Mitsuyoshi Azuma

Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiple sclerosis.

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin‐specific T cells into the CNS. Hallmarks of T helper (Th) cells during active disease are pro‐inflammatory Th1/Th17 cells that predominate over immunoregulatory Th2/Treg cells. Neurodegeneration, a major factor in progressive MS, is often overlooked when considering drug prescription. Here, we show that oral dosing with SNJ‐1945, a novel water‐soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis clinical scores in vivo and has a two pronged effect via anti‐inflammation and protection against neurodegeneration. We also show that SNJ‐1945 treatment down‐regulates Th1/Th17 inflammatory responses, and promotes regulatory T cells (Tregs) and myeloid‐derived suppressor cells in vivo, which are known to have the capacity to suppress helper as well as cytotoxic T cell functions. Through analysis of spinal cord samples, we show a reduction in calpain expression, decreased infiltration of inflammatory cells, and signs of inhibition of neurodegeneration. We also show a marked reduction in neuronal cell death in spinal cord (SC) sections. These results suggest that calpain inhibition attenuates experimental autoimmune encephalomyelitis pathology by reducing both inflammation and neurodegeneration, and could be used in clinical settings to augment the efficacy of standard immunomodulatory agents used to treat MS.

SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP+ and rotenone

Complex pathophysiology of Parkinsons disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinsons disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. To unravel these mechanisms SH‐SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes, respectively, and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP+ and rotenone. SNJ‐1945, a cell‐permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP+ and rotenone dose‐dependently elevated the levels of intracellular free Ca2+ and induced a concomitant rise in the levels of active calpain. SNJ‐1945 pre‐treatment significantly protected cell viability and preserved cellular morphology following MPP+ and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species more profoundly in SH‐SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ‐1945 pre‐treatment. In contrast, significant levels of inflammatory mediators cyclooxygenase‐2 (Cox‐2 and cleaved p10 fragment of caspase‐1) were up‐regulated in the cholinergic phenotype, which could be dose‐dependently attenuated by the calpain inhibitor. Overall, SNJ‐1945 was efficacious against MPP+ or rotenone‐induced reactive oxygen species generation, inflammatory mediators, and proteolysis. A post‐treatment regimen of SNJ‐1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1–3 h after exposure to MPP+ or rotenone. Taken together, these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ‐1945 is an efficacious calpain inhibitor in this context.

Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells

Multiple sclerosis (MS) is a T‐cell mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)‐specific T cells into naïve female SJL/J mice results in a relapsing–remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP‐specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP‐specific T cells would diminish their encephalitogenicity in RR‐EAE mice was tested. Incubating MBP‐specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR‐EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain : calpastatin ratio, production of truncated Bid, and Bax : Bcl‐2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS.

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, Parkinsons disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Calpain inhibition reduces structural and functional impairment of retinal ganglion cells in experimental optic neuritis

Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis. ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells, and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrificing (Day 26). Visual function was determined by electroretinogram recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias toward unilateral OKR impairment among EAE‐ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern electroretinogram amplitudes, and paralysis, with attenuation of retinal ganglion cell death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE‐ON parallels the clinical presentation of ON exacerbations associated with multiple sclerosis. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON.