SUR1, newly expressed in astrocytes, mediates neuropathic pain in a mouse model of peripheral nerve injury.

BACKGROUND: Neuropathic pain following peripheral nerve injury (PNI) is linked to neuroinflammation in the spinal cord marked by astrocyte activation and upregulation of interleukin 6 (IL-6), chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 1 (CXCL1), with inhibition of each individually being beneficial in pain models. METHODS: Wild type (WT) mice and mice with global or pGfap-cre- or pGFAP-cre/ERT2-driven Abcc8/SUR1 deletion or global Trpm4 deletion underwent unilateral sciatic nerve cuffing. WT mice received prophylactic (starting on post-operative day [pod]-0) or therapeutic (starting on pod-21) administration of the SUR1 antagonist, glibenclamide (10 µg IP) daily. We measured mechanical and thermal sensitivity using von Frey filaments and an automated Hargreaves method. Spinal cord tissues were evaluated for SUR1-TRPM4, IL-6, CCL2 and CXCL1. RESULTS: Sciatic nerve cuffing in WT mice resulted in pain behaviors (mechanical allodynia, thermal hyperalgesia) and newly upregulated SUR1-TRPM4 in dorsal horn astrocytes. Global and pGfap-cre-driven Abcc8 deletion and global Trpm4 deletion prevented development of pain behaviors. In mice with Abcc8 deletion regulated by pGFAP-cre/ERT2, after pain behaviors were established, delayed silencing of Abcc8 by tamoxifen resulted in gradual improvement over the next 14 days. After PNI, leakage of the blood-spinal barrier allowed entry of glibenclamide into the affected dorsal horn. Daily repeated administration of glibenclamide, both prophylactically and after allodynia was established, prevented or reduced allodynia. The salutary effects of glibenclamide on pain behaviors correlated with reduced expression of IL-6, CCL2 and CXCL1 by dorsal horn astrocytes. CONCLUSION: SUR1-TRPM4 may represent a novel non-addicting target for neuropathic pain.

Intersecting roles of ER stress, mitochondrial dysfunction, autophagy, and calcium homeostasis in HIV-associated neurocognitive disorder.

HIV-associated neurocognitive disorder (HAND) is a collective term describing the spectrum of neurocognitive deficits that arise from HIV infection. Although the introduction to highly active antiretroviral therapy (HAART) has prolonged the lifespan of HIV patients, neurocognitive impairments remain prevalent, as patients are left perpetually with HIV. Currently, physicians face a challenge in treating HAND patients, so a greater understanding of the mechanisms underlying HAND pathology has been a growing focus in HIV research. Recent research has revealed the role disrupted calcium homeostasis in HIV-mediated neurotoxicity. Calcium plays a well-established role in the crosstalk between the mitochondrion and ER as well as in regulating autophagy, and ER stress, mitochondrial dysfunction, and impaired autophagic activity are considered hallmarks in several neurodegenerative and neurocognitive disorders. Therefore, it is paramount that the intricate inter-organelle signaling in relation to calcium homeostasis during HIV infection and the development of HAND is elucidated. This review consolidates current knowledge regarding the neuropathology of neurocognitive disorders and HIV infection with a focus on the underlying role of calcium during ER stress, mitochondrial dysfunction, and autophagy associated with the progression of HAND. The details of this intricate crosstalk during HAND remain relatively unknown; further research in this field can potentially aid in the development of improved therapy for patients suffering from HAND.

Role of the inflammasomes in HIV-associated neuroinflammation and neurocognitive disorders.

