Activated microglia initiate motor neuron injury by a nitric oxide and glutamate-mediated mechanism

Recent studies suggest that motor neuron (MN) death may be non-cell autonomous, with cell injury mediated by interactions involving non-neuronal cells, such as microglia and astrocytes. To help define these interactions, we used primary MN cultures to investigate the effects of microglia activated by lipopolysaccharide or IgG immune complexes from patients with amyotrophic lateral sclerosis. Following activation, microglia induced MN injury, which was prevented by a microglial iNOS inhibitor as well as by catalase or glutathione. Glutamate was also required since inhibition of the MN AMPA/kainate receptor by CNQX prevented the toxic effects of activated microglia. Peroxynitrite and glutamate were synergistic in producing MN injury. Their toxic effects were also blocked by CNQX and prevented by calcium removal from the media. The addition of astrocytes to cocultures of MN and activated microglia prevented MN injury by removing glutamate from the media. The protective effects could be reversed by inhibiting astrocytic glutamate transport with dihydrokainic acid or pretreating astrocytes with H2O2. Astrocytic glutamate uptake was also decreased by activated microglia or by added peroxynitrite. These data suggest that free radicals released from activated microglia may initiate MN injury by increasing the susceptibility of the MN AMPA/kainate receptor to the toxic effects of glutamate.     

Deglycosylation of anti-beta amyloid antibodies inhibits microglia activation in BV-2 cellular model

Immunotherapy has become a strategy for treatment of Alzheimer's disease, by inducing antibody response to amyloid-beta peptide (AbetaP) or by passive administration of anti-AbetaP antibodies. Clearance of amyloid plaques involves interaction of immunoglobulin Fc receptor (FcR)-expressing microglia and antibodyopsonized Abeta deposits, stimulating phagocytosis but may promote neuroinflammation. Carbohydrate moiety of Fc of the immunoglobulin G molecule plays a significant role in modulating binding to FcR and its effector functions. Here, we enzymatically removed Fc glycan from monoclonal antibody 196 raised against AbetaP Antigen binding ability and in vitro stability of deglycosylated antibody were unaffected by deglycosylation. Moreover, the deglycosylated antibody exhibits low affinity to FcR on microglial BV-2 cells and has limited ability to mediate microglial chemotaxis and antibodydependent cytotoxicity compared to native antibody. These data suggest that deglycosylation of anti-Abeta antibodies before in vivo administration might prevent microglial overactivation, thus reducing the risk of neuroinflammatory response during passive immunization.     

Stress exacerbates neuron loss and microglia proliferation in a rat model of excitotoxic lower motor neuron injury

All individuals experience stress and hormones (e.g., glucocorticoids/GCs) released during stressful events can affect the structure and function of neurons. These effects of stress are best characterized for brain neurons; however, the mechanisms controlling the expression and binding affinity of glucocorticoid receptors in the spinal cord are different than those in the brain. Accordingly, whether stress exerts unique effects on spinal cord neurons, especially in the context of pathology, is unknown. Using a controlled model of focal excitotoxic lower motor neuron injury in rats, we examined the effects of acute or chronic variable stress on spinal cord motor neuron survival and glial activation. New data indicate that stress exacerbates excitotoxic spinal cord motor neuron loss and associated activation of microglia. In contrast, hypertrophy and hyperplasia of astrocytes and NG2+ glia were unaffected or were modestly suppressed by stress. Although excitotoxic lesions cause significant motor neuron loss and stress exacerbates this pathology, overt functional impairment did not develop in the relevant forelimb up to one week post-lesion. These data indicate that stress is a disease-modifying factor capable of altering neuron and glial responses to pathological challenges in the spinal cord.     

Huperzine a provides neuroprotection against several cell death inducers usingin vitro model systems of motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from the progressive loss of motor neurons in the spinal cord and brain. To date, clinically effective neuroprotective agents have not been available. The current study demonstrates for the first time that huperzine A, a potential neuroprotective agent, has the ability to protect a motor neuron-like cell line and motor neurons in spinal cord organotypic cultures from toxin-induced cell death. The neuroblastoma-spinal motor neuron fusion cell line, NSC34 and rat spinal cord organotypic cultures (OTC) were exposed to cell death inducers for 24 h or 14 d, respectively, with and without pre-treatment with huperzine A. The inducers used here include: staurosporine, thapsigargin, hydrogen peroxide (H2O2), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and L-(-)-threo-3-hydroxyaspartic acid (THA). These agents were selected as they induce apoptosis/necrosis via mechanisms implicated in patients with generalized motor neuron disease. Cell death was determined in NSC34 cells by metabolic activity, caspase activity/expression and by nuclear morphology and in the OTCs, using immunohistochemistry and Western blot analysis. Nuclear staining of NSC34 cells revealed cell death induced by staurosporine, thapsigargin, H2O2 and CCCP. This induction was significantly reduced with 2 h pre-treatment with 10 microM huperzine A (maximum, 35% rescue; p 0.05) following exposure to staurosporine, thapsigargin and H2O2 but not with CCCP. These data were supported by the metabolic assays and caspase activity. In addition, pre-treatment with huperzine A dramatically improved motor neuron survival, based on choline acetyltransferase (ChAT) expression analysis in OTCs following exposure to THA, and compared to THA-treated control cultures. These studies are currently being extended to include other inducers and with additional compounds as potential drug therapies that could be used in combination for the treatment of patients with ALS.     

