Muscle-building supplement ß-hydroxy ß-methylbutyrate stimulates the maturation of oligodendroglial progenitor cells to oligodendrocytes.

Oligodendrocytes are the myelinating cells in the CNS and multiple sclerosis (MS) is a demyelinating disorder that is characterized by progressive loss of myelin. Although oligodendroglial progenitor cells (OPCs) should be differentiated into oligodendrocytes, for multiple reasons, OPCs fail to differentiate into oligodendrocytes in MS. Therefore, increasing the maturation of OPCs to oligodendrocytes may be of therapeutic benefit for MS. The ß-hydroxy ß-methylbutyrate (HMB) is a muscle-building supplement in humans and this study underlines the importance of HMB in stimulating the maturation of OPCs to oligodendrocytes. HMB treatment upregulated the expression of different maturation markers including PLP, MBP, and MOG in cultured OPCs. Double-label immunofluorescence followed by immunoblot analyses confirmed the upregulation of OPC maturation by HMB. While investigating mechanisms, we found that HMB increased the maturation of OPCs isolated from peroxisome proliferator-activated receptor ß-/- (PPARß-/-) mice, but not PPARα-/- mice. Similarly, GW6471 (an antagonist of PPARα), but not GSK0660 (an antagonist of PPARß), inhibited HMB-induced maturation of OPCs. GW9662, a specific inhibitor of PPARγ, also could not inhibit HMB-mediated stimulation of OPC maturation. Furthermore, PPARα agonist GW7647, but neither PPARß agonist GW0742 nor PPARγ agonist GW1929, alone increased the maturation of OPCs. Finally, HMB treatment of OPCs led to the recruitment of PPARα, but neither PPARß nor PPARγ, to the PLP gene promoter. These results suggest that HMB stimulates the maturation of OPCs via PPARα and that HMB may have therapeutic prospects in remyelination.

Treadmill workout activates PPARα in the hippocampus to upregulate ADAM10, decrease plaques and improve cognitive functions in 5XFAD mouse model of Alzheimer's disease.

Although liver is rich in peroxisome proliferator-activated receptor α (PPARα), recently we have described the presence of PPARα in hippocampus where it is involved in non-amyloidogenic metabolism of amyloid precursor protein (APP) via ADAM10, decreasing amyloid plaques and improving memory and learning. However, mechanisms to upregulate PPARα in vivo in the hippocampus are poorly understood. Regular exercise has multiple beneficial effects on human health and here, we describe the importance of regular mild treadmill exercise in upregulating PPARα in vivo in the hippocampus of 5XFAD mouse model of Alzheimer's disease. We also demonstrate that treadmill exercise remained unable to stimulate ADAM10, reduce plaque pathology and improve cognitive functions in 5XFADΔPPARα mice (5XFAD mice lacking PPARα). On the other hand, treadmill workout increased ADAM10, decreased plaque pathology and protected memory and learning in 5XFADΔPPARß mice (5XFAD mice lacking PPARß). Moreover, the other PPAR (PPARγ) also did not play any role in the transcription of ADAM10 in vivo in the hippocampus of treadmill exercised 5XFAD mice. These results underline an important role of PPARα in which treadmill exercise remains unable to exhibit neuroprotection in the hippocampus in the absence of PPARα.

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.

Oleamide, a Sleep-Inducing Supplement, Upregulates Doublecortin in Hippocampal Progenitor Cells via PPARα.

