Memantine prolongs survival in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which results from selective loss of upper and lower motor neurons. Mouse models of ALS, such as one carrying the G93A mutant of the human Cu-Zn superoxide dismutase gene[SOD1(G93A)], develop motor neuron pathology and clinical symptoms similar to those observed in ALS patients. There is compelling evidence that both direct and indirect glutamate toxicity contribute to the pathogenesis of motor neuron degeneration. However, the therapeutic effect of various glutamate receptor antagonists has not been clearly demonstrated. Memantine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. It has been shown to protect neurons against NMDA- or glutamate-induced toxicity in vitro and in animal models of neurodegenerative diseases. In the current study, we have examined the therapeutic efficacy of memantine in an ALS mouse model carrying a high copy number of SOD1(G93A). Memantine treatment significantly delayed the disease progression and increased the life span of SOD1(G93A) mice, from 121.4 +/- 5.5 to 129.7 +/- 4.5 days (P = 0.032). Furthermore, NMDA receptor subunits were reliably detected in the spinal cord of SOD1(G93A) mice and their expression levels were similar to those in the wild-type littermate control. Therefore, the neuroprotective effect of memantine in SOD1(G93A) mice is most probably due to the inhibition of spinal cord NMDA receptors. In view of the long-term usage of memantine for dementia patients, with excellent tolerance and safety, these data suggest that memantine may be used in ALS patients alone or in combination with other therapies to prolong survival.     

EMG evaluation of motor neuron sprouting in amyotrophic lateral sclerosis

Collateral sprouting has been evaluated in amyotrophic lateral sclerosis (ALS) patients on the basis of: 1) Motor Unit action potential (M.U.A.P.) parameters evaluated by simultaneous EMG recordings using concentric needle electrodes and surface electrodes and 2) Motor Action Potential (M.A.P.) activated by graded electrical stimulation of nerve and recorded with the same electrodes. Mean values of duration and amplitude were calculated in 40 normal age-matched controls, 42 ALS patients in the early phase of the disease and in 5 muscles of three ALS patients during the whole course of the disease including the last paralytic phase. Percentage of M.U.A.P.s with linked potentials and highest amplitude was also calculated. The study confirms previous suggestions on collateral sprouting occurring in the early, middle and advanced phases of the disease and it shows, at variance with some recent claims, that it fails in the latest phase of paralysis.

---

Sommario è stato valutato lo sprouting collaterale in pazienti con Sclerosi Laterale Amiotrofica (SLA), studiando: 1) i parametri di unità motoria mediante registrazioni EMGrafiche sia ad ago che di superficie e 2) la risposta M ottenuta con stimolazioni graduate del nervo. I valori medi di durata, ampiezza e la percentuale di potenziali linked sono stati misurati in 40 soggetti normali di controllo, 42 pazienti con SLA nella fase iniziale della malattia e in 5 muscoli di 3 pazienti con SLA seguiti fino allo stadio finale paralitico della malattia. Lo studio conferma che lo sprouting collaterale avviene nelle fasi iniziali, medie e avanzate della malattia, mentre avviene meno nella fase terminale paralitica, a differenza di alcune recenti osservazioni.

     

Interplay of upper and lower motor neuron degeneration in amyotrophic lateral sclerosis

Objective

We studied motor unit recruitment to test a new method to identify motor unit firing rate (FR) variability.

Dorfin ameliorates phenotypes in a transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by progressive motor neuron degeneration and leads to death within a few years of diagnosis. One of the pathogenic mechanisms of ALS is proposed to be a dysfunction in the protein quality‐control machinery. Dorfin has been identified as a ubiquitin ligase (E3) that recognizes and ubiquitinates mutant SOD1 proteins, thereby accelerating their degradation and reducing their cellular toxicity. We examined the effects of human Dorfin overexpression in G93A mutant SOD1 transgenic mice, a mouse model of familial ALS. In addition to causing a decrease in the amount of mutant SOD1 protein in the spinal cord, Dorfin overexpression ameliorated neurological phenotypes and motor neuron degeneration. Our results indicate that Dorfin overexpression or the activation or induction of E3 may be a therapeutic avenue for mutant SOD1‐associated ALS. © 2009 Wiley‐Liss, Inc.     

