Monoclonal full-length antibody against TAR DNA binding protein 43 reduces related proteinopathy in neurons.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), 2 incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43, which loses its physiological properties, leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here, we tested the target specificity, in vivo distribution, and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43-related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-κB activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge, this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model.

Mitigation of ALS Pathology by Neuron-Specific Inhibition of Nuclear Factor Kappa B Signaling.

To investigate the role of neuronal NF-κB activity in pathogenesis of amyotrophic lateral sclerosis (ALS), we generated transgenic mice with neuron-specific expression of a super-repressor form of the NF-κB inhibitor (IκBα-SR), which were then crossed with mice of both sexes, expressing ALS-linked gene mutants for TAR DNA-binding protein (TDP-43) and superoxide dismutase 1 (SOD1). Remarkably, neuronal expression of IκBα-SR transgene in mice expressing TDP-43A315T or TDP-43G348C mice led to a decrease in cytoplasmic to nuclear ratio of human TDP-43. The mitigation of TDP-43 neuropathology by IκBα-SR, which is likely due to an induction of autophagy, was associated with amelioration of cognitive and motor deficits as well as reduction of motor neuron loss and gliosis. Neuronal suppression of NF-κB activity in SOD1G93A mice also resulted in neuroprotection with reduction of misfolded SOD1 levels and significant extension of life span. The results suggest that neuronal NF-κB signaling constitutes a novel therapeutic target for ALS disease and related disorders with TDP-43 proteinopathy.SIGNIFICANCE STATEMENT This study reports that neuron-specific expression of IκB super-repressor mitigated behavioral and pathologic changes in transgenic mouse models of amyotrophic lateral sclerosis expressing mutant forms of either Tar DNA-binding protein 43 or superoxide dismutase. The results suggest that neuronal NF-κB signaling constitutes a novel therapeutic target for amyotrophic lateral sclerosis and related disorders with Tar DNA-binding protein 43 proteinopathy.

Protective effects of Withania somnifera extract in SOD1G93A mouse model of amyotrophic lateral sclerosis.

Withania somnifera (WS; commonly known as Ashwagandha or Indian ginseng) is a medicinal plant whose extracts have been in use for centuries in various regions of the world as a rejuvenator. There is now a growing body of evidence documenting neuroprotective functions of the plant extracts or its purified compounds in several models of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Based on the extract's beneficial effect in a mouse model of ALS with TDP-43 proteinopathy, the current study was designed to test its efficacy in another model of familial ALS. Our results show that administration of WS extracts by gavage to mice expressing G93A mutant form of superoxide dismutase (SOD1) resulted in increased longevity, improved motor performance and increased number of motor neurons in lumbar spinal cord. The WS treatment caused substantial reduction in levels of misfolded SOD1whereas it enhanced expression of cellular chaperons in spinal cord of SOD1G93A mice. WS markedly reduced glial activation and prevented phosphorylation of nuclear factor kappaB (NF-κB). The overall immunomodulatory effect of WS was further evidenced by changes in expression of multiple cytokines/chemokines. WS also served as an autophagy inducer which may be beneficial at early stages of the disease. These results suggest that WS extracts might constitute promising therapeutics for treatment of ALS with involvement of misfolded SOD1.

Recent advances in Japanese encephalitis.

Japanese encephalitis is a flaviviral disease that is endemic to the South, Southeast Asia, and Asia Oceania regions. Given that about 60% of the world's population (about 7.4 billion) resides in this region (about 4.4 billion), this disease poses a significant threat to global health. Active vaccination campaigns conducted in endemic countries have led to a decrease in the number of reported cases over the years. In this article, we strive to briefly highlight recent advances in understanding the role of microRNAs in disease pathology, focus on providing brief summaries of recent clinical trials in the field of Japanese encephalitis therapeutics, and review the current prophylactic strategies.

Withania somnifera Reverses Transactive Response DNA Binding Protein 43 Proteinopathy in a Mouse Model of Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration.

