MiR-7-1 potentiated estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons.

Protection of motoneurons is an important goal in the treatment of spinal cord injury (SCI). We tested whether neuroprotective microRNAs (miRs) like miR-206, miR-17, miR-21, miR-7-1, and miR-106a could enhance efficacy of estrogen receptor (ER) agonists such as 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT, ERα agonist), Way200070 (WAY, ERß agonist), and estrogen (EST, ERα and ERß agonist) in preventing apoptosis in the calcium ionophore (CI)-insulted ventral spinal cord 4.1 (VSC4.1) motoneurons. We determined that 200 nM CI induced 70% cell death. Treatment with 50 nM PPT, 100 nM WAY, and 150 nM EST induced overexpression of ERα, ERß, and both receptors, respectively, at mRNA and protein levels. Treatment with ER agonists significantly upregulated miR-206, miR-17, and miR-7-1 in the CI-insulted VSC4.1 motoneurons. Transfection with miR-206, miR-17, or miR-7-1 mimic potentiated WAY or EST to inhibit apoptosis in the CI-insulted VSC4.1 motoneurons. Overexpression of miR-7-1 maximally increased efficacy of WAY and EST for down regulation of pro-apoptotic Bax and upregulation of anti-apoptotic Bcl-2. A search using microRNA database (miRDB) indicated that miR-7-1 could inhibit the expression of L-type Ca(2+) channel protein alpha 1C (CPα1C). miR-7-1 overexpression and WAY or EST treatment down regulated CPα1C but upregulated p-Akt to trigger cell survival signaling. The same therapeutic strategy increased expression of the Ca(2+)/calmodulin-dependent protein kinase II beta (CaMKIIß) and the phosphorylated cAMP response element binding protein (p-CREB) so as to promote Bcl-2 transcription. Whole cell membrane potential and mitochondrial membrane potential studies indicated that miR-7-1 highly potentiated EST to preserve functionality in the CI-insulted VSC4.1 motoneurons. In conclusion, our data indicated that miR-7-1 most significantly potentiated efficacy of EST for functional neuroprotection and this therapeutic strategy could be used in the future to attenuate apoptosis of motoneurons in SCI.

Hippocampal tissue of patients with refractory temporal lobe epilepsy is associated with astrocyte activation, inflammation, and altered expression of channels and receptors.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Previous research has demonstrated several trends in human tissue that, undoubtedly, contribute to the development and progression of TLE. In this study we examined resected human hippocampus tissue for a variety of changes including gliosis that might contribute to the development and presentation of TLE. The study subjects consisted of six TLE patients and three sudden-death controls. Clinicopathological characteristics were evaluated by H&E staining. Immunohistological staining and Western blotting methods were used to analyze the samples. Neuronal hypertrophy was observed in resected epileptic tissue. Immunohistological staining demonstrated that activation of astrocytes was significantly increased in epileptic tissue as compared to corresponding regions of the control group. The Western blot data also showed increased CX43 and AQP4 in the hippocampus and downregulation of Kir4.1, α-syntrophin, and dystrophin, the key constituents of AQP4 multi-molecular complex. These tissues also demonstrated changes in inflammatory factors (COX-2, TGF-ß, NF-κB) suggesting that these molecules may play an important role in TLE pathogenesis. In addition we detected increases in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and kainic acid receptor subunits KA1 (Grik4) and KA2 (Grik5) in patients' hippocampi. We noted increased expression of the α1c subunit comprising class C L-type Ca(2+) channels and calpain expression in these tissues, suggesting that these subunits might have an integral role in TLE pathogenesis. These changes found in the resected tissue suggest that they may contribute to TLE and that the kainic acid receptor (KAR) and deregulation of GluR2 receptor may play an important role in TLE development and disease course. This study identifies alterations in number of commonly studied molecular targets associated with astrogliosis, cellular hypertrophy, water homeostasis, inflammation, and modulation of excitatory neurotransmission in hippocampal tissues from TLE patients.