HIV associated neurocognitive disorders (HAND) is a unique form of neurological impairment that stems from HIV. This disease and its characteristics can be accredited to incorporation of DNA and mRNA of HIV-1 into the CNS. A proper understanding of the intricacies of HAND and the underlying mechanisms associated with corresponding immune reactions are vital for the potential development of a reliable treatment for HAND. A common phenomenon observed in CNS cells, specifically microglia, that are infected with HAND is inflammation, which is a consequence of the activation of innate immune response due to a variety of stimuli, in this case, being the HIV infection. The CNS based inflammation is mediated by the production of cytokines, chemokines, reactive oxygen species, and secondary messengers, which occurs at CNS glia, endothelial cells and peripherally derived immune cells. Inflammasomes play a significant role with regard to neuroinflammation due to their ability to dictate the activation of various inflammatory responses. Certain stimuli can result in the activation of caspase-1; hence, leading to the processing of interleukin-1ß and interleukin-18 pro-inflammatory cytokines. The processed IL-1ß and IL-18 activate signaling pathways that begin the process of neuroinflammation. Due to the fact that the NLRP3 inflammasome is the most abundant in the CNS, it is the most extensively investigated inflammasome with regard to the nervous system. Due to the importance of neuroinflammation in the evolution of HAND and proliferation of neuroinflammation due to HAND, it can be concluded that there exists a relationship between HAND and inflammasomes. The aim of our review is to consolidate current knowledge of important mechanisms in HAND, specifically related to its relationship with neuroinflammation and inflammasomes to shed light on a possible improved treatment for HAND.

Interplay between ER stress and autophagy: A possible mechanism in multiple sclerosis pathology.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system that results in demyelination, neurodegeneration, and axonal loss. During MS pathology, autoreactive T cells specific for self-antigens migrate the blood-brain-barrier and are responsible for the axonal and neuronal damage. ER stress, a disruption in cellular homeostasis due to the accumulation of misfolded proteins, is a hallmark of MS pathology. In response to the homeostatic imbalance, ER stress activates the unfolded protein response, an intricate system of signaling pathways that aims to restore cellular balance. During the UPR, various autophagy pathways are also activated. Autophagy is a diverse network of regulatory catabolic processes which direct the clearance of damaged and unnecessary organelles and proteins while recycling necessary cellular components. In respect to its role in the health of the immune system, autophagy is critical to the survival and proliferation of T cells. This review consolidates current knowledge and recent literature about ER stress, UPR, and autophagy in MS and implicate their crosstalk as a characteristic feature of MS, potentially aiding in the development of novel therapeutic strategies for MS research.

HIVAN associated tubular pathology with reference to ER stress, mitochondrial changes, and autophagy.

Human immunodeficiency virus associated nephropathy (HIVAN) is a unique form of a renal parenchymal disorder. This disease and its characteristics can be accredited to incorporation of DNA and mRNA of human immunodeficiency virus type 1 into the renal parenchymal cells. A proper understanding of the intricacies of HIVAN and the underlying mechanisms associated with renal function and disorders is vital for the potential development of a reliable treatment for HIVAN. Specifically, the renal tubule segment of the kidney is characterized by its transport capabilities and its ability to reabsorb water and salts into the blood. However, the segment is also known for certain disorders, such as renal tubular epithelial cell infection and microcyst formation, which are also closely linked to HIVAN. Furthermore, certain organelles, like the endoplasmic reticulum (ER), mitochondria, and lysosome, are vital for certain underlying mechanisms in kidney cells. A paradigm of the importance of said organelles can be seen in documented cases of HIVAN where the renal disorder results increased ER stress due to HIV viral propagation. This balance can be restored through the synthesis of secretory proteins, but, in return, the secretion requires more energy; therefore, there is a noticeable increase in mitochondrial stress. The increased ER changes and mitochondrial stress will greatly upregulate the process of autophagy, which involves the cell's lysosomes. In conjunction, we found that ER stress and mitochondrial changes are associated in the Tg26 animal model of HIVAN. The aim of our review is to consolidate current knowledge of important mechanisms in HIVAN, specifically related to the renal tubules' association with ER stress, mitochondrial changes and autophagy. Although the specific regulatory mechanism detailing the cross-talk between the various organelles is unknown in HIVAN, the continued research in this field may potentially shed light on a possible improved treatment for HIVAN.

Glomerular mitochondrial changes in HIV associated renal injury.

