. The present results demonstrate for the first time the neuroprotective effects endowed by MLIF after SC injury.

Fasciculation potentials and earliest changes in motor unit physiology in ALS.

BACKGROUND: There is little information on the earliest changes in motor unit (MU) physiology in amyotrophic lateral sclerosis (ALS) and the development of the classical neurophysiological features of ALS over time. OBJECTIVE: We studied the earliest abnormalities in MU physiology in ALS and changes over time. DESIGN: Observational, cross-sectional and longitudinal study. POPULATION AND METHODS: We studied the tibialis anterior (TA) muscle in three groups of subjects; 73 patients with ALS, 10 with benign fasciculation and 37 healthy control subjects. In the ALS group, 61 had normal strength in the TA muscle and 12 had TA muscle strength of 4 on the medical research council scale. In all subjects we evaluated the presence of fasciculation potentials (FPs) and fibrillation/sharp-waves (fibs-sw), and quantified MU potentials (MUPs) and jitter. Twenty-six ALS patients with TA muscle of normal strength were investigated in serial studies. RESULTS: FPs were recorded in TA muscles (medical research council 5) of 21 ALS patients with normal MUPs. Longitudinal studies confirmed that the patients presenting with FPs as the only abnormality progressed to MUP instability before large MUPs associated with fibs-sw were detected. FPs from ALS patients with no other neurophysiological change were simpler than in patients in whom there were also fibs-sw and neurogenic MUPs. The complexity of FPs in patients with weak TA muscle was greater than in the latter group. FPs in patients with benign fasciculations were simpler than FPs in ALS patients with normal TA muscle strength. CONCLUSIONS: FPs are a very early marker of ALS and anticipate MUP instability or reinnervation, consistent with a very early phase of increased axonal excitability. Later, widespread neuronal dysfunction causes widespread fibs-sw and loss of MUPs with compensatory reinnervation. Our results confirm the importance of FP morphology analysis in the differential diagnosis of ALS and other disorders, and indicate that benign FPs represent a different phenomenon.

Neural progenitor cells generate motoneuron-like cells to form functional connections with target muscles after transplantation into the musculocutaneous nerve.

Neural progenitor cells (NPCs) are suggested to be a valuable source of cell transplant in treatment of various neurological diseases because of their distinct attributes. They can be expanded and induced to differentiate in vitro. However, it remains uncertain whether in vitro expanded NPCs have the capacity to give rise to functional motoneurons after transplantation in vivo. Here, we showed that in vitro expanded NPCs, when transplanted into the musculocutaneous nerve, generated motoneuron-like cells that exhibited typical morphology with large cell bodies, expressed specific molecules, and extended axons to form functional connections with the target muscle. In contrast, transplanted NPCs failed to yield motoneurons in the injured ventral horn of the spinal cord. The results of the study demonstrate that NPCs have the potential to generate functional motoneurons in an appropriate environment. The distinct differentiating fate of NPCs in the musculocutaneous nerve and the injured ventral horn suggests the importance and necessity of modifying the host microenvironment in use of NPCs for cell replacement therapies for motoneuron diseases.

Mercury in the spinal cord after inhalation of mercury.

Amyotrophic lateral sclerosis (ALS) affects anterior horn cells of the spinal cord causing an indolent slow and steady deterioration of muscle strength leading inevitably to death in respiratory failure. ALS is a model condition for neurodegenerative disorders. Exposure to different agents dispersed in the environment has been suggested to cause neurodegeneration but no convincing evidence for such a link has yet been presented. Respiratory exposure to metallic mercury (Hg(0)) from different sources may be suspected. Body distribution of metallic mercury is fast and depends on solubility properties. Routes of transport, metabolism, excretion and biological half-life determine the overall toxic effects. Inhalation experiments were performed in 1984 where small marmoset monkeys (Callithrix jacchus) were exposed to (203) Hg(0 vapour) mixed into the breathing air (4-5 µg/l). After 1 hr of exposure, they were killed and whole body autoradiograms prepared to study the distribution of mercury within organs. Autoradiograms showed that Hg was deposited inside the spinal cord. Areas of enhanced accumulation anatomically corresponding to motor nuclei could be observed. This study describes a reinvestigation, with new emphasis on the spinal cord, of these classical metal exposure data in a primate, focusing on their relevance for the causation of neurodegenerative disorders. A comparison with more recent rodent experiments with similar findings is included. The hypothesis that long-time low-dose respiratory exposure to metals, for example, Hg, contributes to neurodegenerative disorders is forwarded and discussed.

