Differential cortico-motoneuron vulnerability after chronic mitochondrial inhibition in vitro and the role of glutamate receptors.

Chronic treatment of rat cortical slices with a relative low concentration of mitochondrial inhibitor malonate leads to cortical motoneuron (CMN) death. In the neurodegenerative disease amyotrophic lateral sclerosis (ALS) corticospinal neurons, CMNs projecting to the spinal cord, degenerate. In the present study we compared the effect of chronic mitochondrial inhibition on the survival of CMNs located in the dorsal cortical areas (including corticospinal neurons) with that on ventrally located CMNs (non-corticospinal neurons) in vitro. In the explant culture model used, the dorsally located CMNs were less vulnerable to a 2-week period of mitochondrial inhibition with malonate as compared to ventrally located CMNs. Treatment with 5 mM malonate resulted in 50% surviving CMNs in the dorsal part and only 16% in the ventral part. Neuroprotection of the CMNs could be achieved with co-administration of the non-NMDA antagonist CNQX, the NMDA antagonist MK-801, or the glutamate release inhibitor riluzole, suggesting that chronic energy shortage leads to excitotoxicity. In the dorsal cortical areas CNQX, MK-801, and riluzole had a neuroprotective effect on the CMNs, whereas in the ventral cortical areas only MK-801 was neuroprotective. The sensitivity to energy depletion and consequently excitotoxicity may be related to glutamate receptor density and subunit composition in various cortical areas, but also to the projection length and input of CMNs in vivo. The present investigation gives insight in mechanisms leading to excitotoxic cell death of CMNs and may therefore be important for the development of treatment strategies in protection and survival of cortical motoneurons in ALS.

Cellular changes in motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis as revealed by monoclonal antibody Py.

Transgenic mice (G93A) carrying the human amyotrophic lateral sclerosis (ALS) linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. The affected motoneurons are characterized by the presence of cellular alterations. The antigen recognized by the monoclonal antibody Py is suggested to be associated with the neurofilamentous and microtubular elements of the cytoskeleton of specific neuron populations including the spinal motoneurons. The aim of the present study was to measure changes in the relative Py-immunoreactivity per identified Choline-Acetyl-Transferase (ChAT)-immunoreactive motoneuron during the disease progression. The relative Py-immunoreactivity of identified spinal motoneurons was measured on double stained (Py and ChAT) motoneurons using a digital imaging system coupled to an inverse microscope. A significant decrease of Py-immunoreactivity was already noted in the pre-symptomatic stages of the disease even before the onset of massive motoneuron degeneration. It is concluded that the Py-antibody detects early intracellular abnormalities related to neurodegenerative changes in spinal motoneurons of transgenic SOD1-(G93A) mice.

Malonate-induced cortico-motoneuron death is attenuated by NT-4, but not by BDNF or NT-3.

Neurotrophins are promising candidates to slow the progression of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease in which spinal and cortical motoneurons selectively degenerate. In a long-term in vitro model, malonate-induced toxicity and cell death of motoneurons have been demonstrated. Here we studied the neuroprotective effect of BDNF, NT-3, and NT-4 on the cell death of cortical motoneurons in an organotypic culture model after chronic mitochondrial inhibition with malonate. Our data show that NT-4 completely prevents malonate-induced toxicity, whereas BDNF or NT-3 had no neuroprotective effect. In clinical trials for ALS, predominantly focussed on the survival of spinal motoneurons, BDNF has already been tested with disappointing results; our results suggest that NT-4 may be a better neurotrophin to prevent motoneuron loss.

Chronic mitochondrial inhibition induces glutamate-mediated corticomotoneuron death in an organotypic culture model.

There is growing evidence that mitochondrial dysfunction is an important factor in a cascade of neurotoxic events as observed during pathogenesis of various neurodegenerative diseases. In the neurodegenerative disease amyotrophic lateral sclerosis (ALS) both spinal and cortical motoneurons degenerate, but in experimental studies most attention so far has been focussed on the spinal motoneurons. In order to study the role of mitochondrial dysfunction in the pathways leading to cortical (upper) motoneuron (CMN) death, a long-term culture system of rat cortical explants was used. CMNs were visualized by immunocytochemical labeling with antibodies directed against nonphosphorylated neurofilament, SMI-32, and for their identification we also used their location in layer V of the explant, their size, and their morphological appearance. In this model the effect of mitochondrial inhibition was studied through chronic malonate treatment. For 2 weeks, low doses of complex II inhibitor malonate were added to the cultures twice a week. The malonate-induced chronic mitochondrial inhibition resulted in a dose-dependent increase of CMN death in the slices. Neuroprotection was achieved with the NMDA antagonist MK-801 and the non-NMDA antagonist CNQX indicating the involvement of glutamate in the malonate-induced CMN death. Furthermore, our data indicate that chronic mitochondrial inhibition results in CMN death, which is mediated by glutamate excitotoxicity via both non-NMDA and NMDA receptors. In this respect the present in vitro approach may act as a model for understanding mechanisms underlying CMN death in ALS.