A novel combination approach to effectively reduce inflammation and neurodegeneration in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated attacks on the central nervous system (CNS), resulting in demyelination and recurring T-cell responses. Unfortunately, there is no cure for it. Current therapies that target immunomodulation and/or immunosuppression show only modest beneficial effects, have many side effects, and do not block neurodegeneration or progression of the disease. Since neurodegeneration and in particular axonal degeneration is implicated in disability in progressive MS, development of novel therapeutic strategies to attenuate the neurodegenerative processes is imperative. This study aims to develop new safe and efficacious treatments that address both the inflammatory and neurodegenerative aspects of MS using its animal model, experimental allergic encephalomyelitis (EAE). In EAE, the cysteine protease calpain is upregulated in CNS tissue, and its activity correlates with neurodegeneration. Our immunologic studies on MS have indicated that increased calpain activity promotes pro-inflammatory T helper (Th)1 cells and the severity of the disease in EAE, suggesting that calpain inhibition could be a novel target to combat neurodegeneration in MS/EAE. While calpain inhibition by SNJ1945 reduced disease severity, treatment of EAE animals with a novel protease-resistant altered small peptide ligand (3aza-APL) that mimic myelin basic protein (MBP), also decreased the incidence of EAE, disease severity, infiltration of inflammatory cells, and protected myelin. A reduction in inflammatory T-cells with an increase in Tregs and myeloid suppressor cells is also found in EAE mice treated with SNJ1945 and 3aza-APL. Thus, a novel combination strategy was tested in chronic EAE mouse model in B10 mice which showed multiple pathological mechanisms could be addressed by simultaneous treatment with calpain inhibitor SNJ1945 and protease-resistant 3aza-APL to achieve a stronger therapeutic effect.

A Novel Aza-MBP Altered Peptide Ligand for the Treatment of Experimental Autoimmune Encephalomyelitis.

Myelin basic protein (MBP) is a major target of T cells in lesions of multiple sclerosis (MS) patients and its animal model, experimental autoimmune encephalomyelitis (EAE). Interactions between the major histocompatibility complex II containing antigenic peptides and the T cell receptor activate CD4+ T cells that perpetuate EAE and MS. Previously reported data has shown that treating with an altered peptide ligand (APL) in which the normal antigenic peptide sequence of MBP has been slightly changed at T cell contact positions is helpful in reducing disease in both rodents and humans. The use of natural peptides, which are susceptible to protease degradation, requires high concentrations that can create hypersensitivity reactions. Our hypothesis is that APL containing aza substitutions, CH(R)-N- > N(R)N, could lead to improved protease resistance, reduced clinical disease scores, and a shift in T cell profile. In this study, several aza-APLs and control peptides were synthesized and screened for the best aza-APL candidate (3aza-APL) based on dissociation half time from major histocompatibility complex (MHC) class II, induction of IL-2 response, and resistance to degradation by proteases. The efficacy was then tested in vivo. Results indicate that 3aza-APL is superior to currently available APLs in terms of protease resistance and disease suppression in EAE mice. The 3aza-APL induced anti-inflammatory immune responses by altering key transcription factors and cytokine genes which regulate T cell subpopulations. These data suggest that the novel 3aza-APL has increased protease resistance property and is effective in reducing clinical and physiological signs of disease in EAE animals.

New targets for treating the underlying pathophysiology and nonmotor aspects of Parkinson's disease.

Parkinson's disease is a common neurodegenerative disease diagnosed by well established clinical motor symptoms. However, the disease also encompasses many nonmotor issues that can impact a myriad of processes such as cardiovascular status, gastrointestinal function, autonomic function, mood and sleep. These issues can be more debilitating and impactful on health status in part because of a lack of effective treatments. The pathophysiology of the disease process is under active investigation with postulated mechanisms involving both the central nervous system and the periphery. More in depth examination of the many nonmotor symptoms may aid in the discovery of the overarching pathological origin and progression of Parkinson's disease. Examining the disease process from the perspective of nonmotor symptoms may also provide additional target pathways and potential drug development options not considered previously.

The Involvement of Calpain in CD4+ T Helper Cell Bias in Multple Sclerosis.

The pathogenesis of multiple sclerosis (MS) is mediated by massive infiltration of myelin-specific T cells into the central nervous system (CNS). Self-reactive CD4+ T helper (Th) cells, specifically Th1 and Th17 cells, are hallmarks of active disease in progression, whereas Th2 cells are predominately in remission stages. Calpain has been shown to be upregulated in the CNS of MS patients and inhibition of calpain has been shown previously to decrease disease in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We investigated calpain involvement in Thcell bias. Here, we show that calpain inhibition in primary myelin basic protein (MBP) Ac1-11-specific T cells and MBP-specific T cell line cultures increase Th2 proliferation, cytokine profile, and transcription and signaling molecules. We also show a relative decrease in Th1 inflammatory factors in these same categories and a relative decrease in Th17 proliferation. These studies provide insight into the various roles that calpain plays in Th cell bias and proliferation and increases our understanding of the role that T cells play in the pathophysiology of EAE and MS. Results also indicate the mechanisms involved by which calpain inhibitor decreases the disease signs of EAE, suggesting that calpain inhibitor can be a possible therapeutic agent for the treatment of EAE and MS.