HIV-associated nephropathy (HIVAN) is an AIDs-related disease of the kidney. HIVAN is characterized by severe proteinuria, podocyte hyperplasia, collapse, glomerular, and tubulointerstitial damage. HIV-1 transgenic (Tg26) mouse is the most popular model to study the HIV manifestations that develop similar renal presentations as HIVAN. Viral proteins, including Tat, Nef, and Vpr play a significant role in renal cell damage. It has been shown that mitochondrial changes are involved in several kidney diseases, and therefore, mitochondrial dysfunction may be implicated in the pathology of HIVAN. In the present study, we investigated the changes of mitochondrial homeostasis, biogenesis, dynamics, mitophagy, and examined the role of reactive oxygen species (ROS) generation and apoptosis in the Tg26 mouse model. The Tg26 mice showed significant impairment of kidney function, which was accompanied by increased blood urea nitrogen (BUN), creatinine and protein urea level. In addition, histological, western blot and PCR analysis of the Tg26 mice kidneys showed a downregulation of NAMPT, SIRT1, and SIRT3 expressions levels. Furthermore, the kidney of the Tg26 mice showed a downregulation of PGC1α, MFN2, and PARKIN, which are coupled with decrease of mitochondrial biogenesis, imbalance of mitochondrial dynamics, and downregulation of mitophagy, respectively. Furthermore, our results indicate that mitochondrial dysfunction were associated with ER stress, ROS generation and apoptosis. These results strongly suggest that the impaired mitochondrial morphology, homeostasis, and function associated with HIVAN. These findings indicated that a new insight on pathological mechanism associated with mitochondrial changes in HIVAN and a potential therapeutic target.

Renal aquaporin-4 associated pathology in TG-26 mice.

Human immunodeficiency virus-associated nephropathy (HIVAN) is a leading cause of end-stage renal disease in HIV patients, which is characterized by glomerulosclerosis and renal tubular dysfunction. Aquaporin-4 (AQP-4) is a membrane bound water channel protein that plays a distinct role in water reabsorption from renal tubular fluid. It has been proven that failure of AQP-4 insertion into the renal tubular membrane leads to renal dysfunction. However, the role of AQP-4 in HIVAN is unclear. We hypothesize that impaired water reabsorption leads to renal injury in HIVAN, where AQP-4 plays a crucial role. Renal function is assessed by urinary protein and serum blood urea nitrogen (BUN). Kidneys from HIV Transgenic (TG26) mice (HIVAN animal model) were compared to wild type mice by immunostaining, immunoblotting and quantitative RT-PCR. TG26 mice had increased proteinuria and BUN. We found decreased AQP-4 levels in the renal medulla, increased endothelin-1, endothelin receptor A and reduced Sirtuin1 (SIRT-1) levels in TG26 mice. Also, oxidative and endoplasmic reticulum stress was enhanced in kidneys of TG26 mice. We provide the first evidence that AQP-4 is inhibited due to induction of HIV associated stress in the kidneys of TG26 mice which limits water reabsorption in the kidney which may be one of the cause associated with HIVAN, impairing kidney physiology. AQP-4 dysregulation in TG26 mice suggests that similar changes may occur in HIVAN patients. This work may identify new therapeutic targets to be evaluated in HIVAN.

Salutary effects of glibenclamide during the chronic phase of murine experimental autoimmune encephalomyelitis.

