Induction of IL-2 by interleukin-12 p40 homodimer and IL-12, but not IL-23, in microglia and macrophages: Implications for multiple sclerosis.

The level of IL-2 increases markedly in serum and central nervous system (CNS) of patients with multiple sclerosis (MS) and animals with experimental allergic encephalomyelitis (EAE). However, mechanisms by which IL-2 is induced under autoimmune demyelinating conditions are poorly understood. The present study underlines the importance of IL-12p40 homodimer (p402), the so-called biologically inactive molecule, in inducing the expression of IL-2 in mouse BV-2 microglial cells, primary mouse and human microglia, mouse peritoneal macrophages, RAW264.7 macrophages, and T cells. Interestingly, we found that p402 and IL-12p70 (IL-12), but not IL-23, dose-dependently induced the production of IL-2 and the expression of IL-2 mRNA in microglial cells. Similarly, p402 also induced the activation of IL-2 promoter in microglial cells and RAW264.7 cells. Among various stimuli tested, p402 was the most potent stimulus followed by IFN-γ, bacterial lipopolysaccharide, HIV-1 gp120, and IL-12 in inducing the activation of IL-2 promoter in microglial cells. Moreover, p402, but not IL-23, increased NFATc2 mRNA expression and the transcriptional activity of NFAT. Furthermore, induction of IL-2 mRNA expression by over-expression of p40, but not by p19, cDNA indicated that p40, but not p19, is responsible for the induction of IL-2 mRNA in microglia. Finally, by using primary microglia from IL to 12 receptor ß1 deficient (IL-12Rß1-/-) and IL-12 receptor ß2 deficient (IL-12Rß2-/-) mice, we demonstrate that p402 induces the expression of IL-2 via IL-12Rß1, but not IL-12Rß2. In experimental autoimmune encephalomyelitis, an animal model of MS, neutralization of p402 by mAb a3-1d led to decrease in clinical symptoms and reduction in IL-2 in T cells and microglia. These results delineate a new biological function of p402, which is missing in the so-called autoimmune cytokine IL-23, and raise the possibility of controlling increased IL-2 and the disease process of MS via neutralization of p402.

Neutralization of p40 Homodimer and p40 Monomer Leads to Tumor Regression in Patient-Derived Xenograft Mice with Pancreatic Cancer.

Pancreatic cancer is a highly aggressive cancer with a high mortality rate and limited treatment options. It is the fourth leading cause of cancer in the US, and mortality is rising rapidly, with a 12% relative 5-year survival rate. Early diagnosis remains a challenge due to vague symptoms, lack of specific biomarkers, and rapid tumor progression. Interleukin-12 (IL-12) is a central cytokine that regulates innate (natural killer cells) and adaptive (cytokine T-lymphocytes) immunity in cancer. We demonstrated that serum levels of IL-12p40 homodimer (p402) and p40 monomer (p40) were elevated and that of IL-12 and IL-23 were lowered in pancreatic cancer patients compared to healthy controls. Comparably, human PDAC cells produced greater levels of p402 and p40 and lower levels of IL-12 and IL-23 compared to normal pancreatic cells. Notably, neutralization of p402 by mAb a3-1d and p40 by mAb a3-3a induced the death of human PDAC cells, but not normal human pancreatic cells. Furthermore, we demonstrated that treatment of PDX mice with p402 mAb and p40 mAb resulted in apoptosis and tumor shrinkage. This study illustrates a new role of p402 and p40 monomer in pancreatic cancer, highlighting possible approaches against this deadly form of cancer with p402 and p40 monomer immunotherapies.

Regression of Lung Cancer in Mice by Intranasal Administration of SARS-CoV-2 Spike S1.

This study underlines the importance of SARS-CoV-2 spike S1 in prompting death in cultured non-small cell lung cancer (NSCLC) cells and in vivo in lung tumors in mice. Interestingly, we found that recombinant spike S1 treatment at very low doses led to death of human A549 NSCLC cells. On the other hand, boiled recombinant SARS-CoV-2 spike S1 remained unable to induce death, suggesting that the induction of cell death in A549 cells was due to native SARS-CoV-2 spike S1 protein. SARS-CoV-2 spike S1-induced A549 cell death was also inhibited by neutralizing antibodies against spike S1 and ACE2. Moreover, our newly designed wild type ACE2-interacting domain of SARS-CoV-2 (wtAIDS), but not mAIDS, peptide also attenuated SARS-CoV-2 spike S1-induced cell death, suggesting that SARS-CoV-2 spike S1-induced death in A549 NSCLC cells depends on its interaction with ACE2 receptor. Similarly, recombinant spike S1 treatment also led to death of human H1299 and H358 NSCLC cells. Finally, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) intoxication led to the formation tumors in lungs of A/J mice and alternate day intranasal treatment with low dose of recombinant SARS-CoV-2 spike S1 from 22-weeks of NNK insult (late stage) induced apoptosis and tumor regression in the lungs. These studies indicate that SARS-CoV-2 spike S1 may have implications for lung cancer treatment.

Regression of Triple-Negative Breast Cancer in a Patient-Derived Xenograft Mouse Model by Monoclonal Antibodies against IL-12 p40 Monomer.

Although some therapies are available for regular breast cancers, there are very few options for triple-negative breast cancer (TNBC). Here, we demonstrated that serum level of IL-12p40 monomer (p40) was much higher in breast cancer patients than healthy controls. On the other hand, levels of IL-12, IL-23 and p40 homodimer (p402) were lower in serum of breast cancer patients as compared to healthy controls. Similarly, human TNBC cells produced greater level of p40 than p402. The level of p40 was also larger than p402 in serum of a patient-derived xenograft (PDX) mouse model. Accordingly, neutralization of p40 by p40 mAb induced death of human TNBC cells and tumor shrinkage in PDX mice. While investigating the mechanism, we found that neutralization of p40 led to upregulation of human CD4+IFNγ+ and CD8+IFNγ+ T cell populations, thereby increasing the level of human IFNγ and decreasing the level of human IL-10 in PDX mice. Finally, we demonstrated the infiltration of human cytotoxic T cells, switching of tumor-associated macrophage M2 (TAM2) to TAM1 and suppression of transforming growth factor ß (TGFß) in tumor tissues of p40 mAb-treated PDX mice. Our studies identify a possible new immunotherapy for TNBC in which p40 mAb inhibits tumor growth in PDX mice.

Selective Inhibition of the Interaction between SARS-CoV-2 Spike S1 and ACE2 by SPIDAR Peptide Induces Anti-Inflammatory Therapeutic Responses.

Many patients with coronavirus disease 2019 in intensive care units suffer from cytokine storm. Although anti-inflammatory therapies are available to treat the problem, very often, these treatments cause immunosuppression. Because angiotensin-converting enzyme 2 (ACE2) on host cells serves as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to delineate a SARS-CoV-2-specific anti-inflammatory molecule, we designed a hexapeptide corresponding to the spike S1-interacting domain of ACE2 receptor (SPIDAR) that inhibited the expression of proinflammatory molecules in human A549 lung cells induced by pseudotyped SARS-CoV-2, but not vesicular stomatitis virus. Accordingly, wild-type (wt), but not mutated (m), SPIDAR inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 and IL-1ß in human lung cells. However, wtSPIDAR remained unable to reduce activation of NF-κB and expression of proinflammatory molecules in lungs cells induced by TNF-α, HIV-1 Tat, and viral dsRNA mimic polyinosinic-polycytidylic acid, indicating the specificity of the effect. The wtSPIDAR, but not mutated SPIDAR, also hindered the association between ACE2 and spike S1 of SARS-CoV-2 and inhibited the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus, into human ACE2-expressing human embryonic kidney 293 cells. Moreover, intranasal treatment with wtSPIDAR, but not mutated SPIDAR, inhibited lung activation of NF-κB, protected lungs, reduced fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1-to-ACE2 interaction by wtSPIDAR may be beneficial for coronavirus disease 2019.

ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy.

COVID-19 is an infectious respiratory illness caused by the virus strain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and until now, there is no effective therapy against COVID-19. Since SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) for entering into host cells, to target COVID-19 from therapeutic angle, we engineered a hexapeptide corresponding to the ACE2-interacting domain of SARS-CoV-2 (AIDS) that inhibits the association between receptor-binding domain-containing spike S1 and ACE-2. Accordingly, wild type (wt), but not mutated (m), AIDS peptide inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 in human lungs cells. Interestingly, intranasal intoxication of C57/BL6 mice with recombinant SARS-CoV-2 spike S1 led to fever, increase in IL-6 in lungs, infiltration of neutrophils into the lungs, arrhythmias, and impairment in locomotor activities, mimicking some of the important symptoms of COVID-19. However, intranasal treatment with wtAIDS, but not mAIDS, peptide reduced fever, protected lungs, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of ACE2-to-SARS-CoV-2 interaction by wtAIDS may be beneficial for COVID-19.

Is COVID-19 Gender-sensitive?

While clinical characteristics exhibit that susceptibility to COVID-19 infection is equally likely between males and females, clinical outcomes show that males experience both a higher severity and fatality for COVID-19 infection than females. This review examines the evidence for these sex and gender differences and aims to illustrate possible mechanisms behind such sensitivity. Successful entry of SARS-CoV-2 into the body is dependent on the angiotensin-converting enzyme 2 (ACE2) receptor and the transmembrane protease serine 2 (TMPRSS2). Thus, sex-based differences in the expression of the ACE2 receptor and TMPRSS2 may explain the disparities in COVID-19 severity and fatality. Furthermore, these disparities may also be attributed to sex-based difference in immunological responses. Finally, the differences in clinical outcomes of COVID-19 infections between men and women may be due to gendered differences in behaviors, such as smoking, and prevalence to comorbidities. An understanding of the sex and gender sensitivities of COVID-19 infection is a necessary component towards the creation of effective treatment options and therapies for the virus. Graphical abstract.

Selective disruption of TLR2-MyD88 interaction inhibits inflammation and attenuates Alzheimer's pathology.

Induction of TLR2 activation depends on its association with the adapter protein MyD88. We have found that TLR2 and MyD88 levels are elevated in the hippocampus and cortex of patients with Alzheimer's disease (AD) and in a 5XFAD mouse model of AD. Since there is no specific inhibitor of TLR2, to target induced TLR2 from a therapeutic angle, we engineered a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that binds to the BB loop of only TLR2, and not other TLRs. Interestingly, WT TIDM peptide inhibited microglial activation induced by fibrillar Aß1-42 and lipoteichoic acid, but not 1-methyl-4-phenylpyridinium, dsRNA, bacterial lipopolysaccharide, flagellin, or CpG DNA. After intranasal administration, WT TIDM peptide reached the hippocampus, reduced hippocampal glial activation, lowered Aß burden, attenuated neuronal apoptosis, and improved memory and learning in 5XFAD mice. However, WT TIDM peptide was not effective in 5XFAD mice lacking TLR2. In addition to its effects in 5XFAD mice, WT TIDM peptide also suppressed the disease process in mice with experimental allergic encephalomyelitis and collagen-induced arthritis. Therefore, selective targeting of the activated status of 1 component of the innate immune system by WT TIDM peptide may be beneficial in AD as well as other disorders in which TLR2/MyD88 signaling plays a role in disease pathogenesis.

Selective neutralization of IL-12 p40 monomer induces death in prostate cancer cells via IL-12-IFN-γ.