Estrogen or estrogen receptor agonist inhibits lipopolysaccharide induced microglial activation and death.

Inflammation is an important pathogenic mechanism in many neurodegenerative disorders. Activated microglia play a pivotal role in releasing pro-inflammatory factors including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) for inducing inflammation. While microglia mediated inflammation is essential in maintaining CNS homeostasis, chronic inflammation results in activation of proteases for cell death. Here, we examined the effect of PPT (estrogen receptor α agonist), DPN (estrogen receptor ß agonist), and estrogen on rat primary microglia following exposure to lipopolysaccharide (LPS). Exposure of microglia to LPS (200 ng/ml) for 24 h induced cell death. After LPS toxicity for 15 min, microglia were treated with 25 nM PPT, 25 nM DPN, or 100 nM estrogen that prevented cell death by attenuating the release of IL-1α, IL-1ß, TNF-α, and COX-2. Treatment of cells with 100 nM fulvestrant (estrogen receptor antagonist) prior to addition of PPT, DPN, or estrogen significantly decreased their ability to prevent cell death, indicating involvement of estrogen receptor (ER) in providing PPT, DPN, or estrogen mediated cytoprotection. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed alterations in mRNA expression of Bax, Bcl-2, calpain, and calpastatin during apoptosis. We also examined mRNA expression of ERß and ERα following exposure of microglia to LPS and subsequent treatment with PPT, DPN, or estrogen. We found that estrogen or estrogen receptor agonists upregulated expression of ERs. Overall, results indicate that estrogen receptor agonist or estrogen uses a receptor mediated pathway to protect microglia from LPS toxicity.

Calpeptin attenuated inflammation, cell death, and axonal damage in animal model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose dependently with the calpain inhibitor calpeptin (50-250 microg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased, with down-regulation of proapoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of proapoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.

N-(4-Hydroxyphenyl) retinamide potentiated paclitaxel for cell cycle arrest and apoptosis in glioblastoma C6 and RG2 cells.

Glioblastoma grows aggressively due to its ability to maintain abnormally high potentials for cell proliferation. The present study examines the synergistic actions of N-(4-hydroxyphenyl) retinamide (4-HPR) and paclitaxel (PTX) to control the growth of rat glioblastoma C6 and RG2 cell lines. 4-HPR induced astrocytic differentiation that was accompanied by increased expression of the tight junction protein e-cadherin and sustained down regulation of Id2 (member of inhibitor of differentiation family), catalytic subunit of rat telomerase reverse transcriptase (rTERT), and proliferating cell nuclear antigen (PCNA). Flow cytometric analysis showed that the microtubule stabilizer PTX caused cell cycle deregulation due to G2/M arrest. This in turn could alter the fate of kinetochore-spindle tube dynamics thereby halting cell cycle progression. An interesting observation was the induction of G1/S arrest by a combination of 4-HPR and PTX, altering the G2/M arrest induced by PTX alone. This was further ratified by the upregulation of tumor suppressor protein retinoblastoma, which repressed the expression of the key signaling moieties to induce G1/S arrest. Collectively, the combination of 4-HPR and PTX diminished the survival factors (e.g., rTERT, PCNA, and Bcl-2) to make glioblastoma cells highly prone to apoptosis with activation of cysteine proteases (e.g., calpain, cathepsins, caspase-8, caspase-3). Hence, the combination of 4-HPR and PTX can be considered as an effective therapeutic strategy for controlling the growth of heterogeneous glioblastoma cell populations.

Involvement of calpain in the process of Jurkat T cell chemotaxis.