BACKGROUND: In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), inflammation is perpetuated by both infiltrating leukocytes and astrocytes. Recent work implicated SUR1-TRPM4 channels, expressed mostly by astrocytes, in murine EAE. We tested the hypothesis that pharmacological inhibition of SUR1 during the chronic phase of EAE would be beneficial. METHODS: EAE was induced in mice using myelin oligodendrocyte glycoprotein (MOG) 35-55. Glibenclamide (10 µg/day) was administered beginning 12 or 24 days later. The effects of treatment were determined by clinical scoring and tissue examination. Drug within EAE lesions was identified using bodipy-glibenclamide. The role of SUR1-TRPM4 in primary astrocytes was characterized using patch clamp and qPCR. Demyelinating lesions from MS patients were studied by immunolabeling and immunoFRET. RESULTS: Administering glibenclamide beginning 24 days after MOG35-55 immunization, well after clinical symptoms had plateaued, improved clinical scores, reduced myelin loss, inflammation (CD45, CD20, CD3, p65), and reactive astrocytosis, improved macrophage phenotype (CD163), and decreased expression of tumor necrosis factor (TNF), B-cell activating factor (BAFF), chemokine (C-C motif) ligand 2 (CCL2) and nitric oxide synthase 2 (NOS2) in lumbar spinal cord white matter. Glibenclamide accumulated within EAE lesions, and had no effect on leukocyte sequestration. In primary astrocyte cultures, activation by TNF plus IFNγ induced de novo expression of SUR1-TRPM4 channels and upregulated Tnf, Baff, Ccl2, and Nos2 mRNA, with glibenclamide blockade of SUR1-TRPM4 reducing these mRNA increases. In demyelinating lesions from MS patients, astrocytes co-expressed SUR1-TRPM4 and BAFF, CCL2, and NOS2. CONCLUSIONS: SUR1-TRPM4 may be a druggable target for disease modification in MS.

Silencing of Abcc8 or inhibition of newly upregulated Sur1-Trpm4 reduce inflammation and disease progression in experimental autoimmune encephalomyelitis.

BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), deletion of transient receptor potential melastatin 4 (Trpm4) and administration of glibenclamide were found to ameliorate disease progression, prompting speculation that glibenclamide acts by directly inhibiting Trpm4. We hypothesized that in EAE, Trpm4 upregulation is accompanied by upregulation of sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which are highly sensitive to glibenclamide, and that Sur1-Trpm4 channels are required for EAE progression. METHODS: EAE was induced in wild-type (WT) and Abcc8-/- mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). Lumbar spinal cords were examined by immunohistochemistry, immuno-Förster resonance energy transfer (immunoFRET), and co-immunoprecipitation for Sur1-Trpm4. WT/EAE mice were administered with the Sur1 inhibitor, glibenclamide, beginning on post-induction day 10. Mice were evaluated for clinical function, inflammatory cells and cytokines, axonal preservation, and white matter damage. RESULTS: Sur1-Trpm4 channels were upregulated in EAE, predominantly in astrocytes. The clinical course and severity of EAE were significantly ameliorated in glibenclamide-treated WT/EAE and in Abcc8-/-/EAE mice. At 30 days, the lumbar spinal cords of glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice showed significantly fewer invading immune cells, including leukocytes (CD45), T cells (CD3), B cells (CD20) and macrophages/microglia (CD11b), and fewer cells expressing pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17). In both glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice, the reduced inflammatory burden correlated with better preservation of myelin, better preservation of axons, and more numerous mature and precursor oligodendrocytes. CONCLUSIONS: Sur-Trpm4 channels are newly upregulated in EAE and may represent a novel target for disease-modifying therapy in multiple sclerosis.

Overexpression of SIRT1 protein in neurons protects against experimental autoimmune encephalomyelitis through activation of multiple SIRT1 targets.

Treatment of experimental autoimmune encephalomyelitis (EAE) with resveratrol, an activator of sirtuin 1 (SIRT1), reduces disease severity. This suggested that activators of SIRT1, a highly conserved NAD-dependent protein deacetylase, might have immune-modulating or neuroprotective therapeutic effects in EAE. Previously, we showed that SIRT1 expression increases in EAE, suggesting that it is an adaptive response. In this study, we investigated the potential function of SIRT1 in regulating EAE using SIRT1-overexpressing mice. The current studies examine potential neuroprotective and immunomodulatory effects of SIRT1 overexpression in chronic EAE induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35-55. SIRT1 suppressed EAE clinical symptoms compared with wild-type EAE mice and prevented or altered the phenotype of inflammation in spinal cords; as a result, demyelination and axonal injury were reduced. Significant neuroprotective effects were observed, with fewer apoptotic cells found in the spinal cords of SIRT1-overexpressing EAE mice associated with increased brain-derived neurotrophic factor and NAD levels. Earlier, we showed that brain-derived neurotrophic factor and NAD play crucial neuroprotective roles in EAE. These results suggest that SIRT1 reduces neuronal loss in this chronic demyelinating disease model and that this is associated with a reduction in inflammation.