Electrophysiological analysis of vulnerability to experimental ischemia in neonatal rat spinal ventral horn neurons.

To clarify the vulnerability of spinal motoneurons to excitotoxicity, we analyzed the agonal current induced by experimental ischemia in ventral lamina IX neurons of spinal cord slices from neonatal rats by using whole-cell patch-clamp. Ischemia was simulated in vitro by oxygen/glucose deprivation. Superfusion with ischemia-simulating medium elicited an agonal inward current, which was initially slow and then became rapid. We compared 8-, 9-, 10-, 11-, and 12-day postnatal rats and found age-dependent shortening of the latency of the rapid inward current. Furthermore, the membrane capacitance (Cm) and resting membrane potential (RMP) of the lamina IX neurons demonstrated significant negative correlations with the latency of the rapid inward current. Logistic regression analysis showed that postnatal age, Cm, and RMP were independent contributing factors to ischemic vulnerability. These results suggest that not only cell volume and ionic balance but also early postnatal maturation of the intracellular environment is vital for developing vulnerability to excitotoxicity.

Clinical features and molecular mechanisms of spinal and bulbar muscular atrophy (SBMA).

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset neurodegenerative disease characterized by slowly progressive muscle weakness and atrophy. The cause of this disease is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine tract, within the first exon of the androgen receptor (AR) gene. SBMA exclusively occurs in adult males, whereas both heterozygous and homozygous females are usually asymptomatic. Lower motor neurons in the anterior horn of the spinal cord and those in the brainstem motor nuclei are predominantly affected in SBMA, and other neuronal and nonneuronal tissues are also widely involved to some extent. Testosterone-dependent nuclear accumulation of the pathogenic AR protein has been considered to be a fundamental step of neurodegenerative process, which is followed by several molecular events such as transcriptional dysregulation, axonal transport disruption and mitochondrial dysfunction. Results of animal studies suggest that androgen deprivation and activation of protein quality control systems are potential therapies for SBMA.

The effects of systemic hypothermia on a murine model of thoracic aortic ischemia reperfusion.

INTRODUCTION: Hypothermia is widely used to mediate ischemia-reperfusion injury associated with repair of the thoracoabdominal aorta. Experiments were designed in a murine model of thoracic aortic ischemia-reperfusion (TAR) to evaluate the effect of moderate systemic hypothermia on neurologic function, spinal cord morphology, and indices of inflammation in critical organs. METHODS: C57BL/6 mice were subjected to TAR under hypothermic (34 degrees C) or normothermic (38 degrees C) conditions, followed by 24 or 48 hours of normothermic reperfusion. Neurologic functions were assessed during reperfusion. Spinal cords were examined at 24 and 48 hours after reperfusion, and the degree of injury qualified by counting the number of viable motor neurons within the anterior horns. Keratinocyte chemokine, interleukin-6, and myeloperoxidase levels were measured from lung, liver, and kidney at 24 and 48 hours. RESULTS: Normothermic TAR resulted in a dense neurologic deficit in all mice throughout the reperfusion period. Mice subjected to TAR under hypothermic conditions had transient, mild neurologic deficit during the initial periods of reperfusion. Between 24 and 48 hours, delayed paralysis developed in half of these mice, whereas the other half remained neurologically intact. Spinal cord histology showed a graded degree of injury that correlated with neurologic function. There was no correlation between markers of inflammation in various organs and neurologic outcomes following TAR. CONCLUSION: Systemic moderate hypothermia was protective against immediate paralysis after TAR in all cases and was associated with delayed paralysis in 50% of mice. This study suggests that delayed-onset paralysis may be the result of a local insult, rather than a systemic inflammatory event, precipitating spinal cord injury.

Region-specific sensitivity of the spinal cord to ischemia/reperfusion: the dynamic of changes in catalytic NOS activity.