Effects of chronic caffeine intake in a mouse model of amyotrophic lateral sclerosis

Caffeine is a nonselective adenosine receptor antagonist; chronic consumption has proved protective toward neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. The present study was designed to determine whether caffeine intake affected survival and/or motor performance in a transgenic model of amyotrophic lateral sclerosis (ALS). SOD1^G93A mice received caffeine through drinking water from 70 days of age until death. Body weight, motor performance and survival were evaluated. Furthermore, the expression of adenosine A_2A receptors (A_2ARs), glial glutamate transporter (GLT1), and glial fibrillar acidic protein (GFAP) were evaluated by Western blotting. The results showed that caffeine intake significantly shortened the survival of SOD1^G93A mice (log rank test, P = 0.01) and induced a nonsignificant advancing of disease onset. The expression of A_2AR, GLT1, and GFAP was altered in the spinal cords of ALS mice, but caffeine did not influence their expression in either wild‐type or SOD1^G93 mice. These data indicate that adenosine receptors may play an important role in ALS. © 2013 Wiley Periodicals, Inc.     

Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte‐mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1^G93A mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1^G93A mice as well as human induced pluripotent stem cell (iPSC)‐derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co‐culture. Increased Cx43 expression led to important functional consequences when tested in SOD1^G93A astrocytes when compared to control astrocytes over‐expressing wild‐type SOD1 (SOD1^WT). We observed SOD1^G93A astrocytes exhibited enhanced gap junction coupling, increased hemichannel‐mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1^G93A astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS‐related motor neuron loss. GLIA 2016;64:1154–1169     

Alterations of the blood‐spinal cord barrier in sporadic amyotrophic lateral sclerosis

The blood‐spinal cord barrier (BSCB) of the spinal cord capillary consists of non‐fenestrated endothelial cells with tight junctions, basal laminae, pericytes and astrocyte feet processes, referred to as a “neurovascular unit.” The primary function of the BSCB is the maintenance and control of homeostasis of the spinal cord parenchyma by the selective transport of molecules and cells from the systemic compartment. Dysfunction of the BSCB shows important function in the etiology or progression of several pathological conditions of the spinal cord, including amyotrophic lateral sclerosis (ALS). However, the role of BSCB in the pathogenesis of ALS is still unclear. Here the changes of BSCB in sporadic ALS patients were studied by electron microscopy to determine whether the BSCB is disrupted and involved in the pathogenesis of motor neuron degeneration. A total of 358 and 366 cross‐sectioned capillaries were quantitatively examined in controls and ALS patients, respectively. The frequency of degenerated endothelia and pericytes, vacuolar changes of the cytoplasm in the endothelia and pericytes, and the replication of basement membranes was significantly higher in ALS patients than in the controls (P = 0.0175). The areas of the capillaries with diameters of ≤ 5 µm in the ALS patients were significantly smaller than those in the controls (P = 0.0124). The frequency of collagen fiber content of more than a moderate degree around the perivascular space was significantly higher in the ALS patients compared to the controls (P = 0.048), although there was no significant difference in the mild degree of accumulation of collagen fibers. Thus, the BSCB may be disrupted in sporadic ALS patients due to increased permeability and reduced microcirculation, leading to motor neuron degeneration and to the progression of the disease.     

Screening for neurotrophic disturbances in amyotrophic lateral sclerosis

Neurotrophic activities in human serum and post-mortem muscle and spinal cord of possible relevance to pathophysiological mechanisms in amyotrophic lateral sclerosis (ALS) were studied. Tests included in vitro assays for nerve fibre outgrowth from sympathetic ganglia and for survival promotion of dissociated ciliary neurons, both types of neurons, of chicken embryo origin. Extracts of postmortem biceps muscle promoted survival of ciliary neurons in a dose-dependent manner. Half-maximum effect was found at a protein concentration of about 450 micrograms/ml for both ALS and control muscle. Ventral horn extracts were about 5 times as efficient as muscle in promoting neuron survival, again with no differences seen between control and ALS samples. Sera from patients suffering from ALS as well as normal sera did not enhance survival of ciliary neurons to any considerable extent, nor did they induce fibre outgrowth from sympathetic ganglia. Both groups of sera, if present above 5% in the medium, suppressed fibre outgrowth induced by added nerve growth factor (NGF). Sera from some of the ALS patients impaired survival in dissociated ciliary neurons supported by a trophic activity in choroid extract. The results do not indicate major neurotrophic deficits as the cause of ALS disease but suggest that a neurotoxic mechanism may be involved.     

A role for the urokinase-type plasminogen activator system in amyotrophic lateral sclerosis

There is substantial evidence, implicating extracellular matrix (ECM) regulating enzymes in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The most important ECM-degrading proteases are serine proteases (plasminogen activators, PA) and matrix metalloproteinases (MMPs). Since the role of MMPs in ALS has been addressed recently, we investigated the expression of the serine protease urokinase-type plasminogen activator (uPA) and its receptor in ALS. Employing rtPCR, zymography and immunohistochemistry we analyzed the expression of uPA and its receptor uPAR in spinal cord tissue of ALS cases and in the G93A SOD1 transgenic mouse. In the ventral horn of the spinal cord of ALS cases we found increased uPAR staining of motor neurons. In G93A mice, the expression profile of uPA and uPAR mRNA was significantly increased starting at the age of 90 days as compared to non-transgenic littermates. The uPA-dependent plasminogen activation in G93A mice at endstage increased markedly compared with controls and immunostaining of the spinal cord from G93A mice revealed increased uPAR immunostaining in neurons. To determine the functional role of uPA, we investigated the effect of intraperitoneal (i.p.) administration of the uPA inhibitor WX-340 (10 mg/kg), starting at the age of 30 days (n=18). Treatment with WX-340 prolonged (p<0.05) survival of the animals (135+/-2 vs. 126+/-3) as well as improving rotarod performance. Our experiments demonstrate that uPA and its receptor are expressed in ALS patients and in an animal model of ALS. Early inhibition with a synthetic uPA inhibitor prolonged the life of the transgenic animals. These findings indicate that the urokinase-type plasminogen activator system may play a role in the complex pathogenesis of ALS.     

Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis

Abstract

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. ER stress is also thought to be involved in the pathogenesis of neurodegenerative diseases. In this study, we investigated whether ER stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) using the anterior part of the lumbar spinal cord of transgenic mice carrying a mutation (G93A) in the superoxide dismutase 1 (SOD1) gene. Western blot and immunohistochemical analyses demonstrated that the expressions of p-PERK and p-eIF2α were increased in the microsome fraction (P3) of the lumbar spinal cord at the pre-symptomatic age of 12 weeks (12W), while the expression of activated caspase-12 was increased in the cytoplasmic fraction (S3) of the lumbar spinal cord at both the pre-symptomatic age of 12W and the late symptomatic age of 20W. In contrast, GRP78 did not show any increases in the microsome fraction (P3) of the lumbar spinal cord at either the pre-symptomatic or symptomatic ages. Thus, the present results strongly suggest that the balance between anti- and pro-apoptotic proteins related to ER stress is impaired from the pre-symptomatic stage in this ALS mouse model, and that this imbalance may be related to the pathogenesis of motor neuron degeneration in ALS.     

Exogenous platelet-derived growth factor improves neurovascular unit recovery after spinal cord injury

The blood-spinal cord barrier plays a vital role in recovery after spinal cord injury. The neurovascular unit concept emphasizes the relationship between nerves and vessels in the brain, while the effect of the blood-spinal cord barrier on the neurovascular unit is rarely reported in spinal cord injury studies. Mouse models of spinal cord injury were established by heavy object impact and then immediately injected with plateletderived growth factor(80 μg/kg) at the injury site. Our results showed that after platelet-derived growth factor administration, spinal cord injury, neuronal apoptosis, and blood-spinal cord barrier permeability were reduced, excessive astrocyte proliferation and the autophagyrelated apoptosis signaling pathway were inhibited, collagen synthesis was increased, and mouse locomotor function was improved. In vitro, human umbilical vein endothelial cells were established by exposure to 200 μM H_2O_2. At 2 hours prior to injury, in vitro cell models were treated with 5 ng/mL platelet-derived growth factor. Our results showed that expression of blood-spinal cord barrier-related proteins, including Occludin, Claudin 5, and β-catenin, was significantly decreased and autophagy was significantly reduced. Additionally, the protective effects of platelet-derived growth factor could be reversed by intraperitoneal injection of 80 mg/kg chloroquine, an autophagy inhibitor, for 3 successive days prior to spinal cord injury. Our findings suggest that platelet-derived growth factor can promote endothelial cell repair by regulating autophagy, improve the function of the blood-spinal cord barrier, and promote the recovery of locomotor function post-spinal cord injury. Approval for animal experiments was obtained from the Animal Ethics Committee, Wenzhou Medical University, China(approval No. wydw2018-0043) in July 2018.     

