Saurav Brahmachari

Induction of Glial Fibrillary Acidic Protein Expression in Astrocytes by Nitric Oxide

Increased expression of glial fibrillary acidic protein (GFAP) represents astroglial activation and gliosis during neurodegeneration. However, the molecular mechanism behind increased expression of GFAP in astrocytes is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of GFAP. Bacterial lipopolysachharides (LPSs) induced the production of NO and the expression of GFAP in mouse primary astrocytes. Either a scavenger of NO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)] or an inhibitor of inducible nitric oxide synthase [l-N6-(I-iminoethyl)-lysine hydrochloride] blocked this induction of GFAP expression. Similarly, other inducers of NO production such as interferon-γ, interleukin-1β, human immunodeficiency virus type 1 gp120, fibrillar amyloid β peptides, and double-stranded RNA (polyinosinic-polycytidilic acid) also induced the expression of GFAP through NO. The role of NO in the expression of GFAP was supported further by increased expression of GFAP by S-nitroso glutathione (GSNO), an NO donor. Interestingly, inhibition of nuclear factor κB (NF-κB) suppressed LPS- but not GSNO-induced expression of GFAP, suggesting that NO does not require NF-κB to induce GFAP and that NF-κB functions upstream of NO production. However, inhibition of LPS- and GSNO-induced expression of GFAP either by NS-2028 [a specific inhibitor of guanylate cyclase (GC)] or by KT5823 [a specific inhibitor of cGMP-activated protein kinase (PKG)], and induction of GFAP expression by either 8-Br cGMP (a cell-permeable cGMP analog) or MY-5445 (a specific inhibitor of cGMP phosphodiesterase) suggests that NO induces GFAP via GC-cGMP-PKG. This study illustrates a novel biological role of NO in regulating the expression of GFAP in astrocytes through the GC-cGMP-PKG pathway that may participate in the pathogenesis of neurodegenerative disorders.

Simvastatin inhibits the activation of p21ras and prevents the loss of dopaminergic neurons in a mouse model of Parkinson's disease.

Parkinsons disease (PD) is second only to Alzheimers disease as the most common devastating human neurodegenerative disorder. Despite intense investigation, no interdictive therapy is available for PD. We investigated whether simvastatin, a Food and Drug Administration-approved cholesterol-lowering drug, could protect against nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication to model PD in mice. First, MPP+ induced the activation of p21ras and nuclear factor-κB (NF-κB) in mouse microglial cells. Inhibition of MPP+-induced activation of NF-κB by Δp21ras, a dominant-negative mutant of p21ras, supported the involvement of p21ras in MPP+-induced microglial activation of NF-κB. Interestingly, simvastatin attenuated activation of both p21ras and NF-κB in MPP+-stimulated microglial cells. Consistently, we found a very rapid activation of p21ras in vivo in the substantia nigra pars compacta of MPTP-intoxicated mice. However, after oral administration, simvastatin entered into the nigra, reduced nigral activation of p21ras, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Similarly, pravastatin, another cholesterol-lowering drug, suppressed microglial inflammatory responses and protected dopaminergic neurons in MPTP-intoxicated mice, but at levels less than simvastatin. Furthermore, both the statins administered 2 d after initiation of the disease were still capable of inhibiting the demise of dopaminergic neurons and concomitant loss of neurotransmitters, suggesting that statins are capable of slowing down the progression of neuronal loss in the MPTP mouse model. Therefore, we conclude that statins may be of therapeutic benefit for PD patients.

Sodium Phenylbutyrate Controls Neuroinflammatory and Antioxidant Activities and Protects Dopaminergic Neurons in Mouse Models of Parkinson’s Disease

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21rac, but not p21ras, attenuated the production of ROS from activated microglia. Inhibition of both p21ras and p21rac activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21ras and p21rac in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. Oral administration of NaPB reduced nigral activation of p21ras and p21rac, protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders.

Sodium benzoate, a metabolite of cinnamon and a food additive, reduces microglial and astroglial inflammatory responses.