Massive T cell infiltration into the central nervous system is a hallmark of multiple sclerosis (MS) and its rodent model experimental autoimmune encephalomyelitis (EAE), resulting in the induction of many of the pathophysiological events that lead to neuroinflammation and neurodegeneration. Thus, blocking T cell migration into the central nervous system may reduce disease severity in MS and EAE. One potential target for reducing T cell migration is inhibition of the Ca(2+)-activated neutral protease calpain. Previous studies in other cell types have demonstrated that migration is reduced by incubation of cells with calpain inhibitors. Thus, we hypothesize that calpain inhibition will reduce migration of T cells in response to and toward the chemokine CCL2. To test this hypothesis, the intracellular free Ca(2+) levels in Jurkat E6-1 T cells was first measured by the fura-2 assay to assess whether the intracellular ion environment would support calpain activation. The intracellular free Ca(2+) levels were found to increase in response to CCL2. The cells were next treated with the calpain inhibitor calpeptin in a multiwelled Boyden chamber with CCL2 used as the chemoattractant. These studies demonstrate that inhibition of calpain with its inhibitor calpeptin produces a dose-dependent inhibition of chemotaxis. Calpain activity, as measured by live cell imaging, was also increased in response to CCL2, providing further evidence of its involvement in the process of chemotaxis and migration. These studies provide evidence for the involvement of calpain in the mechanisms of chemotaxis and warrants further exploration in MS patient and EAE animal samples.

Extranigral neurodegeneration in Parkinson's disease.

It is widely known that the pathophysiology of idiopathic Parkinson's disease (PD) is associated with neurodegeneration and inflammatory responses in the midbrain substantia nigra. However, the possibility of neurodegeneration and inflammatory responses in other areas of the central nervous system (CNS) in course of the pathogenesis of PD remains to be explored. In this investigation, we provide evidence in support of the hypothesis that spinal cord, the final coordinator of movement, is also involved during parkinsonian degeneration using two distinct experimental parkinsonism models induced by the neurotoxin 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) and the environmental toxin rotenone. A key focus of our study is the role that calpain, a Ca(2+)-activated neutral protease, plays in disrupting the structural-functional integrity of the spinal cord in the context of spinal cord degeneration in experimental parkinsonism. We examined the mechanisms of calpain-mediated neuronal death in differentiated spinal cord motoneuron cultures following exposure to the active parkinsonian toxins 1-methyl-4-phenyl-pyridinium ion (MPP(+)) and rotenone and also tested the neuroprotective efficacy of calpeptin, a calpain inhibitor, in these cell culture models of experimental parkinsonism. Our results implied that spinal cord motoneurons could be a potential extranigral target of neurodegeneration during pathogenesis of PD in the CNS and that calpain inhibition could provide neuroprotection.

Activation of calpain and caspase pathways in demyelination and neurodegeneration in animal model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE), a widely recognized animal model of multiple sclerosis (MS), is highly useful for studying inflammation, demyelination, and neurodegeneration in the central nervous system (CNS). EAE exhibits many similarities with MS, which is a chronic inflammatory disease affecting CNS white matter in humans. Various studies have indicated that EAE is a particularly useful animal model for understanding both the mechanisms of immune-mediated CNS pathology and also the progressive clinical course of MS. Demyelination and axonal dysfunction have previously been shown in MS and EAE but current evidences indicate that axonal damage and neuron death also occur, demonstrating that these diseases harbor a neurodegenerative component. Recent studies also have shown that the activation of calpain and caspase pathways contribute to the apoptotic death of oligodendrocytes and neurons, promoting the pathological events leading to neurological deficits. Apoptosis is involved in the disease-regulating as well as in the disease-promoting processes in EAE. This review discusses the major involvement of calpain and caspase pathways in causing demyelination and neurodegeneration in EAE animals.

Spinal cord degeneration in C57BL/6N mice following induction of experimental parkinsonism with MPTP.

We examined neurodegeneration in spinal cord (SC) and role of such extra-nigral degeneration in MPTP-induced experimental parkinsonism in C57BL/6N mice. HPLC-photodiode array analysis confirmed presence of the active neurotoxin MPP+ in SC after single injection of MPTP (25 mg/kg, i.p.). Mitochondrial enzyme monoamine oxidase-B (MAO-B) responsible for in vivo conversion of MPTP to MPP+ was inhibited in SC by pre-treatment with l-deprenyl, a specific inhibitor of MAO-B. Besides in vitro conversion of MPTP to MPP+ occurred by SC mitochondrial preparation, which was inhibited by l-deprenyl implicating SC as a specific target of MPTP-neurotoxicity. Double immunofluorescent labeling and spectrofluorimetric assay via kynuramine oxidation showed MAO-B expression and activity in SC neurons. Localization of dopamine transporter immunoreactivity in SC along with specific uptake of (3)H-MPP+ by SC synaptosomal preparation further confirmed SC as target of MPTP-neurotoxicity. Compared with control, increased neuronal death on the seventh day in SC of mice injected with MPTP (2 x 25 mg/kg, at 6 h interval) strongly suggested SC degeneration in pre-symptomatic phase of MPTP-induced experimental parkinsonism. Such extra-nigral neurodegeneration in Parkinson's disease indicated novel molecular mechanism preceding nigrostriatal degeneration and suggested designing broad therapeutic intervention for this complex movement disorder.