BACKGROUND: Doublecortin (DCX), a microtubule associated protein, has emerged as a central biomarker of hippocampal neurogenesis. However, molecular mechanisms by which DCX is regulated are poorly understood. OBJECTIVE: Since sleep is involved with the acquisition of memory and oleamide or 9-Octadecenamide (OCT) is a sleep-inducing supplement in human, we examined whether OCT could upregulate DCX in hippocampal progenitor cells (HPCs). METHODS: We employed real-time PCR, western blot, immunostaining, chromatin immunoprecipitation, lentiviral transduction in HPCs, and the calcium influx assay. RESULTS: OCT directly upregulated the transcription of Dcx in HPCs via activation of peroxisome proliferator-activated receptor α (PPARα), a lipid-lowering transcription factor. We observed that, HPCs of Ppara-null mice displayed significant impairment in DCX expression and neuronal differentiation as compared to that of wild-type mice. Interestingly, treatment with OCT stimulated the differentiation process of HPCs in wild-type, but not Ppara-null mice. Reconstruction of PPARα in mouse Ppara-null HPCs restored the expression of DCX, which was further stimulated with OCT treatment. In contrast, a dominant-negative mutant of PPARα significantly attenuated the stimulatory effect of OCT on DCX expression and suppressed neuronal differentiation of human neural progenitor cells. Furthermore, RNA microarray, STRING, chromatin immunoprecipitation, site-directed mutagenesis, and promoter reporter assay have identified DCX as a new target of PPARα. CONCLUSION: These results indicate that OCT, a sleep supplement, directly controls the expression of DCX and suggest that OCT may be repurposed for stimulating the hippocampal neurogenesis.

Inhibition of Human Coronaviruses by Antimalarial Peroxides.

As the toll of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues, efforts are ongoing to identify new agents and repurpose safe drugs for its treatment. Antimalarial peroxides have reported antiviral and anticancer activities. Here, we evaluated the in vitro activities of artesunate (AS) and two ozonides (OZ418 and OZ277) against human α-coronavirus NL63 and ß-coronaviruses OC43 and SARS-CoV-2 in several cell lines. OZ418 had the best selectivity index (SI) in NL63-infected Vero cells and MK2 cells. The overall SI of the tested compounds was cell-type dependent. In OC43-infected human foreskin fibroblasts, AS had the best cell-associated SI, ≥17 µM, while the SI of OZ418 and OZ277 was ≥12 µM and ≥7 µM, respectively. AS did not inhibit SARS-CoV-2 in either Vero or Calu-3 cells. A comparison of OZ418 and OZ277 activity in SARS-CoV2-infected Calu-3 cells revealed similar EC50 (5.3 µM and 11.6 µM, respectively), higher than the EC50 of remdesivir (1.0 ± 0.1 µM), but the SI of OZ418 was higher than OZ277. A third ozonide, OZ439, inhibited SARS-CoV-2 efficiently in Vero cells, but compared to OZ418 in Calu-3 cells, it showed higher toxicity. Improved inhibition of SARS-CoV-2 was observed when OZ418 was used together with remdesivir. Although the EC50 of ozonides might be clinically achieved in plasma after intravenous administration, sustained virus suppression in tissues will require further considerations, including drug combination. Our work supports the potential repurposing of ozonides and calls for future in vivo models.

IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rß1 internalization and suppress EAE.

Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system. The IL-12 family of cytokines has four members, which are IL-12 (p40:p35), IL-23 (p40:p19), the p40 monomer (p40), and the p40 homodimer (p402). Since all four members contain p40 in different forms, it is important to use a specific monoclonal antibody (mAb) to characterize these molecules. Here, by using such mAbs, we describe selective loss of p40 in serum of MS patients as compared to healthy controls. Similarly, we also observed decrease in p40 and increase in IL-12, IL-23, and p402 in serum of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as compared to control mice. Interestingly, weekly supplementation of mouse and human recombinant p40 ameliorated clinical symptoms and disease progression of EAE. On the other hand, IL-12, IL-23, and p402 did not exhibit such inhibitory effect. In addition to EAE, p40 also suppressed collagen-induced arthritis in mice. Using IL-12Rß1-/-, IL-12Rß2-/-, and IL-12Rß1+/-/IL-12Rß2-/- mice, we observed that p40 required IL-12Rß1, but not IL-12Rß2, to suppress EAE. Interestingly, p40 arrested IL-12-, IL-23-, or p402-mediated internalization of IL-12Rß1, but neither IL-12Rß2 nor IL-23R, protected regulatory T cells, and suppressed Th1 and Th17 biasness. These studies identify p40 as an anti-autoimmune cytokine with a biological role different from IL-12, IL-23, and p402 in which it attenuates autoimmune signaling via suppression of IL-12Rß1 internalization, which may be beneficial in patients with MS and other autoimmune disorders.

Upregulation of BDNF and hippocampal functions by a hippocampal ligand of PPARα.

