Caspase-11 mediates oligodendrocyte cell death and pathogenesis of autoimmune-mediated demyelination.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS) characterized by localized areas of demyelination. The mechanisms underlying oligodendrocyte (OLG) injury in MS and EAE remain unknown. Here we show that caspase-11 plays crucial roles in OLG death and pathogenesis in EAE. Caspase-11 and activated caspase-3 were both expressed in OLGs in spinal cord EAE lesions. OLGs from caspase-11-deficient mice were highly resistant to the cell death induced by cytotoxic cytokines. EAE susceptibility and cytokine concentrations in the CNS were significantly reduced in caspase-11-deficient mice. Our findings suggest that OLG death is mediated by a pathway that involves caspases-11 and -3 and leads to the demyelination observed in EAE.

Caspases determine the vulnerability of oligodendrocytes in the ischemic brain.

Although oligodendrocytes (OLGs) are thought to be vulnerable to hypoxia and ischemia, little is known about the detailed mechanism by which these insults induce OLG death. From the clinical viewpoint, it is imperative to protect OLGs as well as neurons against ischemic injury (stroke), because they are the only myelin-forming cells of the central nervous system. Using the Cre/loxP system, we have established a transgenic mouse line that selectively expresses p35, a broad-spectrum caspase inhibitor, in OLGs. After hypoxia, cultured OLGs derived from wild-type mice exhibited significant upregulation of caspase-11 and substantial activation of caspase-3, which led to cell loss. Expression of p35 or elimination of caspase-11 suppressed the caspase-3 activation and conferred significant protection against hypoxic injury. Expression of p35 in OLGs in vivo resulted in significant protection from ischemia-induced cell injury, thus indicating that caspases are involved in the ischemia-induced cell death of OLGs. Furthermore, the induction of caspase-11 was evident in the ischemic brains of wild-type mice, and OLGs exhibited resistance to brain ischemia in mice deficient in caspase-11, suggesting that caspase-11 is critically implicated in the mechanism(s) underlying ischemia-induced OLG death. Caspases may therefore offer a good therapeutic target for reducing ischemia-induced damage to OLGs.

Conditional expression of anti-apoptotic protein p35 by Cre-mediated DNA recombination in cardiomyocytes from loxP-p35-transgenic mice.

p35, a viral inhibitor of caspase, prevents cell death induced by various stimuli. We established an experimental system to study the involvement of caspases in cell death, using primary cultured cells from p35 transgenic mice in which the p35 open reading frame (ORF) had been disrupted by the insertion of a DNA segment flanked by loxP sites, the Cre recognition sites. In this system, p35 expression can be initiated by Cre recombinase. Cardiomyocytes, which are highly sensitive to hypoxic stress, were infected with an adenovirus carrying the cre gene (AxCANCre). Expression of p35 by infection with AxCANCre resulted in inhibition of caspase-3 activation and resistance to hypoxia-induced cell death. Hypoxia-induced cytochrome c release was also attenuated in p35-expressing cardiomyocytes. Our transgenic mice can be used as an experimental model for studying the involvement of caspases in various degenerative diseases as well as programmed cell death both in vitro and in vivo.

Role of caspase-1 subfamily in cytotoxic cytokine-induced oligodendrocyte cell death.

Oligodendrocytes are myelin forming cells in mammalian central nervous system. About 50% of oligodendrocytes (OLGs) undergo cell death in normal development. In addition, OLG cell deaths have been observed in demyelinating diseases including multiple sclerosis (MS). Clinical observations and in vitro cell culture studies have suggested that cytokines mediate OLG cell damage in multiple sclerosis (MS). Among the cytokines, tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of OLGs in MS. The administration of TNF-alpha to primary cultures of OLGs induced DNA fragmentation, and significantly decreased the number of live OLGs. Chemical inhibitors Ac-YVAD-CHO (a specific inhibitor of caspase-1 (ICE)-like proteases) enhanced the survival of TNF-alpha treated OLGs better than Ac-DEVD-CHO (a specific inhibitor of caspase-3 (CPP32)-like proteases). These results indicate that caspase-1-mediated cell-death pathway are activated in TNF-induced OLG cell death. Caspase-11 is involved in activation of caspase-1. Oligodendrocytes from caspase-11-deficient mice are partially resistant to TNF-induced OLG cell death. Our results suggest that the inhibition of caspase-1 sufamily may be a novel therapeutic approach to treat MS.