Upon activation, microglia and astrocytes produce a number of proinflammatory molecules that participate in the pathophysiology of several neurodegenerative disorders. This study explores the anti-inflammatory property of cinnamon metabolite sodium benzoate (NaB) in microglia and astrocytes. NaB, but not sodium formate, was found to inhibit LPS-induced expression of inducible NO synthase (iNOS), proinflammatory cytokines (TNF-α and IL-1β) and surface markers (CD11b, CD11c, and CD68) in mouse microglia. Similarly, NaB also inhibited fibrillar amyloid β (Aβ)-, prion peptide-, double-stranded RNA (polyinosinic-polycytidylic acid)-, HIV-1 Tat-, 1-methyl-4-phenylpyridinium+-, IL-1β-, and IL-12 p402-induced microglial expression of iNOS. In addition to microglia, NaB also suppressed the expression of iNOS in mouse peritoneal macrophages and primary human astrocytes. Inhibition of NF-κB activation by NaB suggests that NaB exerts its anti-inflammatory effect through the inhibition of NF-κB. Although NaB reduced the level of cholesterol in vivo in mice, reversal of the inhibitory effect of NaB on iNOS expression, and NF-κB activation by hydroxymethylglutaryl-CoA, mevalonate, and farnesyl pyrophosphate, but not cholesterol and ubiquinone, suggests that depletion of intermediates, but not end products, of the mevalonate pathway is involved in the anti-inflammatory effect of NaB. Furthermore, we demonstrate that an inhibitor of p21ras farnesyl protein transferase suppressed the expression of iNOS, that activation of p21ras alone was sufficient to induce the expression of iNOS, and that NaB suppressed the activation of p21ras in microglia. These results highlight a novel anti-inflammatory role of NaB via modulation of the mevalonate pathway and p21ras.

Sodium benzoate, a food additive and a metabolite of cinnamon, modifies T cells at multiple steps and inhibits adoptive transfer of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. This study explores a novel use of sodium benzoate (NaB), a commonly used food additive and a Food and Drug Administration-approved nontoxic drug for urea cycle disorders, in treating the disease process of relapsing-remitting EAE in female SJL/J mice. NaB, administered through drinking water at physiologically tolerable doses, ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological studies reveal that NaB effectively inhibited infiltration of mononuclear cells and demyelination in the spinal cord of EAE mice. Consequently, NaB also suppressed the expression of proinflammatory molecules and normalized myelin gene expression in the CNS of EAE mice. Furthermore, we observed that NaB switched the differentiation of myelin basic protein-primed T cells from Th1 to Th2 mode, enriched regulatory T cell population, and down-regulated the expression of various contact molecules in T cells. Taken together, our results suggest that NaB modifies encephalitogenic T cells at multiple steps and that NaB may have therapeutic importance in multiple sclerosis.

Suppression of Regulatory T Cells by IL-12p40 Homodimer via Nitric Oxide

Regulatory T cells (Tregs) play a pivotal role in the maintenance of homeostasis between immune response and immune tolerance. The transcription factor Foxp3 and the surface protein CD25 are the two key molecules characterizing Tregs. In autoimmune and various other chronic inflammatory diseases, the expression of Foxp3 is severely down-regulated. However, the molecular mechanism underlying the down-regulation of Foxp3 is not understood yet. Because the IL-12p40 homodimer (p402) is markedly up-regulated in response to various inflammatory stimuli, the present study was undertaken to explore the role of p402 in the regulation of Foxp3 in naive mouse splenocytes. IL-12p402 dose-dependently inhibited the expression of Foxp3 and CD25, but not CD4. Interestingly, this inhibition was absent in splenocytes of IL-12Rβ1−/−, but not IL-12Rβ2−/−, mice. Moreover, suppression of Foxp3 in wild-type and IL-12Rβ2−/− splenocytes was accompanied by production of NO. Consistently, l-N6-(1-iminoethyl)-lysine hydrochloride, an inhibitor of inducible NO synthase (iNOS), and PTIO, a scavenger of NO, restored the expression of Foxp3 and CD25 in p402-stimulated splenocytes, and p402 was unable to down-regulate Foxp3 and CD25 in splenocytes from iNOS−/− mice. Furthermore, NO, but not p402, was able to inhibit Foxp3 in purified CD4+CD25+ T cells in the absence of iNOS-expressing cells. Hence, our results clearly demonstrate that p402 induces NO production via IL-12Rβ1 and that NO subsequently suppresses Tregs in naive mouse splenocytes. This study, therefore, delineates an unprecedented biological function of p402 in the regulation of Foxp3 via IL-12Rβ1-mediated NO production.