Discovery strategies commonly focus on the identification of chemical libraries or natural products, but the modulation of endogenous ligands offers a much better therapeutic strategy due to their low adverse potential. Recently, we found that hexadecanamide (Hex) is present in hippocampal nuclei of normal mice as an endogenous ligand of PPARα. This study underlines the importance of Hex in inducing the expression of brain-derived neurotrophic factor (BDNF) from hippocampal neurons via PPARα. The level of Hex was lower in the hippocampi of 5XFAD mice as compared with that in non-Tg mice. Oral administration of Hex increased the level of this molecule in the hippocampus to stimulate BDNF and its downstream plasticity-associated molecules, promote synaptic functions in the hippocampus, and improve memory and learning in 5XFAD mice. However, oral Hex remained unable to stimulate hippocampal plasticity and improve cognitive behaviors in 5XFADPparα-null and 5XFADPparα-ΔHippo mice, indicating an essential role of hippocampal PPARα in Hex-mediated improvement in hippocampal functions. This is the first demonstration to our knowledge of protection of hippocampal functions by oral administration of a hippocampus-based drug, suggesting that Hex may be explored for therapeutic intervention in AD.

RANTES-induced invasion of Th17 cells into substantia nigra potentiates dopaminergic cell loss in MPTP mouse model of Parkinson's disease.

Although Parkinson's disease (PD) is a progressive neurodegenerative disease, the disease does not progress or persist in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, the most common animal model of PD. Recently, we have described that supplementation of regulated on activation, normal T cell expressed and secreted (RANTES), a chemokine known to drive infiltration of T cells, induces persistent nigrostriatal pathology in MPTP mouse model. However, which particular T cell subsets are recruited to the substantia nigra (SN) by RANTES is not known. Here, by adoptive transfer of different subset of T cells from tomato red transgenic mice to MPTP-intoxicated immunodeficient Rag1-/- mice, we describe that invasion of Th17 cells into the SN is stimulated by exogenous RANTES administration. On the other hand, RANTES supplementation remained unable to influence the infiltration of Th1 and Tregs into the SN of MPTP-insulted Rag1-/- mice. Accordingly, RANTES supplementation increased MPTP-induced TH cell loss in Rag1-/-mice receiving Th17, but neither Th1 nor Tregs. RANTES-mediated aggravation of nigral TH neurons also paralleled with significant DA loss in striatum and locomotor deficits in MPTP-intoxicated Rag1-/- mice receiving Th17 cells. Finally, we demonstrate that levels of IL-17 (a Th17-specific cytokine) and RANTES are higher in serum of PD patients than age-matched controls and that RANTES positively correlated with IL-17 in serum of PD patients. Together, these results highlight the importance of RANTES-Th17 pathway in progressive dopaminergic neuronal loss and associated PD pathology.

Activation of Peroxisome Proliferator-Activated Receptor-α Increases the Expression of Nuclear Receptor Related 1 Protein (Nurr1) in Dopaminergic Neurons.

Nuclear receptor related 1 protein (Nurr1) is an important transcription factor required for differentiation and maintenance of midbrain dopaminergic (DA) neurons. Since decrease in Nurr1 function either due to diminished expression or rare mutation is associated with Parkinson's disease (PD), upregulation of Nurr1 may be beneficial for PD. However, such mechanisms are poorly understood. This study underlines the importance of peroxisome proliferator-activated receptor (PPAR)α in controlling the transcription of Nurr1. Our mRNA analyses followed by different immunoassays clearly indicated that PPARα agonist gemfibrozil strongly upregulated the expression of Nurr1 in wild-type, but not PPARα-/-, DA neurons. Moreover, identification of conserved PPRE in the promoter of Nurr1 gene followed by chromatin immunoprecipitation analysis, PPRE luciferase assay, and manipulation of Nurr1 gene by viral transduction of different PPARα plasmids confirmed that PPARα was indeed involved in the expression of Nurr1. Finally, oral administration of gemfibrozil increased Nurr1 expression in vivo in nigra of wild-type, but not PPARα-/-, mice identifying PPARα as a novel regulator of Nurr1 expression and associated protection of DA neurons.

Upregulation of tripeptidyl-peptidase 1 by 3-hydroxy-(2,2)-dimethyl butyrate, a brain endogenous ligand of PPARα: Implications for late-infantile Batten disease therapy.