Abnormal cytoplasmic mislocalization of transactive response DNA binding protein 43 (TARDBP or TDP-43) in degenerating neurons is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Our previous work suggested that nuclear factor kappa B (NF-κB) may constitute a therapeutic target for TDP-43-mediated disease. Here, we investigated the effects of root extract of Withania somnifera (Ashwagandha), an herbal medicine with anti-inflammatory properties, in transgenic mice expressing a genomic fragment encoding human TDP-43A315T mutant. Ashwagandha extract was administered orally to hTDP-43A315T mice for a period of 8 weeks starting at 64 and 48 weeks of age for males and females, respectively. The treatment of hTDP-43A315T mice ameliorated their motor performance on rotarod test and cognitive function assessed by the passive avoidance test. Microscopy examination of tissue samples revealed that Ashwagandha treatment of hTDP-43A315T mice improved innervation at neuromuscular junctions, attenuated neuroinflammation, and reduced NF-κB activation. Remarkably, Ashwagandha treatment reversed the cytoplasmic mislocalization of hTDP-43 in spinal motor neurons and in brain cortical neurons of hTDP-43A315T mice and it reduced hTDP-43 aggregation. In vitro evidence is presented that the neuronal rescue of TDP-43 mislocalization may be due to the indirect effect of factors released from microglial cells exposed to Ashwagandha. These results suggest that Ashwagandha and its constituents might represent promising therapeutics for TDP-43 proteinopathies.

Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells.

BACKGROUND: Mutations in the gene encoding Ubiquilin-2 (UBQLN2) are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). UBQLN2 plays a central role in ubiquitin proteasome system (UPS) and UBQLN2 mutants can form cytoplasmic aggregates in vitro and in vivo. RESULTS: Here, we report that overexpression of WT or mutant UBQLN2 species enhanced nuclear factor κB (NF-κB) activation in Neuro2A cells. The inhibition of NF-κB stress-mediated activation with SB203580, a p38 MAPK inhibitor, demonstrated a role for MAPK in NF-κB activation by UBQLN2 species. Live cell imaging and microscopy showed that UBQLN2 aggregates are dynamic structures that promote cytoplasmic accumulation of TAR DNA-binding protein (TDP-43), a major component of ALS inclusion bodies. Furthermore, up-regulation of UBQLN2 species in neurons caused an ER-stress response and increased their vulnerability to death by toxic mediator TNF-α. Withaferin A, a known NF-κB inhibitor, reduced mortality of Neuro2A cells overexpressing UBQLN2 species. CONCLUSIONS: These results suggest that UBQLN2 dysregulation in neurons can drive NF-κB activation and cytosolic TDP-43 aggregation, supporting the concept of pathway convergence in ALS pathogenesis. These Ubiquilin-2 pathogenic pathways might represent suitable therapeutic targets for future ALS treatment.

Inflammation Induces TDP-43 Mislocalization and Aggregation.

TAR DNA-binding protein 43 (TDP-43) is a major component in aggregates of ubiquitinated proteins in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we report that lipopolysaccharide (LPS)-induced inflammation can promote TDP-43 mislocalization and aggregation. In culture, microglia and astrocytes exhibited TDP-43 mislocalization after exposure to LPS. Likewise, treatment of the motoneuron-like NSC-34 cells with TNF-alpha (TNF-α) increased the cytoplasmic levels of TDP-43. In addition, the chronic intraperitoneal injection of LPS at a dose of 1mg/kg in TDP-43(A315T) transgenic mice exacerbated the pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and it enhanced the levels of TDP-43 aggregation. These results suggest that inflammation may contribute to development or exacerbation of TDP-43 proteinopathies in neurodegenerative disorders.

TLR7 is a key regulator of innate immunity against Japanese encephalitis virus infection.

Toll-like receptor 7 (TLR7) known to recognize guanidine-rich ssRNA has been shown to mount vital host defense mechanism against many viruses including flaviviruses. Signal transduction through TLR7 has been shown to produce type-1 interferon and proinflammatory mediators, thereby initiating essential innate immune response against ssRNA viruses in hosts. Systemic and brain specific TLR7 knock-down mice (TLR7(KD)) were generated using vivo-morpholinos. These mice were then subcutaneously challenged with lethal dose of JEV (GP78 strain) and were subsequently analyzed for survival. Significant difference in susceptibility to JEV between wild-type and systemic TLR7(KD) mice was observed whereas, no difference in susceptibility to JEV infection was seen in brain-specific TLR7(KD) mice. Significant decreases in IFN-α and antiviral proteins were also observed in both TLR7(KD) mice along with increased viral loads in their brain. Owing to increased viral load, increases in levels of various proinflammatory cyto/chemokines, increased microglial activation and infiltration of peripheral immune cells in brain of TLR7(KD) mice were also observed. Immunocytochemistry and RNA co-immunoprecipitation performed with JEV-infected N2a or HT22 cells indicated endosomal localization and confirmed interaction between JEV ssRNA with TLR7. Treatment of mice with imiquimod, a TLR7 agonist, prior to JEV infection resulted in their increased survival. Overall, our results suggest that the TLR7 response following JEV infection promotes type-1 interferon production and generation of antiviral state which might contribute to protective effect in systemic infection.