Calpain inhibition protected spinal cord motoneurons against 1-methyl-4-phenylpyridinium ion and rotenone.

Parkinson's disease (PD), characterized by selective midbrain nigrostriatal dopaminergic degeneration, is consistently associated with moderate systemic mitochondrial dysfunction. Downstream degeneration of spinal cord has also been suggested in PD, although the mechanisms have not been much investigated. In the present study, two mitochondrial toxicants, 1-methyl-4-phenylpyridinium ion (MPP(+)) and rotenone were tested in ventral spinal cord (VSC 4.1) motoneuronal cells. Cell death was assessed by morphological and biochemical means to discern a lower apoptosis-inducing concentration and lethal concentration of 50% cell death (LC(50)), which were subsequently compared in further cytoprotection experiments. Mitochondrial toxicants dose-dependently induced increase in intracellular free Ca(2+) level, which was conducive for increased expression and activities of Ca(2+)-activated neutral protease calpain and downstream caspase-3. Thus, mitochondrial damage triggered apoptotic mechanisms in spinal cord motoneurons. Inhibition of calpain by calpeptin significantly attenuated damaging effects of MPP(+) and rotenone on motoneurons, especially at low apoptosis-inducing concentrations of toxicants and partly at their LC(50), as demonstrated by absence of DNA ladder formation and decrease in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Cytoprotection by calpeptin was observed with marked decreases in Bax: Bcl-2 ratio and activities of calpain and caspase-3, which affirmed the role of mitochondrial dysfunction and involvement of intrinsic pathway in mediation of apoptosis. These findings strongly suggested that parkinsonian toxicants MPP(+) and rotenone at low doses induced cascade of cell-damaging effects in spinal cord motoneurons, thus, highlighting the possibility of induction of apoptotic mechanisms in these cells, when subjected to mitochondrial stress. Cytoprotection rendered by calpeptin further validated the involvement of calpain in apoptosis and suggested calpain inhibition as a potential neuroprotective strategy.

Combination of N-(4-hydroxyphenyl) retinamide and genistein increased apoptosis in neuroblastoma SK-N-BE2 and SH-SY5Y xenografts.

Neuroblastoma is the childhood malignancy that mainly occurs in adrenal glands and is found also in the neck, chest, abdomen, and pelvis. New therapeutic strategies are urgently needed for successful treatment of this pediatric cancer. In this investigation, we examined efficacy of the retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and the isoflavonoid genistein (GST) alone and also in combination for controlling the growth of human malignant neuroblastoma SK-N-BE2 and SH-SY5Y xenografts in nude mice. Combination of 4-HPR and GST significantly reduced tumor volume in vivo due to overwhelming apoptosis in both neuroblastoma xenografts. Time-dependently, combination of 4-HPR and GST caused reduction in body weight, tumor weight, and tumor volume. Combination of 4-HPR and GST increased Bax:Bcl-2 ratio, mitochondrial release of Smac, downregulation of baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins including BIRC-2 and BIRC-3, and activation of caspase-3 and apoptosis inducing factor (AIF). Further, downregulation of nuclear factor-kappa B (NF-kappaB), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2) was also detected. In situ immunofluorescent labelings of tumor sections showed overexpression of calpain, caspase-12, and caspase-3, and also AIF in the course of apoptosis. Combination therapy increased apoptosis in the xenografts but did not induce kidney and liver toxicities in the animals. Results demonstrated that combination of 4-HPR and GST induced multiple molecular mechanisms for apoptosis and thus could be highly effective for inhibiting growth of malignant neuroblastoma in preclinical animal models.

The parkinsonian neurotoxin rotenone activates calpain and caspase-3 leading to motoneuron degeneration in spinal cord of Lewis rats.