Spinal anterior horn has the capacity to self‐regenerate in amyotrophic lateral sclerosis model mice

The exact host environment necessary for neural regeneration in amyotrophic lateral sclerosis (ALS) has not yet been fully elucidated. We first focused on the extracellular matrix proteins in ALS model mice during development of the disease and then attempted to examine whether regeneration occurs in the ALS spinal cord under regenerative conditions. A progressive increase in γ1 laminin (a promoter of regeneration) and a progressive decrease in semaphorin3A (Sema3A; an inhibitor of regeneration) were observed, mainly in the neuropil of the spinal anterior horn from 15 to 18 weeks, when astrocytes began to express both γ1 laminin and Sema3A. On the other hand, a progressive increase in growth‐associated protein 43 (GAP43; synaptic regeneration site) and a progressive decrease in synaptotagmin1 (actual synaptic bouton) were observed in the same areas of the spinal anterior horn from 15 to 18 weeks. Thus, the present data suggest that, although the spinal anterior horn in ALS models loses motor neurons, it initially possesses the capacity to self‐regenerate but displays a progressive loss of ability to regenerate new effective synapses. © 2009 Wiley‐Liss, Inc.     

Early and progressive impairment of spinal blood flow-glucose metabolism coupling in motor neuron degeneration of ALS model mice

The exact mechanism of selective motor neuron death in amyotrophic lateral sclerosis (ALS) remains still unclear. In the present study, we performed in vivo capillary imaging, directly measured spinal blood flow (SBF) and glucose metabolism, and analyzed whether if a possible flow-metabolism coupling is disturbed in motor neuron degeneration of ALS model mice. In vivo capillary imaging showed progressive decrease of capillary diameter, capillary density, and red blood cell speed during the disease course. Spinal blood flow was progressively decreased in the anterior gray matter (GM) from presymptomatic stage to 0.80-fold of wild-type (WT) mice, 0.61 at early-symptomatic, and 0.49 at end stage of the disease. Local spinal glucose utilization (LSGU) was transiently increased to 1.19-fold in anterior GM at presymptomatic stage, which in turn progressively decreased to 0.84 and 0.60 at early-symptomatic and end stage of the disease. The LSGU/SBF ratio representing flow-metabolism uncoupling (FMU) preceded the sequential pathological changes in the spinal cord of ALS mice and was preferentially found in the affected region of ALS. The present study suggests that this early and progressive FMU could profoundly involve in the whole disease process as a vascular factor of ALS pathology, and could also be a potential target for therapeutic intervention of ALS.     

Reproducibility of the motor unit number index (MUNIX) in normal controls and amyotrophic lateral sclerosis patients

The motor unit number index (MUNIX) refers to an electrophysiologic technique that measures the approximate number of motor units using the surface electromyographic interference pattern (SIP) recorded during voluntary contraction. This study was done to assess the reproducibility of MUNIX performed on hypothenar muscles in 62 normal controls and 22 amyotrophic lateral sclerosis (ALS) patients. Inter- and intraoperator correlation coefficients for MUNIX were 0.74 and 0.86, respectively, in normal controls, and 0.95 and 0.93, respectively, in ALS patients (P < 0.01 in all). Inter- and intraoperator coefficients of variation for MUNIX measurements were 17.5% and 15.3%, respectively, in normal controls, and 23.7% and 24.0%, respectively, in ALS patients. This study shows a good correlation for MUNIX between intra- and interoperator results in both normal controls and ALS patients. The test-retest variability seems to be greater in ALS patients compared with normal controls, but this will need to be confirmed in future studies. Sources of variability should be identified and corrected for clinical use.     

