Inhibition of Calpain Attenuates Degeneration of Substantia Nigra Neurons in the Rotenone Rat Model of Parkinson's Disease.

In the central nervous system (CNS), calcium homeostasis is a critical determinant of neuronal survival. Calpain, a calcium-dependent neutral protease, is widely expressed in the brain, including substantia nigra (SN) dopaminergic (DA) neurons. Though calpain is implicated in human Parkinson's disease (PD) and corresponding animal models, the roles of specific ubiquitous calpain isoforms in PD, calpain-1 and calpain-2, remain poorly understood. In this study, we found that both isoforms are activated in a nigrostriatal pathway with increased phosphorylated synuclein following the administration of rotenone in Lewis rats, but calpain isoforms played different roles in neuronal survival. Although increased expression of calpain-1 and calpain-2 were detected in the SN of rotenone-administered rats, calpain-1 expression was not altered significantly after treatment with calpain inhibitor (calpeptin); this correlated with neuronal survival. By contrast, increased calpain-2 expression in the SN of rotenone rats correlated with neuronal death, and calpeptin treatment significantly attenuated calpain-2 and neuronal death. Calpain inhibition by calpeptin prevented glial (astroglia/microglia) activation in rotenone-treated rats in vivo, promoted M2-type microglia, and protected neurons. These data suggest that enhanced expression of calpain-1 and calpain-2 in PD models differentially affects glial activation and neuronal survival; thus, the attenuation of calpain-2 may be important in reducing SN neuronal loss in PD.

Premarin Reduces Neurodegeneration and Promotes Improvement of Function in an Animal Model of Spinal Cord Injury.

Spinal cord injury (SCI) causes significant mortality and morbidity. Currently, no FDA-approved pharmacotherapy is available for treating SCI. Previously, low doses of estrogen (17ß-estradiol, E2) were shown to improve the post-injury outcome in a rat SCI model. However, the range of associated side effects makes advocating its therapeutic use difficult. Therefore, this study aimed at investigating the therapeutic efficacy of Premarin (PRM) in SCI. PRM is an FDA-approved E2 (10%) formulation, which is used for hormone replacement therapy with minimal risk of serious side effects. The effects of PRM on SCI were examined by magnetic resonance imaging, immunofluorescent staining, and western blot analysis in a rat model. SCI animals treated with vehicle alone, PRM, E2 receptor antagonist (ICI), or PRM + ICI were graded in a blinded way for locomotor function by using the Basso-Beattie-Bresnahan (BBB) locomotor scale. PRM treatment for 7 days decreased post-SCI lesion volume and attenuated neuronal cell death, inflammation, and axonal damage. PRM also altered the balance of pro- and anti-apoptotic proteins in favor of cell survival and improved angiogenesis and microvascular growth. Increased expression of estrogen receptors (ERs) ERα and ERß following PRM treatment and their inhibition by ER inhibitor indicated that the neuroprotection associated with PRM treatment might be E2-receptor mediated. The attenuation of glial activation with decreased inflammation and cell death, and increased angiogenesis by PRM led to improved functional outcome as determined by the BBB locomotor scale. These results suggest that PRM treatment has significant therapeutic implications for the improvement of post-SCI outcome.

Protective Effects of Estrogen via Nanoparticle Delivery to Attenuate Myelin Loss and Neuronal Death after Spinal Cord Injury.

Spinal cord injury (SCI) is associated with devastating neurological deficits affecting more than 11,000 Americans each year. Although several therapeutic agents have been proposed and tested, no FDA-approved pharmacotherapy is available for SCI treatment. We have recently demonstrated that estrogen (E2) acts as an antioxidant and anti-inflammatory agent, attenuating gliosis in SCI. We have also demonstrated that nanoparticle-mediated focal delivery of E2 to the injured spinal cord decreases lesion size, reactive gliosis, and glial scar formation. The current study tested in vitro effects of E2 on reactive oxygen species (ROS) and calpain activity in microglia, astroglia, macrophages, and fibroblasts, which are believed to participate in the inflammatory events and glial scar formation after SCI. E2 treatment decreased ROS production and calpain activity in these glial cells, macrophages, and fibroblast cells in vitro. This study also tested the efficacy of fast- and slow-release nanoparticle-E2 constructs in a rat model of SCI. Focal delivery of E2 via nanoparticles increased tissue distribution of E2 over time, attenuated cell death, and improved myelin preservation in injured spinal cord. Specifically, the fast-release nanoparticle-E2 construct reduced the Bax/Bcl-2 ratio in injured spinal cord tissues, and the slow-release nanoparticle-E2 construct prevented gliosis and penumbral demyelination distal to the lesion site. These data suggest this novel E2 delivery strategy to the lesion site may decrease inflammation and improve functional outcomes following SCI.

