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. As in multiple sclerosis (MS) patients, optic neuritis (ON) and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE, experimental autoimmune encephalomyelitis). Daily oral treatment with the calpain inhibitor SNJ 1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a, brain-specific homeobox/POU domain protein 3A) and their axons (MOSP, myelin oligodendrocyte-specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in ON.

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.

Deletion of 16q24.1 supports a role for the ATP2C2 gene in specific language impairment.

A 10-year-old boy presented with a history of significant delay in language acquisition as well as receptive and expressive language impairment that persisted into elementary school. In school, he exhibited difficulty with reading comprehension, telling and understanding narratives, and making inferences. Other aspects of his neurodevelopment were normal, with no history of significant medical concerns. He did not have hearing impairment, oromotor dysfunction, or specific neurologic abnormalities. He did not meet testing criteria for autism. Chromosomal microarray analysis and quantitative polymerase chain reaction determined that he had a de novo 159-kilobase deletion of chromosome 16q24.1 that included the ATP2C2 gene. ATP2C2 is a known candidate gene for specific language impairment and is postulated to have neurobiological significance in memory-related circuits. Our patient's language deficits were consistent with a global type of specific language impairment impacting language comprehension, formulation, semantics, syntax, and phonology attributed to his de novo chromosome deletion.

Regulation of Th1/Th17 cytokines and IDO gene expression by inhibition of calpain in PBMCs from MS patients.

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin-specific T cells into the central nervous system (CNS). During active disease, pro-inflammatory Th1/Th17 cells predominate over immunoregulatory Th2/Treg cells. Here, we show that calpain inhibition downregulates Th1/Th17 inflammatory cytokines and mRNA in MS patient peripheral blood mononuclear cells (PBMCs) activated with anti-CD3/28 or MBP. Interestingly, calpain inhibition elevated IDO gene expression in MS PBMCs, which was markedly decreased in calpain expressing cells. Functional assay showed that incubation of MS patient PBMCs with calpain inhibitor or recombinant IDO attenuates T cell proliferation. These results suggest that calpain inhibition may attenuate MS pathology and augment the efficacy of standard immunomodulatory agents used to treat this disease.

Calpain inhibition attenuates apoptosis of retinal ganglion cells in acute optic neuritis.

PURPOSE: Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with the pathogenesis of multiple sclerosis (MS) and is initiated by the attack of autoreactive T cells against self-myelin antigens, resulting in demyelination, degeneration of retinal ganglion cells (RGCs), and cumulative visual impairment. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats on day 0, and animals received daily intraperitoneal injections of calpain inhibitor (calpeptin) or vehicle from day 1 until killed. Retinal cell death was analyzed by DNA fragmentation, and surviving ganglion cells were quantified after double labeling of retinal tissue with TUNEL and Brn3a. The expression of apoptotic and inflammatory proteins was determined by Western blotting. RESULTS: It was demonstrated that calpain inhibition downregulates expression of proapoptotic proteins and the proinflammatory molecule nuclear factor-kappa B (NF-κB) in the retina of Lewis rats with acute EAE. Immunofluorescent labeling revealed that apoptotic cells in the RGC layer of vehicle-treated EAE animals were Brn3a positive, and a moderate dose of calpeptin dramatically reduced the frequency of apoptotic RGCs. CONCLUSIONS: These results suggest that calpain inhibition might be a useful supplement to immunomodulatory therapies such as corticosteroids in ON, due to its neuroprotective effect on RGCs.