[Two siblings of galactosialidosis with marked progression of cardiac involvement during 10 years].

Two brothers, aged 34 and 30 showed signs of gargoylism, macular cherry-red spots, angiokeratoma, vertebral deformities, epilepsy, action myoclonus and ataxia. In 1985, they were diagnosed as juvenile galactosialidosis due to the decrease in activity of beta-galactosidase and neuraminidase when they had slight valvular involvement with mild heart murmur. On admission in 1994, their echocardiograms revealed marked progression of the cardiac involvement, i.e. thickening of the valvular and left ventricular wall with valvular regurgitation. These findings suggested that accumulation of abnormal materials was increased during the past 10 years. The change in the echocardiographic findings was more severe in the younger brother who had milder physical and neurological manifestations, although severity of neurological symptoms correlated with those of other symptoms in some reports. This study indicates that an evaluation in cardiac involvement may be very important in patients with galactosialidosis, even those with very mild neurological manifestations. Thus echocardiogram is very useful for this purpose.

[Marked clinical improvement by plasmapheresis in a patient with stiff-man syndrome: a case with a negative anti-GAD antibody].

We described a 56-year-old man with stiff-man syndrome, who was markedly improved after plasmapheresis therapy. He had a 12-year history of progressive painful stiffness of his back and limbs, muscle cramps and difficulty in walking. He had been taking oral diazepam and prednisolone. On examination the abdominal and paraspinal muscles and limbs were continuously contracting, confirmed by surface and needle electromyography. Antibodies against glutamic acid decarboxylase (GAD) and pancreatic islet cells in the serum were negative, but antinuclear antibody and anti-smooth-muscle antibody were present. The patient underwent a course of 4 double filtration plasma exchanges of 3,000 ml each in an 8-day period. Plasmapheresis resulted in marked clinical improvement. The disappearance of muscular cramps and a reduction of stiffness occurred within 24 hours after the first plasmapheresis, and he was able to walk unassisted. The patient's subjective improvement continued over 4 months after the plasma exchange. This case provides additional evidence of the autoimmune mechanism of stiff-man syndrome. Plasmapheresis is one choice in the management for stiff-man syndrome.

[Mononeuropathy multiplex in a patient receiving interferon alpha therapy for chronic hepatitis C].

We report a case of mononeuropathy multiplex due to interferon alpha (IFN alpha) therapy for chronic hepatitis C. A 61-year-old man received IFN alpha (natural type 6 x 10(6) IU), every day for two weeks and three times a week for 22 weeks by intramuscular injection. Seventeen weeks after the initiation of IFN alpha therapy, he noticed painful dysesthesia, muscle weakness and muscular atrophy of the left lower extremity. Neurological examination revealed weakness of the left iliopsoas, quadriceps femoris and femoral adductor muscles, hypesthesia and dysesthesia. Electromyogram showed neuropathic change in the left vastus lateralis muscle. Muscle computed tomography showed muscular atrophy of the left lower extremity. Two months after discontinuation of the IFN alpha therapy, dysesthesia and muscle weakness of the left lower extremity improved spontaneously. He was diagnosed as mononeuropathy multiplex of the left femoral nerve and obturator nerve caused by IFN alpha therapy. Attention must be paid to dysesthesia, muscle weakness and muscular atrophy when IFN alpha therapy is initiated.

[A case of unilateral gustatory disturbance produced by the contralateral midbrain lesion].

We reported a case of unilateral gustatory disturbance produced by a lesion in the contralateral midbrain. A 37-year-old man first noticed dysesthesia in the left side of his face. Later, the patient developed dysesthesia of the left part of the lip, muscular weakness on the left lower extremity and gustatory disturbance on the left side. MR scan disclosed a lesion in the tegmentum of the right midbrain, which showed low intensity on a T1-weighted image and high intensity on a T2-weighted image. This lesion could be due to multiple sclerosis. This case suggests that the unilateral gustatory information ascends via the contralateral pathway at the midbrain level. It is suggested that the central gustatory pathways above the ipsilateral pontine taste are ascend via the contralateral projections to the thalamic taste area (parvicellular portion of the ventral posteromedial thalamic nucleus, VPMpc).

Execution mechanisms of programmed cell death by caspase (ICE/CED-3) family proteases.

Molecular genetic study of Caenorhabditis elegans revealed the cell death-executing gene ced-3. Structural and functional similarities between CED-3 and mammalian ICE family (caspase) gene products provide the evidence that mammalian caspases are common mediators in executing programmed cell death. These findings suggest that the execution mechanisms of cell death by caspase family protease may be evolutionarily conserved from worms to mammals. This report summarizes the unique properties of caspases in apoptosis.