Immunomodulatory therapy with glatiramer acetate reduces endoplasmic reticulum stress and mitochondrial dysfunction in experimental autoimmune encephalomyelitis.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are found in lesions of multiple sclerosis (MS) and animal models of MS such as experimental autoimmune encephalomyelitis (EAE), and may contribute to the neuronal loss that underlies permanent impairment. We investigated whether glatiramer acetate (GA) can reduce these changes in the spinal cords of chronic EAE mice by using routine histology, immunostaining, and electron microscopy. EAE spinal cord tissue exhibited increased inflammation, demyelination, mitochondrial dysfunction, ER stress, downregulation of NAD+ dependent pathways, and increased neuronal death. GA reversed these pathological changes, suggesting that immunomodulating therapy can indirectly induce neuroprotective effects in the CNS by mediating ER stress.

Stem cell based delivery of IFN-beta reduces relapses in experimental autoimmune encephalomyelitis.

Interferon-beta (IFN-beta), an approved treatment of multiple sclerosis (MS), produces only partial clinical responses. IFN-beta therapy has been limited by its short serum half-life and limited ability to cross the blood brain barrier. We have developed a means of delivering the IFN-beta gene both systemically and into the central nervous system (CNS) using bone marrow stem cells (BMSCs) as a vehicle and examined the therapeutic efficacy of this approach in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A retroviral expression vector (pLXSN-IFNbeta) was used to stably transfect virus producer PA317 cells to generate retrovirus containing the IFN-beta gene which then was used to transduce BMSCs. IFN-beta engineered BMSCs were transplanted (i.v.) into mice that then were immunized with proteolipoprotein (PLP) to initiate EAE. IFN-beta-engineered BMSCs transplanted mice showed a significant inhibition of EAE onset, and the overall clinical severity was less compared to control groups. IFN-beta delivery strongly reduced infiltration of mononuclear cells possibly by inhibiting cell adhesion molecules. Reduced demyelination and increased remyelination were also observed in the IFN-beta treated group. Furthermore, inhibition of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and IL-12 and enhanced expression of the anti-inflammatory cytokines IL-10, IL-4 and TGF-beta was observed in CNS tissue. In addition, mice receiving IFN-beta had reduced apoptosis and increases in growth promoting factors including BDNF, CNTF, PDGF and VEGF. These results suggest that BMSCs can be used as vehicles to deliver the IFN-beta into the CNS. This is a potentially novel therapeutic approach which might be used in MS and other diseases of the CNS in which drug access is limited.

Brain derived neurotrophic factor treatment reduces inflammation and apoptosis in experimental allergic encephalomyelitis.

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) which includes a neurodegenerative component. Brain derived neurotrophic factor (BDNF) is a neuroprotective agent which might be useful in preventing neurodegeneration but its application has been limited because the blood brain barrier restricts its access to the CNS. We have developed a novel delivery system for BDNF using transformed bone marrow stem cells (BMSC) and undertook studies of EAE to determine whether the delivery of BDNF could reduce inflammation and apoptosis. Mice receiving BDNF producing BMSC had reduced clinical impairment compared to control mice receiving BMSC that did not produce BDNF. Pathological examination of brain and spinal cord showed a reduction in inflammatory infiltrating cells in treated compared to control mice. Apoptosis was reduced in brain and spinal cord based on TUNEL and cleaved Caspase-3 staining. Consistent with the known mechanism of action of BDNF on apoptosis, Bcl-2 and Akt were increased in treated mice. Further studies suggested that these increases could be mediated by inhibition of both caspase dependent and caspase independent pathways. These results suggest that the BDNF delivered by the transformed bone marrow stem cells reduced clinical severity, inflammation and apoptosis in this model.

SIRT1 and NAD+ precursors: Therapeutic targets in multiple sclerosis a review.