Myelin Basic Protein Priming Reduces the Expression of Foxp3 in T Cells via Nitric Oxide

Regulatory T cells (Tregs) play a vital role in autoimmune disorders. Among several markers, forkhead box p3 (Foxp3) is the most specific with regard to Treg activity. Therefore, understanding mechanisms that regulate Foxp3 expression is a critical step for unraveling the complicacy of autoimmune pathophysiology. The present study was undertaken to investigate the crosstalk between NO and Tregs. Interestingly, after myelin basic protein (MBP) priming, the expression of Foxp3 decreased in MBP-primed T cells. However, blocking NO either by inhibiting inducible NO synthase with l-N6-(1-iminoethyl)-lysine hydrochloride or through scavenging with PTIO or by pharmacological drugs, such as pravastatin, sodium benzoate, or gemfibrozil, restored the expression of Foxp3 in MBP-primed T cells. However, this restoration of Foxp3 by pharmacological drugs was reversed by S-nitrosoglutathione, an NO donor. Similarly, NO also decreased the populations of Tregs characterized by CD4+CD25+ and CD25+FoxP3+ phenotypes. We have further confirmed this inverse relationship between NO and Foxp3 by analyzing the mRNA expression of Foxp3 and characterizing CD25+FoxP3+ or CD4+Foxp3+ phenotypes from inducible NO synthase knockout mice. Moreover, this inverse relation between NO and Foxp3 also was observed during priming with myelin oligodendrocyte glycoprotein, another target neuroantigen in multiple sclerosis, as well as collagen, a target autoantigen in rheumatoid arthritis. Finally, we demonstrate that NO inhibited the expression of Foxp3 in MBP-primed T cells via soluble guanylyl cyclase-mediated production of cGMP. Taken together, our data imply a novel role of NO in suppressing Foxp3+ Tregs via the soluble guanylyl cyclase pathway.

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.

Gender-Specific Expression of β1 Integrin of VLA-4 in Myelin Basic Protein-Primed T Cells: Implications for Gender Bias in Multiple Sclerosis

Susceptibility to multiple sclerosis is higher in females than males. However, the underlying mechanism behind this gender difference is poorly understood. Because the presence of neuroantigen-primed T cells in the CNS is necessary to initiate the neuroinflammatory cascade of multiple sclerosis, we first investigated how these T cells interacted with astroglia, major resident glial cells of the CNS. Interestingly, we found that myelin basic protein (MBP)-primed T cells from female and castrated male mice, but not from male mice, produced proinflammatory molecules, such as NO, IL-1β, and IL-6 in astroglia, and these responses were purely via contact between T cells and astroglia. Because T cell:glia contact requires several integrin molecules, we examined the involvement of integrins in this process. Both α4 and β1, subunits of VLA-4 integrin, were found to be necessary for T cell contact-induced generation of proinflammatory molecules in astroglia. Interestingly, the expression of β1, but not α4, was absent in male MBP-primed T cells. In contrast, female and castrated male MBP-primed T cells expressed both α4 and β1. Similarly, we also detected β1 in spleen of normal young female, but not male, mice. Furthermore, we show that male sex hormones (testosterone and dihydrotestosterone), but not female sex hormones (estrogen and progesterone), were able to suppress the mRNA expression of β1 in female MBP-primed T cells. These studies suggest that β1, but not α4, integrin of VLA-4 is the sex-specific molecule on T cell surface, and that the presence or absence of β1 determines gender-specific T cell contact-mediated glial activation.

Regulation of encephalitogenicity of neuroantigen-primed T cells by nitric oxide: Implications for multiple sclerosis.

Neuroantigen-specific T cells play an important role in the disease process of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). These cells are encephalitogenic and in susceptible animals, these cells alone can cause EAE. However, mechanisms by which encephalitogenicity is controlled are poorly understood. This study underlines the importance of nitric oxide (NO) in the regulation of encephalitogenicity of T cells. Interestingly, reducing NO during myelin basic protein (MBP)-priming of T cells attenuated the ability of these T cells to induce EAE and EAE-associated neuroinflammation and demyelination. Consistently, increasing NO had opposite effect. Similarly scavenging NO reduced the encephalitogenicity of PLP-specific T cells isolated from female PLP-TCR transgenic mice and supplementation of NO broke the tolerance of PLP-specific T cells of male PLP-TCR mice. Reduced encephalitogenicity of neuroantigen-primed T cells isolated from iNOS (-/-) mice compared to that from wild type mice clearly defines an essential role of iNOS-derived NO in controlling the encephalitogenicity of myelin-specific T cells. This study illustrates a novel role of NO in controlling encephalitogenicity of T cells that may participate in the complex pathogenesis of MS.