The late-infantile Batten disease or late-infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive lysosomal storage disorder caused by mutations in the Cln2 gene leading to deficiency of lysosomal enzyme tripeptidyl peptidase 1 (TPP1). At present, available options for this fatal disorder are enzyme replacement therapy and gene therapy, which are extensively invasive and expensive. Our study demonstrates that 3-hydroxy-(2,2)-dimethyl butyrate (HDMB), a brain endogenous molecule, is capable of stimulating TPP1 expression and activity in mouse primary astrocytes and a neuronal cell line. HDMB activated peroxisome proliferator-activated receptor-α (PPARα), which, by forming heterodimer with Retinoid X receptor-α (RXRα), transcriptionally upregulated the Cln2 gene. Moreover, by using primary astrocytes from wild type, PPARα-/- and PPARß-/- mice, we demonstrated that HDMB specifically required PPARα for inducing TPP1 expression. Finally, oral administration of HDMB to Cln2 heterozygous (Cln2+/-) mice led to a marked upregulation of TPP1 expression in the motor cortex and striatum in a PPARα-dependent fashion. Our study suggests that HDMB, a brain endogenous ligand of PPARα, might have therapeutic importance for LINCL treatment.

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.

Aspirin binds to PPARα to stimulate hippocampal plasticity and protect memory.

Despite its long history, until now, no receptor has been identified for aspirin, one of the most widely used medicines worldwide. Here we report that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear hormone receptor involved in fatty acid metabolism, serves as a receptor of aspirin. Detailed proteomic analyses including cheminformatics, thermal shift assays, and TR-FRET revealed that aspirin, but not other structural homologs, acts as a PPARα ligand through direct binding at the Tyr314 residue of the PPARα ligand-binding domain. On binding to PPARα, aspirin stimulated hippocampal plasticity via transcriptional activation of cAMP response element-binding protein (CREB). Finally, hippocampus-dependent behavioral analyses, calcium influx assays in hippocampal slices and quantification of dendritic spines demonstrated that low-dose aspirin treatment improved hippocampal plasticity and memory in FAD5X mice, but not in FAD5X/Ppara-null mice. These findings highlight a property of aspirin: stimulating hippocampal plasticity via direct interaction with PPARα.

Increase in Mitochondrial Biogenesis in Neuronal Cells by RNS60, a Physically-Modified Saline, via Phosphatidylinositol 3-Kinase-Mediated Upregulation of PGC1α.

This study highlights a novel approach to upregulate mitochondrial biogenesis in neuronal cells. 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. 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), increased the expression of Nrf1, Tfam, Mcu, and Tom20 (genes associated with mitochondrial biogenesis) and upregulated mitochondrial biogenesis in MN9D dopaminergic neuronal cells. Similarly RNS60 also increased mitochondrial biogenesis in primary dopaminergic neurons and in the nigra of MPTP-intoxicated mice. However, RNS60 had no effect on lysosomal biogenesis. Interestingly, we found that RNS60 upregulated PGC1α and siRNA knockdown of PGC1α abrogated the ability of RNS60 to increase mitochondrial biogenesis. Furthermore, we delineated that RNS60 increased the transcription of Pgc1a via type IA phosphatidylinositol (PI) 3-kinase-mediated activation of cAMP-response element-binding protein (CREB). Accordingly, knockdown of the PI3K - CREB pathway suppressed RNS60-mediated mitochondrial biogenesis. These results describe a novel property of RNS60 of enhancing mitochondrial biogenesis via PI 3-kinase-CREB-mediated up-regulation of PGC1α, which may be of therapeutic benefit in different neurodegenerative disorders.

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.

Identification and characterization of PPARα ligands in the hippocampus.