Role of pattern recognition receptors in flavivirus infections.

The flaviviral encephalitis has now become a major health concern in global scale. The efficient detection of viral infection and induction of the innate antiviral response by host's innate immune system are crucial to determine the outcome of infection. The intracellular pattern recognition receptors TLRs, RLRs, NLRs and CLRs play a central role in detection and initiation of robust antiviral response against flaviviral infection. Both cytoplasmic RLRs, RIG-I and MDA5 have been shown to be implicated in sensing flaviviral genomic RNA. Similarly among TLRs mainly TLR3 and TLR7 are known to respond in flaviviral infections as they are known to sense dsRNA and ssRNA moiety as their natural cognate ligand. Several studies have also shown the roles of NLRs and CLRs in mounting an innate antiviral response against flavivirus but, it is yet to be completely understood. Until now only few reports have implicated NLRs and CLRs in induction of antiviral and proinflammatory state following flaviviral infection. The current review therefore aims to comprehensively analyze past as well as current understanding on the role of PRRs in flaviviral infections.

Neural stem/progenitor cells induce conversion of encephalitogenic T cells into CD4+-CD25+- FOXP3+ regulatory T cells.

An immune role of neural stem/progenitor cells (NSPCs) has been proposed in many recent studies; however much still remains to be elucidated. In the current investigation, we report that NSPCs possess the ability to convert encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) regulatory T cells (T(reg)). Encephalitogenic and nonencephalitogenic T cells isolated from sham and Japanese encephalitis virus (JEV) infected animals were co-cultured with mouse NSPCs. Post co-culture, significant increase in the number of T(regs) was observed from encephalitogenic T cells co-cultured with NSPCs. This increased conversion was found to be dependent on direct contact between T cells and NSPCs. Neutralization of TGF-ß and IFN-γ in NSPC cultures abrogated this increased conversion of encephalitogenic T cells into T(regs). Flow cytometric, quantitative RT-PCR, and immunoblot analysis of both T cells and NSPCs revealed surface and intracellular changes post co-culture. Co-stimulatory molecules (B7) and ICAM-1 were increased on NSPCs post co-culture; levels of TGFß, IFNγ, and TGFßR1 were also increased in NSPCs. This study provides a basic insight into the interaction between CNS-infiltrating encephalitogenic T cells and NSPCs during viral encephalitis. Conversion of encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) T(regs) through interaction with NSPCs indicates an attempt in regulation of excessive inflammation in the CNS.

Japanese encephalitis virus infection modulates the expression of suppressors of cytokine signaling (SOCS) in macrophages: implications for the hosts' innate immune response.

Viruses have evolved various mechanisms to subvert the host's immune system and one of them is preventing the infected cells from sending out chemotactic signals to activate the adaptive immune response. Japanese encephalitis virus (JEV) is a neuropathologic flavivirus that is responsible for significant number of child mortalities in various parts of South-East Asia. In this study we show that JEV modulates suppressors of cytokine signaling (SOCS)1 and 3 expression in macrophages to bring about changes in the JAK-STAT signaling cascade, so as to inhibit proinflammatory cyto/chemokine release. Using real time PCR, immunoblotting and immunofluorescent staining, we show that the expression of type 1 interferons and intracellular expression of viral genes are also affected over time. Also, following the initial activation of SOCS1 and 3, there is production of interferon-inducible anti-viral proteins in the cells which may be responsible for inhibiting viral replication. However, even at later time points, viral genes were still detected from the macrophages, albeit at lesser quantities, than earlier time points, indicative of intracellular persistence of the virus in a latent form. On knocking down SOCS1 and SOCS3 we found a significant decrease in viral gene expression at an early time point, indicating the dysregulation of the signaling cascade leading to increased production of interferon-inducible anti-viral proteins. Taken together, our study provides an insight into the role of JEV infection in modulating the JAK-STAT pathway with the help of SOCS leading to the generation of an antiviral innate immune response.

Japanese encephalitis in India: risk of an epidemic in the National Capital Region.