Glucocorticoid enhancement of serotonergic facilitation of cat spinal monosynaptic motor neuron excitation

The effects of an intensive short-term triamcinolone diacetate pretreatment regimen (8 mg/kg intramuscularly once daily for 7 days) on the facilitation of neuromuscular transmission produced by either intravenous or direct intraarterial administration of guanidine hydrochloride, were examined in the in vivo cat soleus nerve-muscle preparation. The ability of guanidine to activate a stimulus-bound repetitive after discharge (SBR) of soleus motor axons and to produce an obligatory enhancement of soleus muscle isometric contractile tension was measured in glucocorticoid-treated vs. untreated animals. Triamcinolone pretreatment significantly increased the guanidine-induced soleus contractile potentiation by 3 to 4 times with i.v. injection (P < 0.05) and by more than 30 times with i.a. administration (P < 0.01). The incidence of SBR in normal motor axons after i.v. guanidine was 58.5% in the treated animals and 25.0% in the untreated ones (P < 0.001). Also, the rate of development of the peak SBR incidence was almost doubled as a result of triamcinolone pretreatment (P < 0.05). This glucocorticoid enhancement of the facilitatory actions of guanidine may have relevance for the treatment of certain neuromuscular disorders.     

Effects of anesthetic treatment on motor neuron death in xenopus

Xenopus tadpoles were introduced into anesthetic (chlorbutanol) solutions during a developmental period in which naturally occurring loss of lumbar spinal motor neurons takes place. Animals that developed in these solutions were sacrificed after various lenghts of time and the number of lumbar motor neurons were counted. No significant difference was detected between experimental and control motor neuron counts throughout the period in which most of the cells are normally lost.A similar finding is reported when one or more lumbar spinal nerves were blocked locally with implants of anesthetic (procaine) impregnated silastic chips.     

Protective effect of parvalbumin on excitotoxic motor neuron death

The mechanism responsible for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca(2+) overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca(2+)-permeable AMPA receptors on motor neurons gives rise to higher Ca(2+) transients in motor neurons compared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca(2+) concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Furthermore, we investigated whether increasing the motor neuron's cytosolic Ca(2+)-buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca(2+) buffering capacity, we generated transgenic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thymus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially protected from KA-induced death as compared to those isolated from nontransgenic littermates. PV overexpression also attenuated KA-induced Ca(2+) transients, but not those induced by depolarization. We conclude that the high density of Ca(2+)-permeable AMPA receptors on the motor neuron's surface results in high Ca(2+) transients upon stimulation and that the low cytosolic Ca(2+)-buffering capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpression of a high-affinity Ca(2+) buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca(2+) entry into the cell.     

Influence of reduced neuron pool on the magnitude of naturally occurring motor neuron death

The present investigation was undertaken to examine the role of peripheral competition in survival of motor neurons during development. A loss of approximately half of the trochlear motor neurons in duck and quail occurs during the course of normal embryogenesis. The number of motor neurons in the nucleus of quail prior to the onset of cell death is identical to the final number of survivors in the nucleus of duck embryos (about 1,300 neurons). In the present study competition at the peripheral target was decreased by reducing the number of trochlear motor neurons initially projecting to their target muscle. This was accomplished by substituting the midbrain of duck embryos with the same neural tissue from quail embryos. Midbrain transplantation was performed before motor axon outgrowth and normal cell death begin. The development of the motor neurons and their sole target of innervation, the superior oblique muscle, was examined by using a variety of techniques. The source of the grafted motor neurons and of a reduction in the size of the motor neuron pool was confirmed from histological sections and cell counts. The grafted motor neurons projected their axons into the appropriate peripheral target, which was determined by the use of HRP tracing technique. Counts of muscle fibers, motor endplates, and acetylcholine receptors and measurement of total muscle protein indicated that the size of the superior oblique muscle in the chimera embryos was similar to that of the normal duck but significantly larger than the muscle in quail embryos. Electrophysiological observations indicated that the grafted trochlear motor neurons made functional connections with the superior oblique muscle. Counts of the trochlear motor neurons after the period of cell death indicated an average of 1,310 neurons in the nucleus of duck, 772 in quail, and 690 in the chimera embryos. The number of motor neurons in the chimera embryos is not significantly different from that in the normal quail. In other words, in spite of reduced peripheral competition trochlear motor neuron death of normal magnitude occurred. Lack of increased cell survival in our study suggests that trochlear motor neurons do not compete for survival at the peripheral target.     

