Ceramide is implicated in humoral peripheral and intrathecal autoimmune response in MS patients.

BACKGROUND: The disturbed metabolism of ceramide (Cer) is supposed to evoke the autoimmune response, contributing to MS pathology. OBJECTIVES: To determine levels of anti-Cer immunoglobulins G (IgGs) in the CSF and serum of subjects with various phenotypes of MS, and to investigate relationships between levels of anti-Cer antibodies and MS-related variables. METHODS: IgGs isolated from serum and the CSF of 68 MS patients and appropriate controls were examined for their reactivity to Cer subspecies. Their levels were compared between the studied groups and compartments, and analyzed with regard to clinical variables. RESULTS: Increased levels of anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1-Cer IgGs were detected in the CSF and serum of MS patients in comparison with controls. For IgGs against particular Cer subspecies, correlations were found between their CSF and serum level, as well as with the Link index. Serum and the CSF anti-Cer IgGs differed between patients with clinically isolated syndrome (CIS) and relapsing-remitting MS from those with progressive MS. No correlations were found between anti-Cer IgGs and other MS-related clinical variables. CONCLUSION: Patients with MS have shown altered panels of anti-Cer IgGs in the CSF and serum, which might suggest a relevant, though limited role of Cer as a target for autoimmune humoral response. Utility of antibodies against Cer subspecies as potential markers for MS activity and progression deserves further investigations.

Distinctive sphingolipid patterns in chronic multiple sclerosis lesions.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Autoimmunity in multiple sclerosis: role of sphingolipids, invariant NKT cells and other immune elements in control of inflammation and neurodegeneration.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is classified as being an autoimmune response in the genetically susceptible individual to a persistent but unidentified antigen(s). Both the adaptive and the innate immune systems are likely to contribute significantly to MS pathogenesis. This review summarizes current understanding of the characteristics of MS autoimmunity in the initiation and progression of the disease. In particular we find it timely to classify the autoimmune responses by focusing on the immunogenic features of myelin-derived lipids in MS including molecular mimicry; on alterations of bioactive sphingolipids mediators in MS; and on functional roles for regulatory effector cells, including innate lymphocyte populations, like the invariant NKT (iNKT) cells which bridge adaptive and innate immune systems. Recent progress in identifying the nature of sphingolipids recognition for iNKT cells in immunity and the functional consequences of the lipid-CD1d interaction opens new avenues of access to the pathogenesis of demyelination in MS as well as design of lipid antigen-specific therapeutics.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line.

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated "donor" cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.

Neuron-microglia interaction induced bi-directional cytotoxicity associated with calpain activation.

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Invariant natural killer T cells and their ligands: focus on multiple sclerosis.

Invariant natural killer T (iNKT) cells are an innate population of T cells identified by the expression of an invariant T-cell receptor and reactivity to lipid-based antigens complexed with CD1d. They account for a small percentage of lymphocytes, but are extremely potent and play central roles in immunity to infection, in some cancers, and in autoimmunity. The list of relevant stimulatory lipids and glycolipid antigens now includes a range of endogenous self-antigens including the myelin-derived acetylated galactosylceramides. Recent progress in studies to identify the nature of lipid recognition for iNKT cells in autoimmune diseases like multiple sclerosis is likely to foster the development of therapeutic strategies aimed at harnessing iNKT cell activity.

Invariant Natural Killer T-cell anergy to endogenous myelin acetyl-glycolipids in multiple sclerosis.

To extend our studies on glycolipid-reactive invariant Natural Killer T-cell (iNKT-cell) function in multiple sclerosis (MS), we investigated the stimulatory activities of two myelin-derived glycolipids that are poly-acetylated derivatives of ß-galactosylceramide designated as fast-migrating cerebrosides (FMC) by thin-layer chromatography. In healthy subjects, FMC stimulation of peripheral blood cells significantly expanded iNKT-cells similar to α-GalCer and induced significant increases in Th1, Th2 and Th17 cytokines. In marked contrast, MS patients failed to respond to FMCs or to α-GalCer stimulation indicating an anergic response. We propose that myelin-derived FMC glycolipids stimulate iNKT-cell responses in vivo and this is blocked in MS.

Implications of Lymphocyte Anergy to Glycolipids in Multiple Sclerosis (MS): iNKT Cells May Mediate the MS Infectious Trigger.