Exposure to environmental toxins increases the risk of neurodegenerative diseases including Parkinson's disease (PD). Rotenone is a neurotoxin that has been used to induce experimental Parkinsonism in rats. We used the rotenone model of experimental Parkinsonism to explore a novel aspect of extra-nigral degeneration, the neurodegeneration of spinal cord (SC), in PD. Rotenone administration to male Lewis rats caused significant neuronal cell death in cervical and lumbar SC as compared with control animals. Dying neurons were motoneurons as identified by double immunofluorescent labeling for terminal deoxynucleotidyl transferase, recombinant-mediated dUTP nick-end labeling-positive (TUNEL(+)) cells and choline acetyltransferase (ChAT)-immunoreactivity. Neuronal death was accompanied by abundant astrogliosis and microgliosis as evidenced from glial fibrillary acidic protein (GFAP)-immunoreactivity and OX-42-immunoreactivity, respectively, implicating an inflammatory component during neurodegeneration in SC. However, the integrity of the white matter in SC was not affected by rotenone administration as evidenced from the non co-localization of any TUNEL(+) cells with GFAP-immunoreactivity and myelin basic protein (MBP)-immunoreactivity, the selective markers for astrocytes and oligodendrocytes, respectively. Increased activities of 76 kD active m-calpain and 17/19 kD active caspase-3 further demonstrated involvement of these enzymes in cell death in SC. The finding of ChAT(+) cell death also suggested degeneration of SC motoneurons in rotenone-induced experimental Parkinsonism. Thus, this is the first report of its kind in which the selective vulnerability of a putative parkinsonian target outside of nigrostriatal system has been tested using an environmental toxin to understand the pathophysiology of PD. Moreover, rotenone-induced degeneration of SC motoneuron in this model of experimental Parkinsonism progressed with upregulation of calpain and caspase-3.

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane.

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.

Inhibition of calpain and caspase-3 prevented apoptosis and preserved electrophysiological properties of voltage-gated and ligand-gated ion channels in rat primary cortical neurons exposed to glutamate.

Glutamate toxicity in traumatic brain injury, ischemia, and Huntington's disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 microM glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 microM N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 microM N-benzyloxylcarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases.

Estrogen prevents glutamate-induced apoptosis in C6 glioma cells by a receptor-mediated mechanism.

Estrogen-mediated neuroprotection is well established; however, no single mechanism of action for this effect has yet been established. As glial cells are integral for both the intact and injured nervous system, we hypothesized that estrogen-mediated neuroprotection may partly be attributed to attenuation of glial cell apoptosis, allowing them to protect neurons following injury. To assess the protective effects of estrogen on glia, C6 rat glioma cells were treated for 24 h with 500 microM glutamate. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was confirmed by cell morphology and DNA fragmentation. Pretreatment with 10 nM 17beta-estradiol (estrogen) increased cell viability and attenuated apoptosis. Treatment with the stereoisomer 17alpha-estradiol, or estrogen plus estrogen receptor antagonist ICI 182,780, was significantly less effective, indicating that cytoprotection was receptor-mediated. Estrogen treatment upregulated expression of estrogen receptor alpha. Cell impermeable bovine serum albumin-conjugated estrogen was also protective, indicating activation of estrogen receptors on the cell membrane. Intracellular free [Ca2+] was increased after glutamate treatment. This increase was attenuated in cells pretreated with estrogen. Glutamate increased the activity of pro-apoptotic proteases, such as calpain and caspase-3, and these protease activities were significantly attenuated by estrogen. The mechanism by which estrogen decreased intracellular Ca2+ was examined by assaying cell viability after using inhibitors that either blocked extracellular Ca2+ influx or prevented the release of intracellular Ca2+ stores. While several inhibitors increased cell viability in glutamate-treated cells, none were as protective as estrogen, and estrogen co-treatment significantly increased cell viability. These findings indicate that estrogen-mediated cytoprotection may be related to effects on Ca2+ entry but that these effects are not limited to any one of these Ca2+ entry points alone.

Relatively low levels of calpain expression in juvenile rat correlate with less neuronal apoptosis after spinal cord injury.