Epigenetic regulation of motor neuron cell death through DNA methylation

DNA methylation is an epigenetic mechanism for gene silencing engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to cytosine residues in gene-regulatory regions. It is unknown whether aberrant DNA methylation can cause neurodegeneration. We tested the hypothesis that Dnmts can mediate neuronal cell death. Enforced expression of Dnmt3a induced degeneration of cultured NSC34 cells. During apoptosis of NSC34 cells induced by camptothecin, levels of Dnmt1 and Dnmt3a increased fivefold and twofold, respectively, and 5-methylcytosine accumulated in nuclei. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocked apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with RG108 and procainamide protected cultured neurons from excessive DNA methylation and apoptosis. In vivo, Dnmt1 and Dnmt3a are expressed differentially during mouse brain and spinal cord maturation and in adulthood when Dnmt3a is abundant in synapses and mitochondria. Dnmt1 and Dnmt3a are expressed in motor neurons of adult mouse spinal cord, and, during their apoptosis induced by sciatic nerve avulsion, nuclear and cytoplasmic 5-methylcytosine immunoreactivity, Dnmt3a protein levels and Dnmt enzyme activity increased preapoptotically. Inhibition of Dnmts with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice. In human amyotrophic lateral sclerosis (ALS), motor neurons showed changes in Dnmt1, Dnmt3a, and 5-methylcytosine similar to experimental models. Thus, motor neurons can engage epigenetic mechanisms to drive apoptosis, involving Dnmt upregulation and increased DNA methylation. These cellular mechanisms could be relevant to human ALS pathobiology and disease treatment.     

Molecular effects of activated BV-2 microglia by mitochondrial toxin 1-methyl-4-phenylpyridinium

Microglia plays an important role in inflammation-mediated neurodegeneration. Compelling evidence supports the hypothesis that microglial activation contributes to the pathogenesis of various neurodegenerative diseases. However, little is known about the molecular outcome of activated microglia. In this report, we investigate the molecular consequences of MPP(+) toxin-induced activated BV-2 microglia. Intoxication of specific mitochondrial toxin methyl-4-phenylpyridinium iodide ion (MPP(+)) to BV-2 cells induced significant mitochondrial dysfunction and increased the reactive oxygen species generation, caspase-3 activation, and poly ADP ribose polymerase proteolysis. Further, MAC-1 immunostaining in the midbrain of mice revealed a decrease in activated microglia at day 4 after intoxication with MPP(+). From this study, it was confirmed that BV-2 microglia respond to the mitochondrial toxin MPP(+) which may lead to apoptotic cell death. Understanding of the mechanistic basis of apoptotic elimination of activated microglia may help to develop new strategies for the treatment of neurodegenerative diseases.     

Evidence for facilitation of motor evoked potentials (MEPs) induced by motor imagery

This study examined the extent to which motor imagery can facilitate to specific pools of motoneurons. Motor commands induced by motor imagery were subthreshold for muscle activity and were presumably not associated with any change in background afferent activity. To estimate excitability changes of flexor carpi radialis (FCR) muscle motoneuron in spinal and cortical level, electric stimuli for recording H-reflex and transcranial magnetic stimulation (TMS) for recording motor evoked potentials (MEPs) were used. During motor imagery of wrist flexion, remarkable increases in the amplitude of the MEP of FCR were observed with no change in the H-reflex. Furthermore, facilitation of antagonist (extensor carpi radialis; ECR) was also observed. Therefore, it is concluded that internal motor command can activate precisely cortical excitability with no change in spinal level without recourse to afferent feedback.     