The Pathophysiology of Osteoporosis after Spinal Cord Injury.

Spinal cord injury (SCI) affects approximately 300,000 people in the United States. Most individuals who sustain severe SCI also develop subsequent osteoporosis. However, beyond immobilization-related lack of long bone loading, multiple mechanisms of SCI-related bone density loss are incompletely understood. Recent findings suggest neuronal impairment and disability may lead to an upregulation of receptor activator of nuclear factor-κB ligand (RANKL), which promotes bone resorption. Disruption of Wnt signaling and dysregulation of RANKL may also contribute to the pathogenesis of SCI-related osteoporosis. Estrogenic effects may protect bones from resorption by decreasing the upregulation of RANKL. This review will discuss the current proposed physiological and cellular mechanisms explaining osteoporosis associated with SCI. In addition, we will discuss emerging pharmacological and physiological treatment strategies, including the promising effects of estrogen on cellular protection.

Cellular and molecular pathophysiology in the progression of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder etiologically linked to the loss of substantia nigra (SN) dopaminergic neurons in the mid-brain. The etiopathology of sporadic PD is still unclear; however, the interaction of extrinsic and intrinsic factors may play a critical role in the onset and progression of the disease. Studies in animal models and human post-mortem tissue have identified distinct cellular and molecular changes in the diseased brain, suggesting complex interactions between different glial cell types and various molecular pathways. Small changes in the expression of specific genes in a single pathway or cell type possibly influence others at the cellular and system levels. These molecular and cellular signatures like neuroinflammation, oxidative stress, and autophagy have been observed in PD patients' brain tissue. While the etiopathology of PD is still poorly understood, the interplay between glial cells and molecular events may play a crucial role in disease onset and progression.

Nanoparticle-Based Estrogen Delivery to Spinal Cord Injury Site Reduces Local Parenchymal Destruction and Improves Functional Recovery.

Spinal cord injury (SCI) patients sustain significant functional impairments; this is causally related to restricted neuronal regeneration after injury. The ensuing reactive gliosis, inflammatory cascade, and glial scar formation impede axonal regrowth. Although systemic anti-inflammatory agents (steroids) have been previously administered to counteract this, no current therapeutic is approved for post-injury neuronal regeneration, in part because of related side effects. Likewise, therapeutic systemic estrogen levels exhibit neuroprotective properties, but dose-dependent side effects are prohibitive. The current study thus uses low-dose estrogen delivery to the spinal cord injury (SCI) site using an agarose gel patch embedded with estrogen-loaded nanoparticles. Compared to controls, spinal cords from rodents treated with nanoparticle site-directed estrogen demonstrated significantly decreased post-injury lesion size, reactive gliosis, and glial scar formation. However, axonal regeneration, vascular endothelial growth factor production, and glial-cell-derived neurotrophic factor levels were increased with estrogen administration. Concomitantly improved locomotor and bladder functional recovery were observed with estrogen administration after injury. Therefore, low-dose site-directed estrogen may provide a future approach for enhanced neuronal repair and functional recovery in SCI patients.

Pathophysiology, Biomarkers, and Therapeutic Modalities Associated with Skeletal Muscle Loss Following Spinal Cord Injury.

A spinal cord injury (SCI) may lead to loss of strength, sensation, locomotion and other body functions distal to the lesion site. Individuals with SCI also develop secondary conditions due to the lack of skeletal muscle activity. As SCI case numbers increase, recent studies have attempted to determine the best options to salvage affected musculature before it is lost. These approaches include pharmacotherapeutic options, immunosuppressants, physical activity or a combination thereof. Associated biomarkers are increasingly used to determine if these treatments aid in the protection and reconstruction of affected musculature.

Enolase inhibition alters metabolic hormones and inflammatory factors to promote neuroprotection in spinal cord injury.

