Molecular mechanisms regulating motor neuron development and degeneration

Motor neurons are a well-defined, although heterogeneous group of cells responsible for transmitting information from the central nervous system to the locomotor system. Spinal motor neurons are specified by soluble factors produced by structures adjacent to the primordial spinal cord, signaling through homeodomain proteins. Axonal pathfinding is regulated by cell-surface receptors that interact with extracellular lignads and once synaptic connections have formed, the survival of the somatic motor neuron is dependent on the provision of target-derived growth factors, although nontarget-derived factors, produced by either astrocytes or Schwann cells, are also potentially implicated. Somatic motor neuron degeneration leads to profound disability, and multiple pathogenetic mechanisms including aberrant growth factor signaling, abnormal neurofilament accumulation, excitotoxicity, and autoimmunity have been postulated to be responsible. Even when specific deficits have been identified, for example, mutations of the superoxide dismutase-1 gene in familial amyotrophic sclerosis and polyglutamine expansion of the androgen receptor in spinal and bulbar muscular atrophy, the mechanisms by which somatic motor neuronal degeneration occurs remain unclear. In order to treat motor system degeneration effectively, we will need to understand these mechanisms more thoroughly.     

Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.     

Knockdown of the aryl hydrocarbon receptor attenuates excitotoxicity and enhances NMDA-induced BDNF expression in cortical neurons

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.     

Adult neural stem/progenitor cells reduce NMDA-induced excitotoxicity via the novel neuroprotective peptide pentinin

Although the potential of adult neural stem cells to repair damage via cell replacement has been widely reported, the ability of endogenous stem cells to positively modulate damage is less well studied. We investigated whether medium conditioned by adult hippocampal stem/progenitor cells altered the extent of excitotoxic cell death in hippocampal slice cultures. Conditioned medium significantly reduced cell death following 24 h of exposure to 10 μM NMDA. Neuroprotection was greater in the dentate gyrus, a region neighboring the subgranular zone where stem/progenitor cells reside compared with pyramidal cells of the cornis ammonis. Using mass spectrometric analysis of the conditioned medium, we identified a pentameric peptide fragment that corresponded to residues 26–30 of the insulin B chain which we termed 'pentinin'. The peptide is a putative breakdown product of insulin, a constituent of the culture medium, and may be produced by insulin-degrading enzyme, an enzyme expressed by the stem/progenitor cells. In the presence of 100 pM of synthetic pentinin, the number of mature and immature neurons killed by NMDA-induced toxicity was significantly reduced in the dentate gyrus. These data suggest that progenitors in the subgranular zone may convert exogenous insulin into a peptide capable of protecting neighboring neurons from excitotoxic injury.     

On the relationship between the two branches of the kynurenine pathway in the rat brain in vivo

In the mammalian brain, kynurenine aminotransferase II (KAT II) and kynurenine 3-monooxygenase (KMO), key enzymes of the kynurenine pathway (KP) of tryptophan degradation, form the neuroactive metabolites kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK), respectively. Although physically segregated, both enzymes use the pivotal KP metabolite l -kynurenine as a substrate. We studied the functional consequences of this cellular compartmentalization in vivo using two specific tools, the KAT II inhibitor BFF 122 and the KMO inhibitor UPF 648. The acute effects of selective KAT II or KMO inhibition were studied using a radiotracing method in which the de novo synthesis of KYNA, and of 3-HK and its downstream metabolite quinolinic acid (QUIN), is monitored following an intrastriatal injection of ³H-kynurenine. In naïve rats, intrastriatal BFF 122 decreased newly formed KYNA by 66%, without influencing 3-HK or QUIN production. Conversely, UPF 648 reduced 3-HK synthesis (by 64%) without affecting KYNA formation. Similar, selective effects of KAT II and KMO inhibition were observed when the inhibitors were applied acutely together with the excitotoxin QUIN, which impairs local KP metabolism. Somewhat different effects of KMO (but not KAT II) inhibition were obtained in rats that had received an intrastriatal QUIN injection 7 days earlier. In these neuron-depleted striata, UPF 648 not only decreased both 3-HK and QUIN production (by 77% and 66%, respectively) but also moderately raised KYNA synthesis (by 27%). These results indicate a remarkable functional segregation of the two pathway branches in the brain, boding well for the development of selective KAT II or KMO inhibitors for cognitive enhancement and neuroprotection, respectively.     

Slow and selective death of spinal motor neurons in vivo by intrathecal infusion of kainic acid: implications for AMPA receptor-mediated excitotoxicity in ALS

Excitotoxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been proposed to play a major role in the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), and motor neurons are more vulnerable to AMPA receptor-mediated excitotoxicity than are other neuronal subclasses. On the basis of the above evidence, we aimed to develop a rat model of ALS by the long-term activation of AMPA receptors through continuous infusion of kainic acid (KA), an AMPA receptor agonist, into the spinal subarachnoid space. These rats displayed a progressive motor-selective behavioral deficit with delayed loss of spinal motor neurons, mimicking the clinicopathological characteristics of ALS. These changes were significantly ameliorated by co-infusion with 6-nitro-7-sulfamobenso(f)quinoxaline-2,3-dione (NBQX), but not with d(-)-2-amino-5-phosphonovaleric acid (APV), and were exacerbated by co-infusion with cyclothiazide, indicative of an AMPA receptor-mediated mechanism. Among the four AMPA receptor subunits, expression of GluR3 mRNA was selectively up-regulated in motor neurons but not in dorsal horn neurons of the KA-infused rats. The up-regulation of GluR3 mRNA in this model may cause a molecular change that induces the selective vulnerability of motor neurons to KA by increasing the proportion of GluR2-lacking (i.e. calcium-permeable) AMPA receptors. This rat model may be useful in investigating ALS etiology.     

Methamphetamine-induced spectrin proteolysis in the rat striatum

Methamphetamine (METH) is a widely abused psychostimulant. Multiple high doses of METH cause long-term toxicity to dopamine (DA) and serotonin (5-HT) nerve terminals in the brain, as evidenced by decreases in DA and 5-HT content, decreases in tyrosine and tryptophan hydroxylase activities, decreases in DA and 5-HT re-uptake sites, and nerve terminal degeneration. Multiple high doses of METH are known to elicit a rapid increase in DA release and hyperthermia. Although METH also produces a delayed and sustained rise in glutamate, no studies have shown whether METH produces structural evidence of excitotoxicity in striatum, or identified the receptors that mediate this toxicity directly, independent of alterations in METH-induced hyperthermia. These experiments investigated whether METH can cause excitotoxicity as evidenced by cytoskeletal protein breakdown in a glutamate receptor-dependent manner. METH increased calpain-mediated spectrin proteolysis in the rat striatum 5 and 7 days after METH administration without affecting caspase 3-dependent spectrin breakdown. This effect was completely blocked with the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, GYKI 52466, but not the NMDA receptor antagonist, MK-801. However, AMPA or NMDA receptor antagonism did not attenuate the METH-induced depletions of the dopamine transporter (DAT). Independent mechanisms involved in mediating spectrin proteolysis and DAT protein loss are discussed.     

Mouse brains deficient in neuronal PDGF receptor-beta develop normally but are vulnerable to injury

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null-knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the beta subunit of PDGFR (PDGFR-beta) was genetically deleted in CNS neurons to elucidate the role of PDGFR-beta, particularly in the post-natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR-beta detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild-type mice was depleted, but PDGFR-beta detected in blood vessels remained unaffected. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR-beta expressed in neurons protects them from cryogenic injury and NMDA-induced excitotoxicity.     

Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons

Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.