This study was designed in order to consider whether the release of neuronally derived nitric oxide (NO) in the lumbosacral spinal cord during ischemia/reperfusion is region-specific and whether changes in Ca(2+)-dependent NO synthase (cNOS) activity paralell with functional outcome. The cNOS activity was measured in the spinal cord regions after 13-, 15- and 17-min ischemia alone and that followed by 24 h of reperfusion. In addition, the Tarlov's criteria were applied to define the neurological consequences of ischemia/reperfusion in experimental animals. Based on the results, it is evident that only the 17-min ischemia alone was quite sufficient to cause changes in cNOS activity, however, without alterations in functional outcomes. On the other hand, the ischemic episodes followed by reperfusion caused dynamic, region-specific alterations in cNOS activity and consequently led to deterioration of motor function of hindlimbs in affected animals. Our results indicate that the motoneurons in the ventral horns respond more sensitively to ischemia/reperfusion than do neurons localized in the other spinal cord regions and that changes in cNOS activity may also influence the axonal conductance in the white matter and account for the impairment of motoneuronal activity in affected animals.

Light-induced rescue of breathing after spinal cord injury.

Paralysis is a major consequence of spinal cord injury (SCI). After cervical SCI, respiratory deficits can result through interruption of descending presynaptic inputs to respiratory motor neurons in the spinal cord. Expression of channelrhodopsin-2 (ChR2) and photostimulation in neurons affects neuronal excitability and produces action potentials without any kind of presynaptic inputs. We hypothesized that after transducing spinal neurons in and around the phrenic motor pool to express ChR2, photostimulation would restore respiratory motor function in cervical SCI adult animals. Here we show that light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory diaphragmatic motor activity. Furthermore, robust rhythmic activity persisted long after photostimulation had ceased. This recovery was accomplished through a form of respiratory plasticity and spinal adaptation which is NMDA receptor dependent. These data suggest a novel, minimally invasive therapeutic avenue to exercise denervated circuitry and/or restore motor function after SCI.

Biomechanical study of cervical flexion myelopathy using a three-dimensional finite element method.

OBJECT: The goal of this study was to perform a biomechanical study of cervical flexion myelopathy (CFM) using a finite element method. METHODS: A 3D finite element model of the spinal cord was established consisting of gray matter, white matter, and pia mater. After the application of semi-static compression, the model underwent anterior flexion to simulate CFM. The flexion angles used were 5 degrees and 10 degrees , and stress distributions inside the spinal cord were then evaluated. RESULTS: Stresses on the spinal cord were very low under semi-static compression but increased after 5 degrees of flexion was applied. Stresses were concentrated in the gray matter, especially the anterior and posterior horns. The stresses became much higher after application of 10 degrees of flexion and were observed in the gray matter, posterior funiculus, and a portion of the lateral funiculus. CONCLUSIONS: The 5 degrees model was considered to represent the mild type of CFM. This type corresponds to the cases described in the original report by Hirayama and colleagues. The main symptom of this type of CFM is muscle atrophy and weakness caused by the lesion of the anterior horn. The 10 degrees model was considered to represent a severe type of CFM and was associated with lesions in the posterior fand lateral funiculi. This type of CFM corresponds to the more recently reported clinical cases with combined long tract signs and sensory disturbance.

Hypothermia suppresses excitatory synaptic transmission and neuronal death induced by experimental ischemia in spinal ventral horn neurons.

STUDY DESIGN: Whole-cell patch-clamp recordings were performed from the ventral horn neurons obtained from the rat spinal cord slices. OBJECTIVE: This study investigated the effects of hypothermia on excitatory synaptic transmission and ischemia-induced neuronal death. SUMMARY OF BACKGROUND DATA: Hypothermia has long been recognized as a promising physical strategy against both ischemic and traumatic spinal cord injuries. However, the mechanism of hypothermia-mediated neuroprotective action in the spinal cord is still not fully understood at the single cell level. METHODS: Whole-cell patch-clamp recordings were performed from ventral horn neurons obtained from the spinal cord slices. Ischemia was simulated by superfusing an oxygen- and glucose-deprived medium [ischemia simulating medium (ISM)]. RESULTS: When the temperature of the superfusing artificial cerebrospinal fluid solution was changed from normothermia (36 degrees C) to hypothermia (32 degrees C, 28 degrees C, and 24 degrees C), the frequency of spontaneous excitatory postsynaptic currents was significantly decreased in a temperature-dependent manner. Surperfusing the ISM generated an agonal inward current which consisted of a slow and subsequent rapid inward current in all of the neurons tested. The latencies of the slow and rapid inward currents after the ISM exposures were significantly longer at hypothermia than at normothermia. Hypothermia decreased the slope of the ISM-induced slow inward current, although it did not affect the slope of the rapid inward current. Moreover, the glutamate receptor antagonists slightly prolonged the latencies of the slow and rapid inward currents that were induced by ISM and significantly decreased their slopes. CONCLUSION: These results suggest that hypothermia reduces the excitatory synaptic activities and ischemic neuronal death in the spinal ventral horn. This finding may help in achieving a better understanding of the mechanisms of hypothermia-mediated neuroprotection in the spinal cord.

Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis.

An ubiquitin-binding protein, p62, is one of the components of the ubiquitin-containing inclusions in several human neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is characterized by the presence of skein-like inclusions, Lewy body-like inclusions, and basophilic inclusions in the remaining anterior horn cells, in which these inclusions contain ubiquitin, while the other characteristic inclusions of Bunina type are ubiquitin-negative. We examined the spinal cord from 28 ALS cases including two ALS with dementia and two ALS with basophilic inclusions, using antibody to p62. The results demonstrated that p62 localized in skein-like inclusions, Lewy body-like inclusions and basophilic inclusions. The number of p62-positive inclusions observed in the remaining anterior horn cells of each section was variable among the ALS cases. In contrast, Bunina bodies, that do not contain ubiquitin, were negative for p62. As far as we examined, the 11 non-ALS cases did not show any p62 immunoreactivities in the anterior horn cells. Our results suggested that p62 plays important roles in forming the inclusions and may be associated with the protection of the neurons from degenerative processes involving ubiquitin.

Reflexes in prior polio and their relation to weakness and anterior horn cell loss.

The aim of this study was to evaluate the reflex pattern in patients with prior polio and to relate these findings to the degree of anterior horn cell (AHC) involvement and loss of muscle force. Twenty-five prior polio subjects were investigated with electromyography (EMG), force testing and reflex studies, which included the patellar and Achilles reflex, H-reflex, T-response and interlimb reflex (ILR). The clinical reflexes, H/M-ratio and T-response amplitude at rest were positively correlated with force and negatively correlated with the degree of AHC loss. The H/M-ratio was decreased compared with age matched controls. ILR was present in 68% of the prior polio patients but did not exist in controls. The presence of the ILR was not correlated with the degree of AHC loss or force. The reflex studies gave two main findings. The first is reduced excitability of monosynaptic connections in the motor neuron pool, which is related to weakness. The other is the presence of ILR as an indicator of interneuronal hyper-excitability, which is not related to weakness.

Neuronal intranuclear hyaline inclusion disease showing motor-sensory and autonomic neuropathy.

BACKGROUND: Neuronal intranuclear hyaline inclusion disease (NIHID), a rare neurodegenerative disease in which eosinophilic intranuclear inclusions develop mainly in neurons, has not yet been described to present as hereditary motor-sensory and autonomic neuropathy. METHODS: Patients in two NIHID families showing peripheral neuropathy were evaluated clinically, electrophysiologically, and histopathologically. RESULTS: In both families, patients had severe muscle atrophy and weakness in limbs, limb girdle, and face; sensory impairment in the distal limbs; dysphagia, episodic intestinal pseudoobstruction with vomiting attacks; and urinary and fecal incontinence. No patients developed symptoms suggesting CNS involvement. Electrophysiologic study showed the reduced motor and sensory nerve conduction velocities and amplitudes, and also extensive denervation potentials. In sural nerve specimens, numbers of myelinated and unmyelinated fibers were decreased. In two autopsy cases, eosinophilic intranuclear inclusions were widespread, particularly in sympathetic and myenteric ganglion neurons, dorsal root ganglion neurons, and spinal motor neurons. These neurons also were decreased in number. CONCLUSION: Patients with neuronal intranuclear hyaline inclusion disease (NIHID) can manifest symptoms limited to those of peripheral neuropathy. NIHID therefore is part of the differential diagnosis of hereditary motor-sensory neuropathy associated with autonomic symptoms. Intranuclear hyaline inclusions in Schwann cells and in the myenteric plexus may permit antemortem diagnosis of NIHID.

Functional and electrophysiological changes after graded traumatic spinal cord injury in adult rat.