Simulation studies on the motor unit potentials with satellite components in amyotrophic lateral sclerosis and spinal muscle atrophy

Introduction: We compared motor unit potentials (MUPs) with satellite components recorded in two anterior horn disorders: amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA, types II and III). Methods: We analyzed MUPs recorded from biceps brachii muscle, including 209 associated with ALS (12 patients) and 127 with SMA (5 patients). Simulations were applied to determine the origin of satellites in these processes. Results: MUP parameters differ in ALS and SMA. Simulations indicate that the satellite potential in ALS often originated from a single fiber, whereas in SMA it originated from a group of fibers of smaller diameters than the surrounding ones. Conclusions: These results suggest that, except for neurogenic factors, the variability of muscle fiber diameters also leads to the formation of MUPs with satellites. This variability seems to be responsible for the differences in the shape of the main and satellite MUP components in ALS and SMA. Muscle Nerve, 2012     

Increased binding of3H-L-deprenyI in spinal cords from patients with amyotrophic lateral sclerosis as demonstrated by autoradiography

Summary The present investigation has applied quantitative autoradiography and histochemistry to study the regional distribution of MAO-B and its relation to the number of cells in respective regions. L-deprenyl binds irreversibly and quantitatively to the B-form of monoamine oxidase, MAO, and is an ideal³H-ligand to measure the MAO-B enzyme protein in tissues by means of in vitro autoradiography. The investigation is performed on spinal sections from five controls and five cases with amyotrophic lateral sclerosis (ALS) on cervical, thoracic and lumbar level. The highest density of³H-L-deprenyl binding was found around the central canal (lamina X). MAO-B was markedly increased (up to 2.5 times of values in controls) specifically in regions of neurodegeneration e.g. motor neuron laminae and corticospinal tracts. There was a high correlation between glial cell count and³H-L-deprenyl binding with a relation indicating enhanced MAO-B protein in glial cells within areas of neurodegeneration. In contrast the increased microglial cell number in ALS did not show any correlation with³H-L-deprenyl binding.     

Intra-rater reliability of motor unit number estimation and quantitative motor unit analysis in subjects with amyotrophic lateral sclerosis

ObjectivesTo assess the intra-rater reliability of decomposition-enhanced spike-triggered averaging (DE-STA) motor unit number estimation (MUNE) and quantitative motor unit potential analysis in the upper trapezius (UT) and biceps brachii (BB) of subjects with amyotrophic lateral sclerosis (ALS) and to compare the results from the UT to control data.MethodsPatients diagnosed with clinically probable or definite ALS completed the experimental protocol twice with the same evaluator for the UT (n = 10) and BB (n = 9).ResultsIntra-rater reliability for the UT was good for the maximum compound muscle action potential (CMAP) (ICC = 0.88), mean surface-detected motor unit potential (S-MUP) (ICC = 0.87) and MUNE (ICC = 0.88), and for the BB was moderate for maximum CMAP (ICC = 0.61), and excellent for mean S-MUP (ICC = 0.94) and MUNE (ICC = 0.93). A significant difference between tests was found for UT MUNE. Comparing subjects with ALS to control subjects, UT maximum CMAP (p < 0.01) and MUNE (p < 0.001) values were significantly lower, and mean S-MUP values significantly greater (p < 0.05) in subjects with ALS.ConclusionsThis study has demonstrated the ability of the DE-STA MUNE technique to collect highly reliable data from two separate muscle groups and to detect the underlying pathophysiology of the disease.SignificanceThis was the first study to examine the reliability of this technique in subjects with ALS, and demonstrates its potential for future use as an outcome measure in ALS clinical trials and studies of ALS disease severity and natural history.     

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     

Involvement of cathepsin B in the motor neuron degeneration of amyotrophic lateral sclerosis

Abnormal proteolysis may be involved in the motor neuron degeneration of amyotrophic lateral sclerosis (ALS). Although several studies of the ubiquitin-proteasome system in ALS have been reported, the endosome-lysosome system has not been investigated in detail. To clarify the association of neurodegeneration with the endosome-lysosome system in ALS, we examined the pathological expression of cysteine proteases such as cathepsins B, H and L and an aspartate protease, cathepsin D, in the anterior horns of 15 ALS cases and 5 controls. In the ALS cases, cathepsin B immunoreactivity was preferentially decreased in the lateral parts of the anterior gray horns compared with the controls. Its immunoreactivity was increased in the cytoplasm of both shrunken and pigmented neurons but was weak in the neurons containing Bunina bodies. In addition, reactive astrocytes were also immunolabeled with cathepsin B. Cathepsin H and cathepsin L were detected in the cytoplasm of a small number of shrunken and pigmented neurons. Cathepsin D immunoreactivity was strong in the cytoplasm of all motor neurons. The immunoreactivity of cathepsins H, L and D was not significantly different between control and ALS cases. Western blot analysis showed that the 25-kDa activated form of cathepsin B was down-regulated in ALS. Our results suggest that cathepsin B is involved in the motor neuron degeneration in ALS.