Objective: Degeneration of chronically demyelinated axons is a major cause of irreversible neurological disability in multiple sclerosis (MS) patients. Development of neuroprotective therapies will require elucidation of the molecular mechanisms by which neurons and axons degenerate. Methods: We report ultrastructural changes that support Ca2؉-mediated destruction of chronically demyelinated axons in MS patients. We compared expression levels of 33,000 characterized genes in postmortem motor cortex from six control and six MS brains matched for age, sex, and postmortem interval. As reduced energy production is a major contributor to Ca2؉-mediated axonal degeneration, we focused on changes in oxidative phosphorylation and inhibitory neurotransmission. Results: Compared with controls, 488 transcripts were decreased and 67 were increased (p < 0.05, 1.5-fold) in the MS cortex. Twenty-six nuclear-encoded mitochondrial genes and the functional activities of mitochondrial respiratory chain complexes I and III were decreased in the MS motor cortex. Reduced mitochondrial gene expression was specific for neurons. In addition, pre-synaptic and postsynaptic components of GABAergic neurotransmission and the density of inhibitory interneuron processes also were decreased in the MS cortex. Interpretation: Our data supports a mechanism whereby reduced ATP production in demyelinated segments of upper motor neuron axons impacts ion homeostasis, induces Ca2؉-mediated axonal degeneration, and contributes to progressive neurological disability in MS patients. Neurol 2006;59:478 -489 Rapid communication between neurons requires energy and the insulation of axons by discontinuous segments of myelin. Voltage-gated Na ϩ channels produce nerve impulses and are concentrated at the nodes of Ranvier, 1 the short unmyelinated axon segment between individual myelin internodes. The nerve impulse rapidly jumps from node to node by a process called saltatory conduction. Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that destroys myelin, oligodendrocytes, axons, and neurons. Ann2 Pathologically, demyelination predominates during early stages of MS. Neurological disability associated with demyelinating lesions is initially reversible because of a variety of adaptive changes in the MS brain. As part of these changes, Na ϩ channels are distributed diffusely along the surface of demyelinated axons, 3 resulting in slow but effective nerve communication. This also increases the energy demands of neuronal communication and renders the demyelinated axon more susceptible to hypoxic/ischemic damage (for review, see Stys 4 ). After an initial stage (commonly 10 -15 years) of relapses and remissions (RRMS), most MS patients enter a course of irreversible and continuous neurological decline, termed secondary progressive multiple sclerosis (SPMS).2 During SPMS, new inflammatory brain lesions substantially decrease with age, 5 but neurological decline continues due in part to degeneration of chronically...

Ceramide is a lipid second messenger that mediates the effects of tumor necrosis factor ␣ and other agents on cell growth and differentiation. Ceramide is believed to act via activation of protein phosphatase, prolinedirected protein kinase, or protein kinase C. Tumor necrosis factor ␣-induced common pathway of apoptosis is associated with an early impairment of mitochondria. Herein, we demonstrate that ceramide can directly inhibit mitochondrial respiratory chain function. In isolated mitochondria, a rapid decline of mitochondrial oxidative phosphorylation occurs in the presence of Nacetylsphingosine (C 2 -ceramide), a synthetic cell-permeable ceramide analog. An investigation of the site of ceramide action revealed that the activity of respiratory chain complex III is reduced by C 2 -ceramide with halfmaximum effect at 5-7 M. In contrast, N-acetylsphinganine (C 2 -dihydroceramide), which lacks a functionally critical double bond and is ineffective in cells, did not alter mitochondrial respiration or complex III activity. We suggest that these in vitro observations may set the stage for identifying a novel mechanism of regulation of mitochondrial function in vivo.The pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) 1 elicits a wide variety of cellular responses including profound alterations in transcriptional programs, perturbation of mitochondrial function, and apoptosis in a number of cell types (1, 2). Strong evidence supports a pivotal role for TNF-␣ in the genesis of septic shock (2), and it also has been implicated in ischemia reperfusion injury of heart (3). A recent report demonstrates that a physiologically relevant concentration of TNF-␣ induced apoptosis in rat cardiomyocytes as quantified by single cell microgel electrophoresis of nuclei and in situ 3Ј nick end labeling assay (4). Mitochondria are considered an early target in TNF-␣-induced cytotoxicity because they appear swollen with a reduced number of cristae, in association with profound inhibition of mitochondrial respiration (1,5,6). A growing body of evidence suggests that treatment of cells with TNF-␣ results in an electron transport inhibition at the level of complex III (1, 6) followed by an increased generation of oxygen radicals in mitochondria (7-9). However, the mechanism of TNF-␣-induced inhibition of mitochondrial respiration has not been elucidated.The sphingomyelin pathway has been implicated as a major signaling mechanism mediating the action of a number of extracellular agents (such as TNF-␣, Fas ligands, and chemotherapeutic agents) causing the activation of sphingomyelinases that cleave membrane sphingomyelin resulting in the formation of ceramide (10, 11). Synthetic cell-permeable ceramide analogs have been shown to mimic many TNF-␣-induced cell responses (10 -13). In malignant and nonmalignant cell lines, ceramides specifically induce apoptosis that involves activation of interleukin-1␤-converting enzyme-like proteases, whereas closely related dihydro-analogs are inactive (10). The intracellular targets for ceramid...

Mastoparan facilitates opening of the mitochondrial permeability transition pore through an apparent bimodal mechanism of action. In the submicromolar concentration range, the action of mastoparan is dependent upon the medium Ca2+ and phosphate concentration and is subject to inhibition by cyclosporin A. At concentrations above 1 microM, pore induction by mastoparan occurs without an apparent Ca2+ requirement and in a cyclosporin A insensitive manner. Studies utilizing phospholipid vesicles show that mastoparan perturbs bilayer membranes across both concentration ranges, through a mechanism which is strongly dependent upon transmembrane potential. However, solute size exclusion studies suggest that the pores formed in mitochondria in response to both low and high concentrations of mastoparan are the permeability transition pore. It is proposed that low concentrations of mastoparan influence the pore per se, with higher concentrations having the additional effect of depolarizing the mitochondrial inner membrane through an action exerted upon the lipid phase. It may be the combination of these effects which allow pore opening in the absence of Ca2+ and in the presence of cyclosporin A, although other interpretations remain viable. A comparison of the activities of mastoparan and its analog, MP14, on mitochondria and phospholipid vesicles provides an initial indication that a G-protein may participate in regulation of the permeability transition pore. These studies draw attention to peptides, in a broad sense, as potential pore regulators in cells, under both physiological and pathological conditions.