A graded contusion spinal cord injury (SCI) was created in the adult rat spinal cord using the Infinite Horizons (IH) impactor to study the correlation between injury severity and anatomical, behavioral, and electrophysiological outcomes. Adult Fisher rats were equally divided into five groups and received contusion injuries at the ninth thoracic level (T9) with 100, 125, 150, 175, or 200 kdyn impact forces, respectively. Transcranial magnetic motor-evoked potentials (tcMMEPs) and BBB open-field locomotor analyses were performed weekly for 4 weeks postinjury. Our results demonstrated that hindlimb locomotor function decreased in accordance with an increase in injury severity. The locomotor deficits were proportional to the amount of damage to the ventral and lateral white matter (WM). Locomotor function was strongly correlated to the amount of spared WM, which contains the reticulospinal and propriospinal tracts. Normal tcMMEP latencies were recorded in control, all of 100-kdyn-injured and half of 125-kdyn-injured animals. Delayed latency responses were recorded in some of 125-kdyn-injured and all of 150-kdyn-injured animals. No tcMMEP responses were recorded in 175- and 200-kdyn-injured animals. Comparison of tcMMEP responses with areas of WM loss or demyelination identified the medial ventrolateral funiculus (VLF) as the location of the tcMMEP pathway. Immunohistochemical and electromicroscopic (EM) analyses showed the presence of demyelinated axons in WM tracts surrounding the lesion cavities at 28 days postinjury. These data support the notion that widespread WM damage in the ventral and lateral funiculi may be a major cause for locomotor deficits and lack of tcMMEP responses after SCI.

HyperIgEaemia in patients with juvenile muscular atrophy of the distal upper extremity (Hirayama disease).

BACKGROUND: Juvenile muscular atrophy of the distal upper extremity (Hirayama disease) is characterised by anterior horn cell loss in the lower cervical cord, presumably caused by anterior displacement of the dural sac during neck flexion. A recent report suggests that atopy and IgE may contribute to anterior horn damage. OBJECTIVE: To investigate whether IgE is a contributing factor in Hirayama disease. METHODS: Serum total IgE and allergen specific IgE were examined in 20 consecutive patients, and their correlations with clinical profiles investigated. RESULTS: Past or present history of allergy/atopy was found in only four patients (20%), but serum IgE was raised in 14 (70%). Patients with hyperIgEaemia had more severe clinical disabilities than those without (p = 0.01). In patients whose history of Hirayama disease was less than five years, serum total IgE was higher than in those with the disease for five years or more (p = 0.05). CONCLUSIONS: The results suggest that hyperIgEaemia is often associated with Hirayama disease and can facilitate its pathophysiology, particularly in the early phases of the disease. HyperIgEaemia does not appear to involve the anterior horn cells primarily.

Life span extension and reduced neuronal death after weekly intraventricular cyclosporin injections in the G93A transgenic mouse model of amyotrophic lateral sclerosis.

OBJECT: The authors investigated whether cyclosporin A (CsA), a cyclophilin ligand with mitochondrial permeability transition pore-blocking and calcineurin-inhibiting properties, affects motor function, neuronal death, and life span in the G93A transgenic mouse model of familial amyotrophic lateral sclerosis (FALS). METHODS: The G93A mice received weekly intracerebroventricular injections of CsA (20 microg/mouse/week) starting at the age of 65 days, and physical performance on an exercise wheel was monitored beginning at 84 days of age. Mice were allowed to survive for clinical observation of body weight, hindlimb weakness, and life span or until a defined end stage or were killed at 110 days of age for histological analysis. CONCLUSIONS: Treatment with CsA significantly delayed the onset of hindlimb weakness and also extended the time from its onset to paralysis. The overall life span of CsA-treated G93A mice was significantly extended, by 12% compared with vehicle-treated transgenic littermates. The CsA also prolonged physical performance on the exercise wheel and delayed weight loss. Histologically, there was significant preservation of both cervical and lumbar spine motor neurons and also tyrosine hydroxylase-positive dopaminergic substantia nigra neurons in 110-day-old CsA-treated mice compared with their transgenic littermates. The local administration of CsA directly into the brain ventricles is an effective means of central nervous system drug delivery (because CsA does not readily cross the blood-brain barrier), which in this study ameliorated clinical and neuropathological features of FALS in G93A mice. The remarkably low intrathecal CsA dose required for neuroprotection reduces potential adverse effects of systemic immunosuppression or nephrotoxicity seen with chronic systemic delivery of the drug.