Immunogenic lipids may play key roles in host defenses against infection and in generating autoimmune inflammation and organ-specific damage. In multiple sclerosis (MS) there are unequivocal autoimmune features and vulnerability to aggravation or induction by microbial or viral infection. We have found glycolipid-driven anergy of circulating lymphocytes in MS indicating that this immune response is affected in MS and the robust effects of iNKT activation with potent cellular and cytokine activities emphasizes its potential importance. Diverse glycolipids including the endogenous myelin acetylated-galactosylceramides (AcGalCer) can drive activation that could be critical to the inflammatory demyelination in the central nervous system and clinical consequences. The iNKT cells and their invariant or iTCR (Vα24Jα18Vß11) receptor an innate defense-a discrete immune arm that is separate from peptide-driven acquired immune responses. This offers new possibilities for insight including a likelihood that the pattern recognition of exogenous microbial and myelin immunogens can overlap and cross-react especially in an inflammatory milieu.

Myelin recovery in multiple sclerosis: the challenge of remyelination.

Multiple sclerosis (MS) is the most common demyelinating and an autoimmune disease of the central nervous system characterized by immune-mediated myelin and axonal damage, and chronic axonal loss attributable to the absence of myelin sheaths. T cell subsets (Th1, Th2, Th17, CD8+, NKT, CD4+CD25+ T regulatory cells) and B cells are involved in this disorder, thus new MS therapies seek damage prevention by resetting multiple components of the immune system. The currently approved therapies are immunoregulatory and reduce the number and rate of lesion formation but are only partially effective. This review summarizes current understanding of the processes at issue: myelination, demyelination and remyelination-with emphasis upon myelin composition/ architecture and oligodendrocyte maturation and differentiation. The translational options target oligodendrocyte protection and myelin repair in animal models and assess their relevance in human. Remyelination may be enhanced by signals that promote myelin formation and repair. The crucial question of why remyelination fails is approached is several ways by examining the role in remyelination of available MS medications and avenues being actively pursued to promote remyelination including: (i) cytokine-based immune-intervention (targeting calpain inhibition), (ii) antigen-based immunomodulation (targeting glycolipid-reactive iNKT cells and sphingoid mediated inflammation) and (iii) recombinant monoclonal antibodies-induced remyelination.

Signaling and regulatory functions of bioactive sphingolipids as therapeutic targets in multiple sclerosis.

Spingolipids (SLs) are an important component of central nervous system (CNS) myelin sheaths and affect the viability of brain cells (oligodendrocytes, neurons and astrocytes) that is determined by signaling mediated by bioactive sphingoids (lyso-SLs). Recent studies indicate that two lipids, ceramide and sphingosine 1-phosphate (S1P), are particularly involved in many human diseases including the autoimmune inflammatory demyelination of multiple sclerosis (MS). In this review we: (1) Discuss possible sources of ceramide in CNS; (2) Summarize the features of the metabolism of S1P and its downstream signaling through G-protein-coupled receptors; (3) Link perturbations in bioactive SLs metabolism to MS neurodegeneration and (4) Compile ceramide and S1P relationships to this process. In addition, we described recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod) as well as proposed intervention to specify critical SL levels that tilt balances of apoptotic/active ceramide versus anti-apoptotic/inactive dihydroceramide that may offer a novel and important therapeutic approach to MS.

The structural and functional role of myelin fast-migrating cerebrosides: pathological importance in multiple sclerosis.

A family of neutral glycosphingolipids containing a 3-O-acetyl-sphingosine galactosylceramide (3-SAG) has been characterized. Seven new derivatives of galactosylceramide (GalCer), designated as fast-migrating cerebrosides (FMCs) by TLC retention factor, have been identified. The simplest compounds - FMC-1 and FMC-2 - of this series have been characterized as the 3-SAG containing nonhydroxy and hydroxy fatty acyl, respectively. The next two - FMC-3 and FMC-4 - add 6-O-acetyl-galactose and the most complex glycosphingolipids, FMC-5, -6 and -7, are 2,3,4,6-tetra-O-acetyl-3-SAG. These hydrophobic myelin lipid biomarkers coappear with GalCer during myelinogenesis and disappear along with GalCer in de- or dys-myelinating disorders. Myelin lipid antigens, including FMCs, are keys to myelin biology, opening the possibility of new and novel immune modulatory tools for treatment of autoimmune diseases including multiple sclerosis.

Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid.

Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O-acetylation of the 2-hydroxy-fatty acid. The immuno-reactivity in human cerebrospinal fluid (CSF) to these acetylated glycolipids was examined in central nervous system (CNS) infectious disease, noninflammatory disorders, and multiple sclerosis (MS). Screening for lipid binding in MS and other neurological disease groups revealed that the greatest anti-hydrophobic FMC reactivity was observed in the inflammatory CNS diseases (meningitis, meningo-encephalitis, and subacute sclerosing panencephalitis). Some MS patients had increased reactivity with the hydrophobic FMCs and with glycoglycerophospholipid MfGL-II from Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked.

Ceramide and neurodegeneration: susceptibility of neurons and oligodendrocytes to cell damage and death.

Neurodegenerative disorders are marked by extensive neuronal apoptosis and gliosis. Although several apoptosis-inducing agents have been described, understanding of the regulatory mechanisms underlying modes of cell death is incomplete. A major breakthrough in delineation of the mechanism of cell death came from elucidation of the sphingomyelin (SM)-ceramide pathway that has received worldwide attention in recent years. The SM pathway induces apoptosis, differentiation, proliferation, and growth arrest depending upon cell and receptor types, and on downstream targets. Sphingomyelin, a plasma membrane constituent, is abundant in mammalian nervous system, and ceramide, its primary catabolic product released by activation of either neutral or acidic sphingomyelinase, serves as a potential lipid second messenger or mediator molecule modulating diverse cellular signaling pathways. Neutral sphingomyelinase (NSMase) is a key enzyme in the regulated activation of the SM cycle and is particularly sensitive to oxidative stress. In a context of increasing clarification of the mechanisms of neurodegeneration, we thought that it would be useful to review details of recent findings that we and others have made concerning different pro-apoptotic neurotoxins including proinflammatory cytokines, hypoxia-induced SM hydrolysis and ceramide production that induce cell death in human primary neurons and primary oligodendrocytes: redox sensitive events. What has and is emerging is a vista of therapeutically important ceramide regulation affecting a variety of different neurodegenerative and neuroinflammatory disorders.

T-cells expressing natural killer (NK) receptors are altered in multiple sclerosis and responses to alpha-galactosylceramide are impaired.

Multiple sclerosis (MS) is an autoimmune disorder characterised by clinical relapse and remission and pathological demyelination with varying inflammation. Because it is suggested that T-cells expressing natural killer cell receptors (NKR) play important roles in regulating human autoimmune diseases, we have quantified populations of T-cells expressing the NKR CD56, CD161 and CD94 in the peripheral blood of MS patients, in healthy control subjects (HS) and in patients with other neurological diseases (OND). CD161(+) T-cells and CD94(+) T-cells were significantly decreased in MS patients with primary progressive disease and secondary progressive disease respectively whereas CD56(+) T-cell numbers were unchanged. In contrast NKT-cells that express the invariant Valpha24-Jalpha18(+) T-cell receptor identified here by specific receptor antibody and CD1d-tetrameric PBS57-loaded complexes, were increased in MS patients compared with HS. Reductions in CD161(+) T-cells and CD94(+) T-cells relative to HS were also observed in the OND group and this was particularly prominent in Parkinsonian patients. A striking functional finding was that while NKT-cells in unfractionated peripheral blood from healthy subjects expanded in number and produced IFN-gamma upon stimulation with alpha-galactosylceramide, NKT-cells from MS patients did not. Thus we have identified alterations in a number of potentially important lymphocyte sub-populations warranting further investigation in the immune response in MS.

Cell-specific expression of neutral glycosphingolipids in vertebrate brain: immunochemical localization of 3-O-acetyl-sphingosine-series glycolipid(s) in myelin and oligodendrocytes.

The tissue- and cell-specific expression of three neutral glycosphingolipids, gangliotetraosylceramide (GA1), gangliopentaosylceramide (GalNAc-GA1), and the novel 3-O-acetyl-sphingosine-series glycolipid (FMC-5), were examined with monospecific polyclonal antibodies. Immunohistochemical studies of rodent brain cross-sections indicated that both GA1 and FMC-5 antibodies stained myelin. In contrast, GalNAc-GA1 antibody distinctly stained neurons in cerebral cortex, but only partially delineated Purkinje cells and other neurons in cerebellum. Preliminary studies of mixed glial cultures suggested the following: 1) both FMC-5 and GA1 antibodies stained oligodendrocytes and oligo progenitors, and 2) GalNAc-GA1 antibody did not stain any cells in the culture. Because the GalNAc-GA1 was associated with neurons, we examined the immunoreactivity of GalNAc-GA1 antibody in primary neuronal cultures. Further studies using primary cultures of rat brain oligodendrocytes, and dissociated cerebellar neuronal cultures indicated that both GA1 and FMC-5 are specifically expressed by oligodendrocytes, whereas GalNAc-GA1 is primarily localized in interneurons and to some extent in Purkinje neurons.