Reductions in motor unit number estimates (MUNE) precede motor neuron loss in the plasma membrane calcium ATPase 2 (PMCA2)-heterozygous mice

The potential of MUNE as a unique electrophysiological tool to detect early motor unit abnormalities during a clinically silent period was investigated in the plasma membrane calcium ATPase 2 (PMCA2)-heterozygous mice. There was a significant reduction in MUNE in the PMCA2-heterozygous mice as compared to the wild type littermates at two months of age. In contrast, the compound motor action potential (CMAP) was not altered. The conduction velocity (CV) of the sensory nerve and sensory nerve action potentials (SNAP) were not modified indicating lack of major sensory deficits. Interestingly, despite a decline in MUNE at this age, no changes were detected in choline acetyl transferase (ChAT) positive motor neuron number in the ventral horn of the lumbar spinal cord. Hindlimb grip strength, a test that evaluates clinical dysfunction, was also similar to that of the wild type controls. However, motor neuron number significantly decreased by five months suggesting that a drop in MUNE preceded motor neuron loss. In the two-month-old PMCA2-null mice, reduced MUNE measurements coincided with lower motor neuron number and decreased hindlimb grip strength. The fall in motor neuron number was already detectable at three weeks, the earliest time studied, and became more pronounced by five months. Our results show that even partial reductions in PMCA2 levels are sufficient to cause delayed death of motor neurons and that MUNE may be a reliable and sensitive approach to detect pathology prior to cell loss and in the absence of overt clinical signs.     

Proteasome inhibition induces selective motor neuron death in organotypic slice cultures

A dysfunctional ubiquitin-proteasome system recently has been proposed to play a role in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We have shown previously that spinal motor neurons are more vulnerable to proteasome inhibition-induced neurotoxicity, using a dissociated culture system. To confirm this toxicity, we used organotypic slice cultures from rat neonatal spinal cords, which conserve the structure of the spinal cord in a horizontal plane, enabling us to identify motor neurons more accurately than in dissociated cultures. Furthermore, such easy identifications make it possible to follow up the course of the degeneration of motor neurons. When a specific proteasome inhibitor, lactacystin (5 microM), was applied to slice cultures, proteasome activity of a whole slice was suppressed below 30% of control. Motor neurons were selectively damaged, especially in neurites, with the increase of phosphorylated neurofilaments. They were eventually lost in a dose-dependent manner (1 microM, P < 0.05; 5 microM, P < 0.01). The low capacity of Ca(2+) buffering is believed to be one of the factors of selectivity for damaged motor neurons in ALS. In our system, negative staining of Ca(2+)-binding proteins supported this notion. An intracellular Ca(2+) chelator, BAPTA-AM (10 microM), exerted a significant protective effect when it was applied with lactacystin simultaneously (P < 0.01). We postulate that proteasome inhibition is an excellent model for studying the mechanisms underlying selective motor neuron death and searching for new therapeutic strategies in the treatment of ALS.     

胰岛素样生长因子(IGF-1)对体外培养的脊髓和皮层运动神经元的保护作用

目的研究胰岛素样生长因子(IGF-1)对THA诱导的运动神经元损伤的保护作用。方法选用生后7d和1d的SD乳鼠,7d龄乳鼠用于脊髓片培养,1d龄乳鼠用于脑片培养。在无菌条件下断头取脊髓腰膨大部分或脑组织,将脊髓腰膨大部分和包含运动皮层的脑组织切成薄片进行体外培养,对照组加入正常培养基,模型组给予谷氨酸转运体抑制剂-THA进行干预,IGF-1组于培养液中同时加入THA和不同浓度的IGF-1。药物干预3w后,应用免疫组织化学方法显示运动神经元并计数。结果 THA能够选择性诱导脊髓前角运动神经元和皮层运动神经元死亡,IGF-1能阻止THA诱导的运动神经元的死亡。结论 IGF-1对THA诱导的慢性运动神经元损伤具有保护作用,IGF-1可能有益于ALS的治疗。     

Development of the topographical projection of motor neurons to amphibian muscle accompanies motor neuron death

The topographical projection of segmental nerves 8 and 9 to the glutaeus muscle of developing frogs was determined during the motor neuron death period (stages 54 to 59). Electrophysiological techniques showed that at stage 54 the entire glutaeus muscle received an innervation from both nerves 8 and 9. By stage 59 the muscle received an innervation from nerve 8 only on one side and by nerve 9 only on the opposite side. The emergence of this mature innervation pattern is accompanied by motor neuron death, with which it may be causally related.     

Spontaneous lower motor neuron disease in rabbits (Oryctolagus cuniculus)

Summary Spontaneous neurologic disease was observed in 6 to 8-week-old rabbits. Both males and females from several different litters were affected but all were sired by the same male. Clinically, the disease was characterized initially by posterior weakness and incoordination which progressed to tetraplegia within 3–4 weeks. With light microscopy there was neuronal degeneration and loss within the ventral horns of the spinal cord and brain stem and type-II fiber atrophy of skeletal muscles. Ultrastructurally the neuronal degeneration was charactered by accumulations of 100 Å neurofilaments within the perikaryon. These findings are compared to diseases with neurofibrillary accumulation in animals and man.This work was supported in part by NIH grant RR00685-05 and by the Scott-Ritchy Research Fund of Auburn University