Neurodegeneration is an important determinant of disability in multiple sclerosis (MS) but while currently approved treatments reduce inflammation, they have not been shown to reduce neurodegeneration. SIRT1, a NAD dependent protein deacetylase, has been implicated in the pathogenesis of neurodegeneration in neurological diseases including MS. We have studied the role of SIRT1 in experimental autoimmune encephalomyelitis (EAE) and found evidence for a neuroprotective role. In this review we summarize the most recent findings from the use of SIRT1 activators and SIRT1 overexpression in transgenic mice. These data support provide a rational for the use of SIRT1 activators in MS.

TrkB agonist, 7,8-dihydroxyflavone, reduces the clinical and pathological severity of a murine model of multiple sclerosis.

7,8-Dihydroxyflavone (DHF), is a recently described TrkB agonist that readily crosses the blood brain barrier. We treated C57Bl/6 mice with MOG--induced EAE daily with DHF starting on the day of disease induction. Clinical severity of impairment was reduced throughout the course of disease. Pathological examination of brains and spinal cords on day 28 showed that DHF treatment increased the phosphorylation of TrkB and activated downstream signaling pathways including AKT and STAT3 and reduced inflammation, demyelination and axonal loss compared to EAE controls. DHF treatment duplicated the central nervous system effects of brain derived neurotrophic factor in the EAE.

Cell-based delivery of brain-derived neurotrophic factor in experimental allergic encephalomyelitis.

Brain-derived neurotrophic factor (BDNF) is a pleiotropic cytokine with neuroprotective properties that has been identified as a potential therapeutic agent for diseases of the central nervous system (CNS). The use of BDNF has been limited by a short serum half-life and poor penetration of the blood-brain barrier. To address this limitation we have explored cell-based approaches to delivery. We have used experimental allergic encephalomyelitis (EAE), an inflammatory disease of the CNS, as a model system. We engineered hematopoietic stem cells to produce BDNF to determine the feasibility and effectiveness of cell-based delivery of BDNF into the CNS in EAE. We review those studies here.

Use of engineered bone marrow stem cells to deliver brain derived neurotrophic factor under the control of a tetracycline sensitive response element in experimental allergic encephalomyelitis.

Brain derived neurotrophic factor (BDNF) has neuroprotective properties but its use has been limited by poor penetration of the blood brain barrier. Treatment using bone marrow stem cells (BMSC) or retroviruses as vectors reduces the clinical and pathological severity of experimental allergic encephalomyelitis (EAE). We have refined the BMSC based delivery system by introducing a tetracycline sensitive response element to control BDNF expression. We have now tested that construct in EAE and have shown a reduction in both the clinical and pathological severity of the disease. Further, we looked for changes in sirtuin1 and nicotinamide phosphoribosyltransferase expression that would be consistent with a neuroprotective effect.

Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is neuroprotective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the central nervous system (CNS), when delivered peripherally, is limited due to the blood-brain barrier (BBB). We have developed a means of delivering BDNF into the CNS using genetically engineered bone marrow stem cells (BMSCs) as a vehicle, and have explored the clinical effects of BDNF on outcomes in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). BDNF-engineered-BMSCs were transplanted (i.v.) into irradiated 2-week-old SJL/J female mice. Eight weeks after transplantation, mice were immunized with a peptide of proteolipid protein (PLP(139-151)). Mice, which had received BDNFengineered BMSCs, showed a significant delay in EAE onset and a reduction in overall clinical severity compared to mice receiving BMSC transfected with an empty vector lacking the BDNF gene. In addition, pathological examination showed that BDNF delivery reduced demyelination and increased remyelination. Inhibition of pro-inflammatory cytokines TNF-alpha and IFN-gamma and enhanced expression of the antiinflammatory cytokines IL-4, IL-10, and IL-11 were found in the CNS tissues of the BDNF transplanted group. These results support the use of BMSCs as vehicles to deliver BDNF into the CNS of EAE animals. This is a potentially novel therapeutic approach that might be used to deliver BDNF gene or genes for other therapeutic proteins into the CNS in MS or in other diseases of the CNS in which accessibility of therapeutic proteins is limited due to the BBB.