Cancer cells are adept at evading cell death, but the underlying mechanisms are poorly understood. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of T-helper type 1 cells, favoring cell-mediated immunity. IL-12 is composed of two different subunits, p40 and p35. This study underlines the importance of IL-12 p40 monomer (p40) in helping cancer cells to escape cell death. We found that different mouse and human cancer cells produced greater levels of p40 than p40 homodimer (p402), IL-12, or IL-23. Similarly, the serum level of p40 was much greater in patients with prostate cancer than in healthy control subjects. Selective neutralization of p40, but not p402, by mAb stimulated death in different cancer cells in vitro and in vivo in a tumor model. Interestingly, p40 was involved in the arrest of IL-12 receptor (IL-12R) IL-12Rß1, but not IL-12Rß2, in the membrane, and that p40 neutralization induced the internalization of IL-12Rß1 via caveolin and caused cancer cell death via the IL-12-IFN-γ pathway. These studies identify a role of p40 monomer in helping cancer cells to escape cell death via suppression of IL-12Rß1 internalization.

Induction of Adaptive Immunity Leads to Nigrostriatal Disease Progression in MPTP Mouse Model of Parkinson's Disease.

Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model is the most widely used animal model for Parkinson's disease (PD), it is known that nigrostriatal pathologies do not persist in the acute MPTP mouse model. This study highlights the importance of adaptive immunity in driving persistent and progressive disease in acute MPTP-intoxicated mice. Although marked infiltration of T cells into the nigra was found on 1 d of MPTP insult, T cell infiltration decreased afterward, becoming normal on 30 d of insult. Interestingly, twice-weekly supplementation of RANTES and eotaxin, chemokines that are involved in T cell trafficking, drove continuous T cell infiltration to the nigra and incessant glial inflammation. Supplementation of RANTES and eotaxin was also associated with the induction of nigral α-synuclein pathology, persistent loss of dopaminergic neurons and striatal neurotransmitters, and continuous impairment of motor functions in MPTP-intoxicated mice. In contrast, supplementation of TNF-α and IL-1ß, widely studied proinflammatory cytokines, did not induce persistent disease in MPTP-insulted mice. Our results suggest that induction of adaptive immunity by RANTES and eotaxin could hold the key for driving persistent nigrostriatal pathologies in the MPTP mouse model, and that targeting these factors may halt disease progression in PD patients.

BPOZ-2 Gene Delivery Ameliorates Alpha-Synucleinopathy in A53T Transgenic Mouse Model of Parkinson's Disease.

Ankyrin-rich BTB/POZ domain containing protein-2 or BPOZ-2, a scaffold protein, has been recently shown to control the degradation of many biological proteins ranging from embryonic development to tumor progression. However, its role in the process of neuronal diseases has not been properly explored. Since, abnormal clearance of metabolic proteins contributes to the development of alpha-synuclein (α-syn) pathologies in Parkinson's disease (PD), we are interested to explore if BPOZ-2 participates in the amelioration of α-syn in vivo in basal ganglia. Here we report that lentiviral administration of bpoz-2 gene indeed lowers the burden of α-syn in DA neurons in the nigra of A53T transgenic (A53T-Tg) mouse. Our detailed immunological analyses have shown that the overexpression of bpoz-2 dramatically improves both somatic and neuritic α-syn pathologies in the nigral DA neurons. Similarly, the specific ablation of bpoz-2 by lentiviral-shRNA stimulates the load of monomeric and polymeric forms of α-syn in the nigral DA neurons of A53T-Tg. While investigating the mechanism, we observed that BPOZ-2 was involved in a protein-protein association with PINK1 and therefore could stimulate PINK1-dependent autophagic clearance of α-syn. Our results have demonstrated that bpoz-2 gene delivery could have prospect in the amelioration of alpha-synucleinopathy in PD and other Lewy body diseases.