Enolase inhibition is a potential therapeutic strategy currently being investigated for treatment of spinal cord injury (SCI) as it reduces pro-inflammatory cytokines and chemokines, alters metabolic factors, and reduces gliosis in acute SCI. Herein, the role of enolase in SCI has been examined to better understand the effects of this enzyme on inflammation, metabolic hormones, glial cell activation, and neuroprotection under these shorter injury conditions. Immunohistochemical analyses of inflammatory markers vimentin, Cox-2, and caspase-1 indicated that enolase inhibition attenuated the elevated levels of inflammation seen following SCI. Iba1, GFAP, NFP, and CSPG staining indicated that enolase inhibition with prolonged administration of ENOblock reduced microglia/astrocyte activation and lead to enhanced neuroprotection in SCI. An analysis of metabolic hormones revealed that ENOblock treatment significantly upregulated plasma concentrations of peptide YY, glucagon-like peptide 1, glucose-dependent insulinotropic peptide, glucagon, and insulin hormones as compared to vehicle-treated controls (Mann-Whitney, p ≤ 0.05). ENOblock did not have a significant effect on plasma concentrations of pancreatic polypeptide. Interestingly, ENOblock treatment inhibited chondroitin sulfate proteoglycan (CSPG), which is produced by activated glia and serves to block regrowth of axons across the lesion site following injury. An increased level of NeuN and MBP with reduced caspase-1 was detected in SCI tissues after ENOblock treatment, suggesting preservation of myelin and induction of neuroprotection. ENOblock also induced improved motor function in SCI rats, indicating a role for enolase in modulating inflammatory and metabolic factors in SCI with important implications for clinical consideration.

Calpain mediated expansion of CD4+ cytotoxic T cells in rodent models of Parkinson's disease.

Parkinson's disease (PD), a debilitating progressive degenerative movement disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra (SN), afflicts approximately one million people in the U.S., including a significant number of Veterans. Disease characteristics include tremor, rigidity, postural instability, bradykinesia, and at a cellular level, glial cell activation and Lewy body inclusions in DA neurons. The most potent medical/surgical treatments do not ultimately prevent disease progression. Therefore, new therapies must be developed to halt progression of the disease. While the mechanisms of the degenerative process in PD remain elusive, chronic inflammation, a common factor in many neurodegenerative diseases, has been implicated with associated accumulation of toxic aggregated α-synuclein in neurons. Calpain, a calcium-activated cysteine neutral protease, plays a pivotal role in SN and spinal cord degeneration in PD via its role in α-synuclein aggregation, activation/migration of microglia and T cells, and upregulation of inflammatory processes. Here we report an increased expression of a subset of CD4+ T cells in rodent models of PD, including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mice and DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride]/6-hydroxydopamine rats, which produced higher levels of perforin and granzyme B - typically found in cytotoxic T cells. Importantly, the CD4+ cytotoxic subtype was attenuated following calpain inhibition in MPTP mice, suggesting that calpain and this distinct CD4+ T cell subset may have critical roles in the inflammatory process, disease progression, and neurodegeneration in PD.

Cytokine/chemokine dysregulation in progressive MS patient is apparent and can be modulated by calpain inhibition.

This study examines the cytokine/chemokine profile of a 62-year-old African American male with progressive multiple sclerosis (MS). MRI images of the MS patient demonstrated generalized white matter involvement with multiple lesions in the periventricular area. A 42-plex Discovery Assay® (Eve Technologies) of the patient's plasma and peripheral blood mononuclear cells (PBMCs) supernatant or PBMC-derived T cell supernatant samples from two separate clinic visits revealed vastly differing cytokine/chemokine levels. In addition, certain cytokine/chemokine profiles had notable differences when compared to the larger patient group or patients' PBMCs treated with a calpain inhibitor in vitro. Interestingly, large numbers of cytokines/chemokines and growth factors in MS PBMCs are modulated by calpain inhibition, suggesting the clinical significance of these findings in designing better therapeutics against progressive MS.

Distinct Cytokine and Chemokine Expression in Plasma and Calpeptin-Treated PBMCs of a Relapsing-Remitting Multiple Sclerosis Patient: A Case Report.

The cytokine/chemokine expression signature of a 60-year-old African American male with relapsing-remitting multiple sclerosis (RRMS) was analyzed using patient blood samples obtained from two separate visits to the clinic. Thirty-six different cytokines, chemokines, and growth factors were detected in the plasma of the RRMS patient using a multiplexed bead-based immunoassay. Results indicated that at least ten of these factors with a concentration of > 100 pg/mL are identified as pro-inflammatory. Calpain inhibition led to an anti-inflammatory effect, as indicated by a decrease in expression of pro-inflammatory cytokines/chemokines such as GM-CSF, IFNγ, and IL-17A, and a relative increase in two of the anti-inflammatory cytokines (IL-13 and IL-4) in the peripheral blood mononuclear cells activated with anti-CD3/CD28. Overall, these results suggest that the unique cytokine/chemokine pattern observed in the plasma of the RRMS patient can be used as a prognostic marker and calpain inhibition may be used as a novel therapeutic strategy for treating excessive inflammatory response specific to RRMS patients.