Approximately 5% of spinal cord injuries in the US occur in patients younger than 16 years. These young patients have an increased mortality within the 24 h after trauma but have a greater capacity for functional recovery than adults, suggesting age-related differences in injury tolerance. Unfortunately, the response of the developing cord to secondary injury has not been thoroughly investigated. Calpain, a Ca(2+)-dependent protease, has been implicated in the pathogenesis of spinal cord injury (SCI) in rats. Our current investigation revealed that following SCI, calpain upregulation was qualitatively less in the 21-day-old rats than in adult rats, as shown by immunofluorescent labeling. Decreased levels of TUNEL+ neurons were also noted in juvenile rat spinal cord, indicating that the developing cord may have an increased resistance to injury.

Molecular mechanisms in the pathogenesis of traumatic brain injury.

Traumatic brain injury (TBI) is a serious neurodisorder commonly caused by car accidents, sports related events or violence. Preventive measures are highly recommended to reduce the risk and number of TBI cases. The primary injury to the brain initiates a secondary injury process that spreads via multiple molecular mechanisms in the pathogenesis of TBI. The events leading to both neurodegeneration and functional recovery after TBI are generalized into four categories: (i) primary injury that disrupts brain tissues; (ii) secondary injury that causes pathophysiology in the brain; (iii) inflammatory response that adds to neurodegeneration; and (iv) repair-regeneration that may contribute to neuronal repair and regeneration to some extent following TBI. Destructive multiple mediators of the secondary injury process ultimately dominate over a few intrinsic protective measures, leading to activation of cysteine proteases such as calpain and caspase-3 that cleave key cellular substrates and cause cell death. Experimental studies in rodent models of TBI suggest that treatment with calpain inhibitors (e.g., AK295, SJA6017) and neurotrophic factors (e.g., NGF, BDNF) can prevent neuronal death and dysfunction in TBI. Currently, there is still no precise therapeutic strategy for the prevention of pathogenesis and neurodegeneration following TBI in humans. The search continues to explore new therapeutic targets and development of promising drugs for the treatment of TBI.

Calpain expression and infiltration of activated T cells in experimental allergic encephalomyelitis over time: increased calpain activity begins with onset of disease.

Calpain activity and expression at the protein level were examined in inflammatory cells, activated microglia, and astrocytes prior to or at onset of symptomatic experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). EAE was induced in Lewis rats by injection of guinea pig spinal cord homogenate and myelin basic protein (MBP) emulsified with Complete Freund's Adjuvant (CFA). Calpain translational expression, determined by Western blot and immunocytochemistry, was correlated with calpain activity, infiltration of inflammatory cells, and myelin loss at 2-11 days following challenge with antigen. Controls (CFA only) did not show any changes over time in these parameters and very few changes (CD11+ microglia/mononuclear phagocytes) were seen in either group from days 2 to 8 post-induction. In contrast, from days 9 to 11, the animals that developed the disease (at least grade 1) demonstrated extensive cellular infiltration (CD4+, CD25+, and CD11+ as well as increased calpain expression (content) and activity. This study demonstrates that cell infiltration and increased calpain activity do not begin in the CNS until the onset of clinical signs.

Inhibition of calpain-mediated apoptosis by E-64 d-reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats.

Upregulation of calpain, a Ca(2+)-activated cysteine protease, has been implicated in apoptosis and tissue degeneration in spinal cord injury (SCI) that over time spreads from the site of injury to the surrounding regions. We examined calpain content and activity, regulation of immediate early genes (IEGs) such as c-jun and c-fos, reactive astrogliosis as the expression of glial fibrillary acidic protein (GFAP), and apoptosis-related features such as caspase-3 mRNA expression and internucleosomal DNA fragmentation in 1-cm long spinal cord segments (S1, distant rostral; S2, adjacent rostral; S3, lesion or injury; S4, adjacent caudal; and S5, distant caudal) following SCI in rats. Calpain content and production of 150 kD calpain-cleaved alpha-fodrin fragment, expression of IEGs, reactive astrogliosis, and apoptotic features were highly increased in the lesion (S3), moderately in adjacent areas (S2 and S4), and slightly in distant areas (S1 and S5) in SCI rats when compared to sham animals. Administration of the calpain-specific inhibitor E-64-d (1 mg/kg) to SCI rats continuously for 24 h inhibited calpain activity and other factors contributing to apoptosis in the lesion and surrounding areas, indicating that calpain played a key role in the pathophysiology of SCI. The results obtained from this animal model of SCI suggest that calpain inhibitor can provide neuroprotection in patients with SCI.

The effects of calpain inhibition upon IL-2 and CD25 expression in human peripheral blood mononuclear cells.

Calcium is an important contributor to T cell activation; it is also the major factor in the activation of the calcium-activated neutral proteinase, calpain. For this reason, we wanted to investigate if calpain has a role in T cell activation and what aspects of this activation calpain affects. As measured by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), calpain inhibition decreased interleukin-2 (IL-2) and CD25 mRNA expression in a dose-dependent manner, at early time points following the initial activation, and over extended periods of time in activated human peripheral blood mononuclear cells (PBMCs). Using an enzyme-linked immuno-sorbent assay (ELISA) specific for human IL-2, we found that calpain inhibition decreased IL-2 secretion in a dose-dependent manner, shortly after activation, and continuously over time. Inhibiting calpain caused a dose-dependent inhibition of CD25 cell surface expression and also inhibited expression shortly after activation and for at least 48 h. This study showed that calpain has an integral role in the synthesis of the two important T cell activation factors, IL-2 and CD25.

Mechanism of myelin breakdown in experimental demyelination: a putative role for calpain.

Although calpain has been extensively studied, its physiological function is poorly understood. In contrast, its role in the pathophysiology of various diseases has been implicated, including that of experimental allergic encephalomyelitis (EAE), an animal model of the demyelinating disease multiple sclerosis (MS). In EAE, calpain degrades myelin proteins, including myelin basic protein (MBP), suggesting a role for calpain in the breakdown of myelin in this disease. Subsequent studies revealed increased calpain activity and expression in the glial and inflammatory cells concomitant with loss of axon and myelin proteins. This suggested a crucial role for calpain in demyelinating diseases.

Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.

Degradation of cytoskeletal proteins by calpain, a Ca(2+)-dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g-cm force) SCI in rats was induced at T12 by a standardized weight-drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24-h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell-permeable calpain inhibitor E-64-d (1 mg/kg). Following 24-h treatment, a 5-cm-long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68-kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E-64-d-treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E-64-d providing neuroprotective benefit.

Inhibition of proteolysis by a cyclooxygenase inhibitor, indomethacin.

The effect of indomethacin, a non-steroidal anti-inflammatory drug upon purified calpain has been studied. Also, its effects upon Ca2+-mediated degradation of cytoskeletal proteins (neurofilament) in spinal cord homogenate has been investigated. A dose-dependent inhibition of purified calpain activity was observed. A 50% inhibition of 14C-caseinolytic activity was obtained with less than 1.1 mM of indomethacin while the activity was completely inhibited at 3.3 mM concentration. The inhibitory effect of ketorlac, another non-steroidal anti-inflammatory drug, upon calpain was weaker than that of indomethacin. The degradation of myelin basic protein (MBP) by cathepsin B, a lysosomal cysteine protease, was significantly inhibited by indomethacin. It also inhibited the Ca2+-mediated degradation of neurofilament protein (NFP) in spinal cord homogenate. The extent of NFP degradation was analyzed by SDS-PAGE and the inhibition shown by indomethacin was weaker than that observed with leupeptin and the calpain inhibitor E64-d. The inhibitory effect of indomethacin on the activity of multicatalytic proteinase complex was negligible. These results suggest that indomethacin, a non-steroidal anti-inflammatory drug and cyclooxygenase inhibitor also inhibits proteinases, including cathepsin B and calpain.