Characterization of novel myelin components 3-O-acetyl-sphingosine galactosylceramides by electrospray ionization Q-TOF MS and MS/CID-MS of Li+ adducts.

Glycosphingolipids with R(f) values higher than those of monoglycosylceramides (MGCs) in normal phase HPTLC appear to be normal components of myelin. A series of such low polarity components, referred to as 'fast moving cerebrosides' (FMCs), have been isolated from rat brain, and two of these fractions (FMC-1 and FMC-2) were found to be novel derivatives of galactosylceramide (GalCer) exhibiting O-acetylation at the 3-hydroxy group of the sphingoid moiety, and incorporating either non-hydroxy or 2-hydroxy fatty-N-acylation (Dasgupta S, Levery SB, Hogan EL. J. Lipid Res. 2002; 43: 751-761). Similar to the parent compounds, the 3-O-acetyl-sphingoid derivatives exhibit considerable diversity with respect to fatty-N-acyl chain length, manifested by heterogeneous molecular ion (Li(+) adduct) profiles. However, a detailed analysis of the individual molecular variants ('lipoforms'), e.g. by tandem MS/CID-MS analysis, was not carried out. In addition, several other FMCs distinguished by even lower polarity (higher HPTLC R(f) values) were isolated but have remained uncharacterized. For this study, analysis of both the known and unknown FMC components was carried out by positive ion ESI-MS and MS/CID-MS of their Li(+) adducts on a Q-TOF mass spectrometer. Since a Q-TOF instrument has not yet been applied to MS of lithiated cerebrosides and FMCs, MS/CID-MS spectra of bovine brain GalCer (both types) and the previously characterized rat brain FMCs (FMC-1 and FMC-2), having 3-O-acetylation of the sphingoid, were systematically acquired and their fragmentation behavior compared. This was followed by systematic analysis of previously uncharacterized FMC fractions (FMC-3 through FMC-5/6/7). The GalCer and FMC components proved to be amenable to analysis by this technique, and the data confirm that the latter are all related 3-O-acetyl-sphingoid derivatives, with the higher R(f) components carrying additional O-acetyl modifications on the galactosyl residue, which further reduce their polarity. The utility of the technique, the structures of unknown FMCs, and their characteristic fragmentation patterns are described.

Absence of Mycoplasma-specific DNA sequence in brain, blood and CSF of patients with multiple sclerosis (MS): a study by PCR and real-time PCR.

Mycoplasmas are the smallest of the known self-replicating organisms. They lack cell walls and are associated with numerous diseases in humans and animals. We are exploring the possibility that infection by Mycoplasma may induce the inflammatory demyelinating disease of the central nervous system (CNS) that is MS. The presence of specific Mycoplasma species DNA was sought in brain, serum and cerebrospinal fluid (CSF) of patients diagnosed with multiple sclerosis (MS) and other neurological diseases (OND) including inflammatory disorders. The MS samples from patients with active and progressive MS, as well as in remission, a variety of other neurological disease controls, including inflammatory CNS diseases such as meningitis, cryptococcal meningitis and encephalitis and other neurological disorders such as migraine were also examined. Clinical samples were provided by the National Neurological Research Specimen Bank and the Human Brain and Spinal Fluid Resource Centre, Los Angeles. Analysis was carried out by conventional PCR using Mycoplasma-specific primers (McAuliffe et al., 2005) that target the 16S rDNA gene in Mycoplasma species. The Mycoplasma-specific primers could detect 102 Mycoplasma species. In this study, 30 samples of human brain and 57 pairs of serum and CSF and were examined. No Mycoplasma-specific nucleic acid sequence was detected, and the consistent observation of an endogenous gene, human serum albumin (HSA), as a positive control documented the adequacy of the method. Real-time PCR analysis of serum and CSF was done also targeting utilizing the Mycoplasma 16S rDNA gene, and this also demonstrated the lack of Mycoplasma in these samples. The presence of Mycoplasma at extraneural sites in MS patients is now being explored.

Early induction of secondary injury factors causing activation of calpain and mitochondria-mediated neuronal apoptosis following spinal cord injury in rats.