New Insights into the Role of Neuron-Specific Enolase in Neuro-Inflammation, Neurodegeneration, and Neuroprotection.

Neurodegeneration is a complex process that leads to irreversible neuronal damage and death in spinal cord injury (SCI) and various neurodegenerative diseases, which are serious, debilitating conditions. Despite exhaustive research, the cause of neuronal damage in these degenerative disorders is not completely understood. Elevation of cell surface α-enolase activates various inflammatory pathways, including the production of pro-inflammatory cytokines, chemokines, and some growth factors that are detrimental to neuronal cells. While α-enolase is present in all neurological tissues, it can also be converted to neuron specific enolase (NSE). NSE is a glycolytic enzyme found in neuronal and neuroendocrine tissues that may play a dual role in promoting both neuroinflammation and neuroprotection in SCI and other neurodegenerative events. Elevated NSE can promote ECM degradation, inflammatory glial cell proliferation, and actin remodeling, thereby affecting migration of activated macrophages and microglia to the injury site and promoting neuronal cell death. Thus, NSE could be a reliable, quantitative, and specific marker of neuronal injury. Depending on the injury, disease, and microenvironment, NSE may also show neurotrophic function as it controls neuronal survival, differentiation, and neurite regeneration via activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. This review discusses possible implications of NSE expression and activity in neuroinflammation, neurodegeneration, and neuroprotection in SCI and various neurodegenerative diseases for prognostic and therapeutic potential.

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague-Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.

Administration of low dose estrogen attenuates persistent inflammation, promotes angiogenesis, and improves locomotor function following chronic spinal cord injury in rats.

Spinal cord injury (SCI) causes loss of neurological function and, depending upon the severity of injury, may lead to paralysis. Currently, no FDA-approved pharmacotherapy is available for SCI. High-dose methylprednisolone is widely used, but this treatment is controversial. We have previously shown that low doses of estrogen reduces inflammation, attenuates cell death, and protects axon and myelin in SCI rats, but its effectiveness in recovery of function is not known. Therefore, the goal of this study was to investigate whether low doses of estrogen in post-SCI would reduce inflammation, protect cells and axons, and improve locomotor function during the chronic phase of injury. Injury (40 g.cm force) was induced at thoracic 10 in young adult male rats. Rats were treated with 10 or 100 µg 17ß-estradiol (estrogen) for 7 days following SCI and compared with vehicle-treated injury and laminectomy (sham) controls. Histology (H&E staining), immunohistofluorescence, Doppler laser technique, and Western blotting were used to monitor tissue integrity, gliosis, blood flow, angiogenesis, the expression of angiogenic factors, axonal degeneration, and locomotor function (Basso, Beattie, and Bresnahan rating) following injury. To assess the progression of recovery, rats were sacrificed at 7, 14, or 42 days post injury. A reduction in glial reactivity, attenuation of axonal and myelin damage, protection of cells, increased expression of angiogenic factors and microvessel growth, and improved locomotor function were found following estrogen treatment compared with vehicle-treated SCI rats. These results suggest that treatment with a very low dose of estrogen has significant therapeutic implications for the improvement of locomotor function in chronic SCI. Experimental studies with low dose estrogen therapy in chronic spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes that could ameliorate the degenerative pathways in chronic SCI as shown in (a). Furthermore, the alterations in local spinal blood flow could be significantly alleviated with low dose estrogen therapy. This therapy led to the preservation of the structural integrity of the spinal cord (b), which in turn led to the improved functional recovery as shown (c).

Administration of low dose estrogen attenuates gliosis and protects neurons in acute spinal cord injury in rats.