Acute multiple sclerosis characterized by extensive mononuclear phagocyte infiltration.

In this study, we examine the clinical, neuroradiological, and immunohistochemical findings of a 51 year old white female who died 27 months after onset of acute multiple sclerosis despite treatment with interferon-beta, azathioprine, corticosteroids, and cyclophosphamide. Immunohistochemical studies revealed extensive gliosis and mononuclear phagocyte infiltration with corresponding upregulation of proinflammatory cytokines (eg. IFN-alpha, TNF-alpha). The significance of immunohistochemical findings with respect to clinical presentation is discussed.

Increased calpain expression is associated with apoptosis in rat spinal cord injury: calpain inhibitor provides neuroprotection.

Calpain content was investigated in the lesion of rat spinal cord at 1, 4, 24, and 72 h following injury induced by the weight-drop (40 g-cm force) technique. Calpain content was increased in the lesion, and was highest at 24 h following injury. microCalpain mRNA level in the lesion was increased by 58.4% (p = 0.0135) at 24 h following trauma, compared to sham. Alterations in mRNA expression in the lesion increased bax/bcl-2 ratio by 20.8% (p = 0.0395) at this time point, indicating a commitment to apoptosis. Therapeutic effect of the calpain inhibitor E-64-d (1 mg/kg) was studied in SCI rats following administration for 24 h. Internucleosomal DNA fragmentation (apoptosis) was observed in SCI rats, but not in sham or E-64-d treated rats. These results indicate a new information that E-64-d has the therapeutic potential for inhibiting apoptosis in SCI.

Calpain upregulation in spinal cords of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a heroin analogue, is a neurotoxin that undergoes in vivo oxidation by monoamine oxidase-B (MAO-B) to 1-methyl-4-phenylpyridinium ion (MPP+) which preferentially exerts its toxic effects on the dopaminergic neurons of the substantia nigra in brain. Spinal interneuronal pathways are also likely to be affected in the course of MPP+ neurotoxicity. The primary effect of MPP+ is mediated by irreversible inhibition of mitochondrial complex I, releasing free radicals. MPP+ may also activate N-methyl-D-aspartate (NMDA) receptors, increasing the cytosolic concentration of free Ca2+. Intracellular free radicals indirectly and free Ca2+ directly can activate Ca2+-dependent proteases such as calpain. We investigated involvement of calpain in spinal cord degeneration due to neurotoxin by subjecting male C57BL/6N mice (17 months old) to MPTP administration (12.5 mg/kg for 0.5 h; 25 mg/kg for 0.25 h; and 50 mg/kg for 0.25, 0.5, 1, 2, and 24 h). RT-PCR and Western blot analysis were performed using the thoracic segment of spinal cords from control and MPTP-administered mice. The administration of MPTP caused calpain upregulation at the mRNA and protein levels to various extents, compared to control mice. Calpain activity was measured by 68 kDa neurofilament protein (NFP) degradation, which was increased in MPTP-induced PD mice. These results suggest that calpain may play a role in spinal cord degeneration in mice with MPTP-induced PD.

Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease.

Apoptosis is usually associated with genomic DNA fragmentation which can be detected in situ by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. We describe a combined TUNEL and double immunofluorescent labeling technique to determine the fate of inflammatory infiltrates and resident glial cells in the central nervous system following the onset of an autoimmune demyelinating disease such as experimental allergic encephalomyelitis (EAE) in rats. Anti-digoxigenin (anti-DIG) antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) emitting blue fluorescence was used to detect apoptotic cell DNA, which was already labeled by modified TUNEL using alkali-stable DIG-11-dUTP. Anti-mouse IgG secondary antibody conjugated with Texas Red emitting red fluorescence was used to detect anti-rat CD11b primary antibody (clone OX-42) directed to the surface antigen of mononuclear phagocytes including microglia. Using this technique, we detected apoptotic mononuclear phagocytes (co-labeled with blue and red fluorescences) in the spinal cord sections of rats with EAE.