Intranasal Delivery of NEMO-Binding Domain Peptide Prevents Memory Loss in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia. Despite intense investigations, no effective therapy is available to halt its progression. We found that NF-κB was activated within the hippocampus and cortex of AD subjects and that activated forms of NF-κB negatively correlated with cognitive function monitored by Mini-Mental State Examination and global cognitive z score. Accordingly, NF-κB activation was also observed in the hippocampus of a transgenic (5XFAD) mouse model of AD. It has been shown that peptides corresponding to the NF-κB essential modifier (NEMO)-binding domain (NBD) of IκB kinase α (IKKα) or IκB kinase ß (IKKß) specifically inhibit the induction of NF-κB activation without inhibiting the basal NF-κB activity. Interestingly, after intranasal administration, wild-type NBD peptide entered into the hippocampus, reduced hippocampal activation of NF-κB, suppressed hippocampal microglial activation, lowered the burden of Aß in the hippocampus, attenuated apoptosis of hippocampal neurons, protected plasticity-related molecules, and improved memory and learning in 5XFAD mice. Mutated NBD peptide had no such protective effect, indicating the specificity of our finding. These results suggest that selective targeting of NF-κB activation by intranasal administration of NBD peptide may be of therapeutic benefit for AD patients.

Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Upregulates Ciliary Neurotrophic Factor in Astrocytes and Oligodendrocytes.

Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that plays an important role in multiple sclerosis (MS). However, mechanisms by which CNTF expression could be increased in the brain are poorly understood. Recently we have discovered anti-inflammatory and immunomodulatory activities of sodium benzoate (NaB), a metabolite of cinnamon and a widely-used food additive. Here, we delineate that NaB is also capable of increasing the mRNA and protein expression of CNTF in primary mouse astrocytes and oligodendrocytes and primary human astrocytes. Accordingly, oral administration of NaB and cinnamon led to the upregulation of astroglial and oligodendroglial CNTF in vivo in mouse brain. Induction of experimental allergic encephalomyelitis, an animal model of MS, reduced the level of CNTF in the brain, which was restored by oral administration of cinnamon. While investigating underlying mechanisms, we observed that NaB induced the activation of protein kinase A (PKA) and H-89, an inhibitor of PKA, abrogated NaB-induced expression of CNTF. The activation of cAMP response element binding (CREB) protein by NaB, the recruitment of CREB and CREB-binding protein to the CNTF promoter by NaB and the abrogation of NaB-induced expression of CNTF in astrocytes by siRNA knockdown of CREB suggest that NaB increases the expression of CNTF via the activation of CREB. These results highlight a novel myelinogenic property of NaB and cinnamon, which may be of benefit for MS and other demyelinating disorders.

A physically-modified saline suppresses neuronal apoptosis, attenuates tau phosphorylation and protects memory in an animal model of Alzheimer's disease.

Alzheimer's disease (AD), the leading cause of dementia in the aging population, is characterized by the presence of neuritic plaques, neurofibrillary tangles and extensive neuronal apoptosis. Neuritic plaques are mainly composed of aggregates of amyloid-ß (Aß) protein while neurofibrillary tangles are composed of the hyperphosphorylated tau protein. Despite intense investigations, no effective therapy is currently available to halt the progression of this disease. Here, we have undertaken a novel approach to attenuate apoptosis and tau phosphorylation in cultured neuronal cells and in a transgenic animal model of AD. RNS60 is a 0.9% saline solution containing oxygenated nanobubbles that is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. In our experiments, fibrillar Aß1-42, but not the reverse peptide Aß42-1, induced apoptosis and cell death in human SHSY5Y neuronal cells. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), attenuated Aß(1-42)-induced cell death. RNS60 inhibited neuronal cell death via activation of the type 1A phosphatidylinositol-3 (PI-3) kinase-Akt-BAD pathway. Furthermore, RNS60 also decreased Aß(1-42)-induced tau phosphorylation via (PI-3 kinase-Akt)-mediated inhibition of GSK-3ß. Similarly, RNS60 treatment suppressed neuronal apoptosis, attenuated Tau phosphorylation, inhibited glial activation, and reduced the burden of Aß in the hippocampus and protected memory and learning in 5XFAD transgenic mouse model of AD. Therefore, RNS60 may be a promising pharmaceutical candidate in halting or delaying the progression of AD.

Enhancement of morphological plasticity in hippocampal neurons by a physically modified saline via phosphatidylinositol-3 kinase.

Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer's disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.