Spinal cord injury (SCI) is a debilitating condition with neurological deficits and loss of motor function that, depending on the severity, may lead to paralysis. The only treatment currently available is methylprednisolone, which is widely used and renders limited efficacy in SCI. Therefore, other therapeutic agents must be developed. The neuroprotective efficacy of estrogen in SCI was studied with a pre-clinical and pro-translational perspective. Acute SCI was induced in rats that were treated with low doses of estrogen (1, 5, 10, or 100 µg/kg) and compared with vehicle-treated injured rats or laminectomy control (sham) rats at 48 h post-SCI. Changes in gliosis and other pro-inflammatory responses, expression and activity of proteolytic enzymes (e.g., calpain, caspase-3), apoptosis of neurons in SCI, and cell death were monitored via Western blotting and immunohistochemistry. Negligible pro-inflammatory responses or proteolytic events and very low levels of neuronal death were found in sham rats. In contrast, vehicle-treated SCI rats showed profound pro-inflammatory responses with reactive gliosis, elevated expression and activity of calpain and caspase-3, elevated Bax:Bcl-2 ratio, and high levels of neuronal death in lesion and caudal regions of the injured spinal cord. Estrogen treatment at each dose reduced pro-inflammatory and proteolytic activities and protected neurons in the caudal penumbra in acute SCI. Estrogen treatment at 10 µg was found to be as effective as 100 µg in ameliorating the above parameters in injured animals. Results from this investigation indicated that estrogen at a low dose could be a promising therapeutic agent for treating acute SCI. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript.

Calpain inhibitor attenuated optic nerve damage in acute optic neuritis in rats.

Optic neuritis (ON), which is an acute inflammatory autoimmune demyelinating disease of the central nervous system (CNS), often occurs in multiple sclerosis (MS). ON is an early diagnostic sign in most MS patients caused by damage to the optic nerve leading to visual dysfunction. Various features of both MS and ON can be studied following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in Lewis rats. Inflammation and cell death in the optic nerve, with subsequent damage to the retinal ganglion cells in the retina, are thought to correlate with visual dysfunction. Thus, characterizing the pathophysiological changes that lead to visual dysfunction in EAE animals may help develop novel targets for therapeutic intervention. We treated EAE animals with and without the calpain inhibitor calpeptin (CP). Our studies demonstrated that the Ca(2+)-activated neutral protease calpain was upregulated in the optic nerve following induction of EAE at the onset of clinical signs (OCS) of the disease, and these changes were attenuated following treatment with CP. These reductions correlated with decreases in inflammation (cytokines, iNOS, COX-2, and NF-κB), and microgliosis (i.e. activated microglia). We observed that calpain inhibition reduced astrogliosis (reactive astroglia) and expression of aquaporin 4 (AQP4). The balance of Th1/Th2 cytokine production and also expression of the Th1-related CCR5 and CXCR3 chemokine receptors influence many pathological processes and play both causative and protective roles in neuron damage. Our data indicated that CP suppressed cytokine imbalances. Also, Bax:Bcl-2 ratio, production of tBid, PARP-1, expression and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated after treatment with CP. Our results demonstrated that CP decreased demyelination [loss of myelin basic protein (MBP)] and axonal damage [increase in dephosphorylated neurofilament protein (de-NFP)], and also promoted intracellular neuroprotective pathways in optic nerve in EAE rats. Thus, these data suggest that calpain is involved in inflammatory as well as in neurodegenerative aspects of the disease and may be a promising target for treating ON in EAE and MS.

Low dose estrogen prevents neuronal degeneration and microglial reactivity in an acute model of spinal cord injury: effect of dosing, route of administration, and therapy delay.

Spinal cord injury (SCI), depending on the severity of injury, leads to neurological dysfunction and paralysis. Methylprednisolone, the only currently available therapy renders limited protection in SCI. Therefore, other therapeutic agents must be tested to maximize neuroprotection and functional recovery. Previous data from our laboratory indicate that estrogen (17ß-estradiol) at a high dose may attenuate multiple damaging pathways involved in SCI and improve locomotor outcome. Since use of high dose estrogen may have detrimental side effects and therefore may never be used in the clinic, the current study investigated the efficacy of this steroid hormone at very low doses in SCI. In particular, we tested the impact of dosing (1-10 µg/kg), mode of delivery (intravenous vs. osmotic pump), and delay in estrogen application (15 min-4 h post-SCI) on microgliosis and neuronal death in acute SCI in rats. Treatment with 17ß-estradiol (1-10 µg/kg) significantly reduced microglial activation and also attenuated apoptosis of neurons compared to untreated SCI animals. The attenuation of cell death and inflammation by estrogen was observed regardless of mode and time of delivery following injury. These findings suggest estrogen as a potential agent for the treatment of individuals with SCI.