ICE/CED-3 family executes oligodendrocyte apoptosis by tumor necrosis factor.

Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes (OLGs) in multiple sclerosis (MS). We report here the involvement of the interleukin 1beta-converting enzyme (ICE)/Caenorhabditis elegans gene ced-3 (CED-3) family in TNF-mediated cell death of OLGs. The addition of TNF-alpha to primary cultures of OLGs that express ice and cpp32 significantly decreased the number of live OLGs in 72 h. DNA fragmentation was detected in TNF-treated OLGs at 36 h with the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene, an inhibitor of the ICE/CED-3 family that shows p35-like inhibitory specificity, protected against the TNF-induced cell death of OLGs. Furthermore, acetyl-YVAD-CHO (a specific inhibitor of ICE-like proteases) as well as acetyl-DEVD-CHO (a specific inhibitor of CPP32-like proteases) enhanced the survival of OLGs treated with TNF-alpha, indicating that ICE- and the CPP32-mediated cell death pathways are activated in TNF-induced OLG cell death. Our results suggest that the inhibition of ICE/CED-3 proteases may be a novel approach to treat neurodegenerative diseases such as MS.

Nerve growth factor protects oligodendrocytes from tumor necrosis factor-alpha-induced injury through Akt-mediated signaling mechanisms.

Tumor necrosis factor-alpha is thought to be one of the most important inflammatory cytokines associated with the demyelinating disease multiple sclerosis. We determined whether neurotrophins could protect oligodendrocytes from tumor necrosis factor-alpha-mediated cytotoxicity. Among the neurotrophins tested, nerve growth factor was most effective at preventing cell death. Nerve growth factor also prevented the tumor necrosis factor-induced loss of mitochondrial membrane potential. Overexpression of constitutively active Akt, a downstream target of phosphatidylinositol 3-kinase, but not of constitutively active MEK, protected oligodendrocytes from tumor necrosis factor-induced injury. Moreover, overexpression of dominant-negative Akt negated the protective effects of nerve growth factor on tumor necrosis factor-mediated oligodendrocyte cytotoxicity. These findings indicate that the Akt pathway is crucial in nerve growth factor-mediated oligodendrocyte protection.

Polymyalgia rheumatica in a patient with acute tubulointerstitial nephritis due to Sjögren's syndrome.

A 68-year-old Japanese woman with polymyalgia rheumatica associated with acute tubulointerstitial nephritis and subclinical Sjögren's syndrome is described. Gallium scintigraphy showed marked accumulation in both kidneys and the salivary glands. Renal biopsy revealed lymphocytic infiltration in the tubulointerstitium. She was treated with intravenous high-dose methylprednisolone followed by oral prednisolone. Her clinical symptoms improved. This is the first report of this particular association.

Caenorhabditis elegans anti-apoptotic gene ced-9 prevents ced-3-induced cell death in Drosophila cells.

ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central roles in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. We demonstrated that the CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protected cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduced the cell death induced by ced-3 in Drosophila SL2 cells. Furthermore, apoptosis of SL2 cells that was induced by a Drosophila cell-death gene, reaper, was shown to be partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death.

Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: implicated mechanisms for caspase activation.

CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis showed that both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially prevented cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.

Targeted expression of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against autoimmune-mediated demyelination.

The mechanisms underlying oligodendrocyte (OLG) loss and the precise roles played by OLG death in human demyelinating diseases such as multiple sclerosis (MS), and in the rodent model of MS, experimental autoimmune encephalomyelitis (EAE), remain to be elucidated. To clarify the involvement of OLG death in EAE, we have generated transgenic mice that express the baculovirus anti-apoptotic protein p35 in OLGs through the Cre-loxP system. OLGs from cre/p35 transgenic mice were resistant to tumor necrosis factor-alpha-, anti-Fas antibody- and interferon-gamma-induced cell death. cre/p35 transgenic mice were resistant to EAE induction by immunization with the myelin oligodendrocyte glycoprotein. The numbers of infiltrating T cells and macrophages/microglia in the EAE lesions were significantly reduced, as were the numbers of apoptotic OLGs expressing the activated form of caspase-3. Thus, inhibition of apoptosis in OLGs by p35 expression alleviated the severity of the neurological manifestations observed in autoimmune demyelinating diseases.