Cinnamon treatment upregulates neuroprotective proteins Parkin and DJ-1 and protects dopaminergic neurons in a mouse model of Parkinson's disease.

Upregulation and/or maintenance of Parkinson's disease (PD)-related beneficial proteins such as Parkin and DJ-1 in astrocytes during neurodegenerative insults may have therapeutic efficacy in PD. Cinnamon is a commonly used natural spice and flavoring material throughout the world. Here we have explored a novel use of cinnamon in upregulating Parkin and DJ-1 and protecting dopaminergic neurons in MPTP mouse model of PD. Recently we have delineated that oral feeding of cinnamon (Cinnamonum verum) powder produces sodium benzoate (NaB) in blood and brain of mice. Proinflammatory cytokine IL-1ß decreased the level of Parkin/DJ-1 in mouse astrocytes. However, cinnamon metabolite NaB abrogated IL-1ß-induced loss of these proteins. Inability of TNF-α to produce nitric oxide (NO) and decrease the level of Parkin/DJ-1 in wild type (WT) astrocytes, failure of IL-1ß to reduce Parkin/DJ-1 in astrocytes isolated from iNOS (-/-) mice, and decrease in Parkin/DJ-1 in WT astrocytes by NO donor DETA-NONOate suggest that NO is a negative regulator of Parkin/DJ-1. Furthermore, suppression of IL-1ß-induced expression of iNOS in astrocytes by NaB and reversal of NaB-mediated protection of Parkin/DJ-1 by DETA-NONOate in astrocytes indicate that NaB protects Parkin/DJ-1 in activated astrocytes via suppressing iNOS. Similarly MPTP intoxication also increased the level of iNOS and decreased the level of Parkin/DJ-1 in vivo in the nigra. However, oral treatment of MPTP-intoxicated mice with cinnamon powder and NaB reduced the expression of iNOS and protected Parkin/DJ-1 in the nigra. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions by cinnamon in MPTP-intoxicated mice. These results suggest that cinnamon may be beneficial for PD patients.

Ankyrin repeat and BTB/POZ domain containing protein-2 inhibits the aggregation of alpha-synuclein: implications for Parkinson's disease.

Aggregation of α-synuclein is a pathological hallmark of sporadic or familial PD. However, the detailed molecular mechanism responsible for the aggregation of α-synuclein has not been properly explored. In the present study, we have identified a novel role of an anti-tumorigenic BTB/POZ domain containing protein-2 (BPOZ-2) in the regulation of α-synuclein accumulation in dopaminergic (DA) neurons. MPP(+), an etiological factor for PD, significantly downregulated the expression of BPOZ-2 ahead of α-synuclein upregulation. Moreover, siRNA knockdown of BPOZ-2 alone stimulated the aggregation of α-synuclein protein; the effect was further induced in presence of MPP(+) in mouse primary DA neurons. Finally, the absence of BPOZ-2 in α-synuclein expressing neuronal populations of MPTP-intoxicated mouse and primate nigra indicates that the suppression of BPOZ-2 could be involved in the accumulation of α-synuclein protein.

Altered spinal microRNA-146a and the microRNA-183 cluster contribute to osteoarthritic pain in knee joints.

The objective of this study was to examine whether altered expression of microRNAs in central nervous system components is pathologically linked to chronic knee joint pain in osteoarthritis. A surgical animal model for knee joint OA was generated by medial meniscus transection in rats followed by behavioral pain tests. Relationships between pathological changes in knee joint and development of chronic joint pain were examined by histology and imaging analyses. Alterations in microRNAs associated with OA-evoked pain sensation were determined in bilateral lumbar dorsal root ganglia (DRG) and the spinal dorsal horn by microRNA array followed by individual microRNA analyses. Gain- and loss-of-function studies of selected microRNAs (miR-146a and miR-183 cluster) were conducted to identify target pain mediators regulated by these selective microRNAs in glial cells. The ipsilateral hind leg displayed significantly increased hyperalgesia after 4 weeks of surgery, and sensitivity was sustained for the remainder of the 8-week experimental period (F = 341, p < 0.001). The development of OA-induced chronic pain was correlated with pathological changes in the knee joints as assessed by histological and imaging analyses. MicroRNA analyses showed that miR-146a and the miR-183 cluster were markedly reduced in the sensory neurons in DRG (L4/L5) and spinal cord from animals experiencing knee joint OA pain. The downregulation of miR-146a and/or the miR-183 cluster in the central compartments (DRG and spinal cord) are closely associated with the upregulation of inflammatory pain mediators. The corroboration between decreases in these signature microRNAs and their specific target pain mediators were further confirmed by gain- and loss-of-function analyses in glia, the major cellular component of the central nervous system (CNS). MicroRNA therapy using miR-146a and the miR-183 cluster could be powerful therapeutic intervention for OA in alleviating joint pain and concomitantly regenerating peripheral knee joint cartilage.

Up-regulation of ciliary neurotrophic factor in astrocytes by aspirin: implications for remyelination in multiple sclerosis.

Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor, and the mechanisms by which CNTF expression could be increased in the brain are poorly understood. Acetylsalicylic acid (aspirin) is one of the most widely used analgesics. Interestingly, aspirin increased mRNA and protein expression of CNTF in primary mouse and human astrocytes in a dose- and time-dependent manner. Aspirin induced the activation of protein kinase A (PKA) but not protein kinase C (PKC). H-89, an inhibitor of PKA, abrogated aspirin-induced expression of CNTF. The activation of cAMP-response element-binding protein (CREB), but not NF-κB, by aspirin, the abrogation of aspirin-induced expression of CNTF by siRNA knockdown of CREB, the presence of a consensus cAMP-response element in the promoter of CNTF, and the recruitment of CREB and CREB-binding protein to the CNTF promoter by aspirin suggest that aspirin increases the expression of the Cntf gene via the activation of CREB. Furthermore, we demonstrate that aspirin-induced astroglial CNTF was also functionally active and that supernatants of aspirin-treated astrocytes of wild type, but not Cntf null, mice increased myelin-associated proteins in oligodendrocytes and protected oligodendrocytes from TNF-α insult. These results highlight a new and novel myelinogenic property of aspirin, which may be of benefit for multiple sclerosis and other demyelinating disorders.

Suppression of nuclear factor-κB activation and inflammation in microglia by physically modified saline.

Chronic inflammation involving activated microglia and astroglia is becoming a hallmark of many human diseases, including neurodegenerative disorders. Although NF-κB is a multifunctional transcription factor, it is an important target for controlling inflammation as the transcription of many proinflammatory molecules depends on the activation of NF-κB. Here, we have undertaken a novel approach to attenuate NF-κB activation and associated inflammation in activated glial cells. RNS60 is a 0.9% saline solution containing charge-stabilized nanostructures that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not normal saline, RNS10.3 (TCP-modified saline without excess oxygen), and PNS60 (saline containing excess oxygen without TCP modification) were found to inhibit the production of nitric oxide (NO) and the expression of inducible NO synthase in activated microglia. Similarly, RNS60 also inhibited the expression of inducible NO synthase in activated astroglia. Inhibition of NF-κB activation by RNS60 suggests that RNS60 exerts its anti-inflammatory effect through the inhibition of NF-κB. Interestingly, RNS60 induced the activation of type IA phosphatidylinositol (PI) 3-kinase and Akt and rapidly up-regulated IκBα, a specific endogenous inhibitor of NF-κB. Inhibition of PI 3-kinase and Akt by either chemical inhibitors or dominant-negative mutants abrogated the RNS60-mediated up-regulation of IκBα. Furthermore, we demonstrate that RNS60 induced the activation of cAMP-response element-binding protein (CREB) via the PI 3-kinase-Akt pathway and that RNS60 up-regulated IκBα via CREB. These results describe a novel anti-inflammatory property of RNS60 via type IA PI 3-kinase-Akt-CREB-mediated up-regulation of IκBα, which may be of therapeutic benefit in neurodegenerative disorders.