Japanese encephalitis (JE) is a mosquitoborne viral disease that is the primary cause of acute encephalitis syndrome in India. This virus mainly infects the central nervous system and causes massive inflammation which, if left unchecked, may prove fatal. Survivors often suffer from mild to severe neuropsychiatric sequelae. JE is a major cause of death in many parts of India and there is a possibility of it spreading into the National Capital Region from highly endemic neighbouring states. Fourteen cases of JE were reported in Delhi in 2011 compared with none in the previous 4 years from 2007 to 2010. Unless immediate preventive measures are taken this trend could continue and the disease could spread with increasing prevalence.

Chandipura virus induces neuronal death through Fas-mediated extrinsic apoptotic pathway.

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.

Histone deacetylase inhibition by Japanese encephalitis virus in monocyte/macrophages: a novel viral immune evasion strategy.

Japanese encephalitis virus (JEV) is a common cause of encephalitis in humans who are dead-end hosts producing negligible viremia. The virus reaches the brain and causes massive inflammation. Our study seeks to understand the virus-host interaction using the murine monocyte/macrophage cell line RAW264.7, an antigen presenting cell involved in eliciting an innate immune response. We have discovered several interesting phenomena occurring in JEV-infected RAW264.7 cells which diverge from established observations. JEV remains inside RAW264.7 and appears to have little negative effect on cell viability. Expression studies of major histocompatibility complexes (MHC) and co-stimulatory molecules show inhibition of antigen presentation. There is enhanced immune suppression creating an anti-viral milieu. Expression of pro-inflammatory cytokines and chemokines is suppressed along with increased expression of anti-inflammatory molecules. Histone deacetylases (HDACs) have known inflammatory properties. In our study, through modulation of HDACs JEV seems to induce a crucial anti-inflammatory and anti-viral role in host macrophages.

Increased resistance of immobilized-stressed mice to infection: correlation with behavioral alterations.

Immobilization is an easy and convenient method to induce both psychological and physical stress resulting in restricted motility and aggression and is believed to be the most severe type of stress in rodent models. Although it has been generally accepted that chronic stress often results in immunosuppression while acute stress has been shown to enhance immune responses, the effects of IS on the host resistance to Escherichia coli (E. coli) infection and associated behavioral changes are still not clear. In a series of experiments aimed at determining the level of hypothalamic COX-2, HSP-90, HSP-70, SOD-1 and plasma level of corticosterone, cytokine, antibody titer and their association with behavioral activities, mice were infected with viable E. coli during acute and chronic IS by taping their paws. In this study we show that acute and chronic IS enhances the resistance of mice to E. coli infection via inhibiting the production of pro-inflammatory cytokines, free radicals, and by improving the exploratory behavior. Altogether, our findings support the notion that cytokines released during immune activation and under the influence of corticosterone can modulate the open field behavior both in terms of locomotor activity as well as exploration. One of the features observed with chronic stressor was a lower ability to resist bacterial infection, although in case of acute stress, a better clearance of bacterial infection was observed in vivo with improvement of exploratory behavior and cognitive functions.

Azithromycin in combination with riboflavin decreases the severity of Staphylococcus aureus infection induced septic arthritis by modulating the production of free radicals and endogenous cytokines.

OBJECTIVE AND DESIGN: To determine alternate therapeutic measures to combat Staphylococcus aureus induced arthritis. Thus, azithromycin was combined with riboflavin, which may combat the ROS production and inflammation. METHODS: An in vivo model of S. aureus infection-induced arthritis was set up by infecting mice with 5 × 106 bacterial cell/mouse. S. aureus was administered intravenously. Azithromycin and riboflavin was injected intraperitoneally at a single dose of 100 and 20 mg/kg body, respectively. The mice were sacrificed at 3, 9, 15 days post infection (dpi). TNF-α, IFN-γ, IL-6 and IL-10 from serum and SOD, catalase and reduced glutathione concentration were observed in hepatic, cardiac, renal and splenic tissue. RESULTS: CFU was found very prominent in spleen and joints and reduced in blood at 3 and 9 dpi. However, treatment with azithromycin and riboflavin completely eradicated the bacteria from blood and spleen. TNF-α, IFN-γ, IL-6, and MCP-1 were induced due to infection which were downregulated by treatment with azithromycin and riboflavin. Infected mice were also found to have altered antioxidant status, measured in terms of reduced glutathione and anti-oxidant enzymes such as SOD and catalase. CONCLUSION: These changes were found to be ameliorated when the animals were co-treated with azithromycin and riboflavin.

Ampicillin alone and in combination with riboflavin modulates Staphylococcus aureus infection induced septic arthritis in mice.