To investigate a potential relationship between calpain and mitochondrial damage in spinal cord injury (SCI), a 40 gram-centimeter force (g-cm) injury was induced in rats by a weight-drop method and allowed to progress for 4 hr. One-centimeter segments of spinal cord tissue representing the adjacent rostral, lesion, and adjacent caudal areas were then removed for various analyses. Calcium green 2-AM staining of the lesion and penumbra sections showed an increase in intracellular free calcium (Ca(2+)) levels following injury, compared with corresponding tissue sections from sham-operated (control) animals. Western blot analysis showed increased calpain expression and activity in the lesion and penumbra segments following SCI. Double-immunofluorescent labeling indicated that increased calpain expression occurred in neurons in injured segments. Western blot analysis also showed an increased Bax:Bcl-2 ratio, indicating the induction of the mitochondria-mediated cell death pathway in the lesion and penumbra. The morphology of mitochondria was altered in lesion and penumbra following SCI: mostly hydropic change (swelling) in the lesion, with the penumbra shrunken or normal. At 4 hr after induction of injury, a substantial amount of cytochrome c had been released into the cytoplasm, suggesting a trigger for apoptosis through caspase 3 activation. Neuronal death after 4 hr of injury was detected by a combined TUNEL and double-immunofluoresence assay in the lesion and penumbra sections of injured cord, compared with sham controls. These results suggest that an early induction of secondary factors is involved in the pathogenesis of SCI. The increased Ca(2+) levels could activate calpain and mediate mitochondrial damage leading to neuronal death in lesion and penumbra following injury. Thus, secondary injury processes mediating cell death are induced as early as 4 hr after the injury, and calpain and caspase inhibitors may provide neuroprotection.

Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors.

Spinal cord injury (SCI) evokes an increase in intracellular free Ca(2+) level resulting in activation of calpain, a Ca(2+)-dependent cysteine protease, which cleaves many cytoskeletal and myelin proteins. Calpain is widely expressed in the central nervous system (CNS) and regulated by calpastatin, an endogenous calpain-specific inhibitor. Calpastatin degraded by overactivation of calpain after SCI may lose its regulatory efficiency. Evidence accumulated over the years indicates that uncontrolled calpain activity mediates the degradation of many cytoskeletal and membrane proteins in the course of neuronal death and contributes to the pathophysiology of SCI. Cleavage of the key cytoskeletal and membrane proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells leading to cell death. Calpain in conjunction with caspases, most notably caspase-3, can cause apoptosis of the CNS cells following trauma. Aberrant Ca(2+) homeostasis following SCI inevitably activates calpain, which has been shown to play a crucial role in the pathophysiology of SCI. Therefore, calpain appears to be a potential therapeutic target in SCI. Substantial research effort has been focused upon the development of highly specific inhibitors of calpain and caspase-3 for therapeutic applications. Administration of cell permeable and specific inhibitors of calpain and caspase-3 in experimental animal models of SCI has provided significant neuroprotection, raising the hope that humans suffering from SCI may be treated with these inhibitors in the near future.

Molecular evidence of apoptotic death in malignant brain tumors including glioblastoma multiforme: upregulation of calpain and caspase-3.

Cell death in the core of human brain tumors is triggered by hypoxia and lack of nutrients, but the mode of cell death whether necrosis or apoptosis is not clearly defined. To identify the role of apoptosis in brain tumor cell death, we investigated macromolecular (RNA and protein) synthesis and activity in the central to peripheral region of benign [desmoplastic infantile ganglioglioma (DIG) and transitional meningioma (TMG)] and malignant [ependymoma (END), anaplastic astrocytoma (APA), and glioblastoma multiforme (GBM)] brain tumors derived from five patients who had not received previously radiotherapy or chemotherapy. Normal brain tissue (NBT) served as control. RT-PCR analysis of tumor tissues covering central to peripheral regions detected mRNA overexpression of pro-apoptotic gene bax in malignant tumors, indicating a commitment to apoptosis. The mRNA expression of calpain (a Ca(2+)-dependent cysteine protease) and calpastatin (endogenous calpain inhibitor) was altered resulting in an elevated calpain/calpastatin ratio. Calpain content and activity were increased, suggesting a role for calpain in cell death. In the mitochondria-dependent death pathway, caspase-9 and caspase-3 were also overexpressed in tumors. The increased caspase-3 activity cleaved poly(ADP-ribose) polymerase (PARP). Agarose gel electrophoresis detected a mixture of random and internucleosomal DNA fragmentation in malignant brain tumors. Overexpression of pro-apoptotic bax, upregulation of calpain and caspase-3, and occurrence of internucleosomal DNA fragmentation are now presented indicating that one mechanism of cell death in malignant brain tumors is apoptosis, and that enhancement of this process therapeutically may promote decreased tumor growth.