Effects of ampicillin (Amp) in combination with riboflavin on septic arthritis in mice infected with Staphylococcus aureus have been reported. Ampicillin was given at 100 mg/kg after 24 h of infection, followed by riboflavin (Ribo) at 20 mg/kg body wt, after 2 h of Amp treatment. Mice were sacrificed at 3, 9, 15 days post infection (dpi). Combined treatment of infected mice with ampicillin and riboflavin eradicated the bacteria from blood, spleen and synovial tissue and showed a significant gross reduction in arthritis, reduced serum levels of TNF-alpha and IFN-gamma. S. aureus infected mice exhibited higher synovial TNF-alpha and IL-6, which was also reduced by ampicillin and riboflavin treatment. S. aureus infected mice showed a disturbed antioxidant status measured in terms of cellular anti-oxidants like reduced glutathione and anti-oxidant enzymes such as superoxide dismutase and catalase and were ameliorated when the animals were co-treated with ampicillin along with riboflavin. Results of the study showed that combined treatment with anti-oxidant and antibiotic may protect from staphylococcal arthritis and may ameliorate oxidative stress caused by S. aureus infection.

STING mediates neuronal innate immune response following Japanese encephalitis virus infection.

Flavivirus-mediated inflammation causes neuronal death, but whether the infected neurons can evoke an innate immune response to elicit their own protection, is unknown. In an earlier study we have shown that neuronal RIG-I, play a significant role in inducing production and release of molecules that are related to inflammation. In this study, using a neuronal cell line, we show that RIG-I acts with STING in a concerted manner following its interaction with Japanese encephalitis viral RNA to induce a type 1 interferon response. Knock-down of STING showed that the expressions of various inflammatory signaling molecules were down-regulated along with increased intracellular viral load. Alternatively, over-expressing STING decreased intracellular viral load. Our results indicate that at the sub-cellular level, interaction between the pattern recognition receptor RIG-I and the adapter molecule STING, is a major contributor to elicit immunological responses involving the type 1 interferons in neurons following JEV infections.

RIG-I mediates innate immune response in mouse neurons following Japanese encephalitis virus infection.

BACKGROUND: Neuroinflammation associated with Japanese encephalitis (JE) is mainly due to the activation of glial cells with subsequent release of proinflammatory mediators from them. The recognition of viral RNA, in part, by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been indicated to have a role in such processes. Even though neurons are also known to express this receptor, its role after JE virus (JEV) infections is yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Upon infecting murine neuroblastoma cells and primary cortical neurons with JEV the expression profile of key proinflammatory cyto/chemokines were analyzed by qRT-PCR and bead array, both before and after ablation of RIG-I. Immunoblotting was performed to evaluate the levels of key molecules downstream to RIG-I leading to production of proinflammatory mediators. Changes in the intracellular viral antigen expression were confirmed by intracellular staining and immunoblotting. JEV infection induced neuronal expression of IL-6, IL-12p70, MCP-1, IP-10 and TNF-α in a time-dependent manner, which showed significant reduction upon RIG-I ablation. Molecules downstream to RIG-I showed significant changes upon JEV-infection, that were modulated following RIG-I ablation. Ablation of RIG-I in neurons also increased their susceptibility to JEV. CONCLUSIONS/SIGNIFICANCE: In this study we propose that neurons are one of the potential sources of proinflammatory cyto/chemokines in JEV-infected brain that are produced via RIG-I dependent pathways. Ablation of RIG-I in neurons leads to increased viral load and reduced release of the cyto/chemokines.

Use of minocycline in viral infections.

Repurposing of old drugs is a useful concept as it helps to minimize costs associated with the research and development of a new drug. Minocycline, a common second generation antibiotic, has been shown to possess several other beneficial effects other than its intended uses. The antiviral role of minocycline has generated considerable interest from the last decade. It was first shown to be beneficial in preventing human immunodeficiency virus (HIV) infections and later it was reported to improve cognitive deficiencies associate with neuroAIDS. However, its antiviral efficacies are not limited to retroviruses alone. In animal models or in vitro systems of flaviviral infections (especially Japanese encephalitis virus), minocycline has been shown to be highly effective. However, not all effects are based on direct inhibition of viral replication. The general anti-inflammatory and immunomodulatory properties of minocycline are also responsible in part, in imparting the protective effects. Owing to the fact that minocycline is well tolerated by most people and that the drug has nearly 40 years history of usage, it is an exciting prospect to try out in other viral infections.