Peroxisome proliferator-activated receptor-α (PPARα) regulates hepatic fatty acid catabolism and mediates the metabolic response to starvation. Recently we found that PPARα is constitutively activated in nuclei of hippocampal neurons and controls plasticity via direct transcriptional activation of CREB. Here we report the discovery of three endogenous PPARα ligands-3-hydroxy-(2,2)-dimethyl butyrate, hexadecanamide, and 9-octadecenamide-in mouse brain hippocampus. Mass spectrometric detection of these compounds in mouse hippocampal nuclear extracts, in silico interaction studies, time-resolved FRET analyses, and thermal shift assay results clearly indicated that these three compounds served as ligands of PPARα. Site-directed mutagenesis studies further revealed that PPARα Y464 and Y314 are involved in binding these hippocampal ligands. Moreover, these ligands activated PPARα and upregulated the synaptic function of hippocampal neurons. These results highlight the discovery of hippocampal ligands of PPARα capable of modulating synaptic functions.

Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Enriches Regulatory T Cells via STAT6-Mediated Upregulation of TGF-ß.

Upregulation and/or maintenance of regulatory T cells (Tregs) during autoimmune insults may have therapeutic efficacy in autoimmune diseases. Earlier we have reported that sodium benzoate (NaB), a metabolite of cinnamon and a Food and Drug Administration-approved drug against urea cycle disorders, upregulates Tregs and protects mice from experimental allergic encephalomyelitis, an animal model of multiple sclerosis. However, mechanisms by which NaB increases Tregs are poorly understood. Because TGF-ß is an important inducer of Tregs, we examined the effect of NaB on the status of TGF-ß. In this study, we demonstrated that NaB induced the expression of TGF-ß mRNA and protein in normal as well as proteolipid protein-primed splenocytes. The presence of a consensus STAT6 binding site in the promoter of the TGF-ß gene, activation of STAT6 in splenocytes by NaB, recruitment of STAT6 to the TGF-ß promoter by NaB, and abrogation of NaB-induced expression of TGF-ß in splenocytes by small interfering RNA knockdown of STAT6 suggest that NaB induces the expression of TGF-ß via activation of STAT6. Furthermore, we demonstrated that blocking of TGF-ß by neutralizing Abs abrogated NaB-mediated protection of Tregs and experimental allergic encephalomyelitis. These studies identify a new function of NaB in upregulating TGF-ß via activation of STAT6, which may be beneficial in MS 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.

HMG-CoA Reductase Inhibitors Bind to PPARα to Upregulate Neurotrophin Expression in the Brain and Improve Memory in Mice.

Neurotrophins are important for neuronal health and function. Here, statins, inhibitors of HMG-CoA reductase and cholesterol lowering drugs, were found to stimulate expression of neurotrophins in brain cells independent of the mevalonate pathway. Time-resolved fluorescence resonance energy transfer (FRET) analyses, computer-derived simulation, site-directed mutagenesis, thermal shift assay, and de novo binding followed by electrospray ionization tandem mass spectrometry (ESI-MS) demonstrates that statins serve as ligands of PPARα and that Leu331 and Tyr 334 residues of PPARα are important for statin binding. Upon binding, statins upregulate neurotrophins via PPARα-mediated transcriptional activation of cAMP-response element binding protein (CREB). Accordingly, simvastatin increases CREB and brain-derived neurotrophic factor (BDNF) in the hippocampus of Ppara null mice receiving full-length lentiviral PPARα, but not L331M/Y334D statin-binding domain-mutated lentiviral PPARα. This study identifies statins as ligands of PPARα, describes neurotrophic function of statins via the PPARα-CREB pathway, and analyzes the importance of PPARα in the therapeutic success of simvastatin in an animal model of Alzheimer's disease.

Unfolding studies of Escherichia coli maltodextrin glucosidase monitored by fluorescence spectroscopy.

Equilibrium unfolding of a 69-kDa monomeric Escherichia coli maltodextrin glucosidase (MalZ) was studied using intrinsic and extrinsic fluorescence spectroscopy. The unfolding transition of MalZ followed a three-state process, involving the formation of a stable intermediate state having more exposed hydrophobic surface. It was found that the protein structure can be easily perturbed by low concentration of guanidium hydrochloride (GdnHCl) and, at a GdnHCl concentration of 2 M, MalZ was denatured completely. The active site of the protein also has been proved to be sensitive to a low concentration of GdnHCl since MalZ deactivated at 0.5 M GdnHCl completely. The surface hydrophobicity and ANS-binding site of the protein have been determined to be 150.7 and 0.24, respectively. Perhaps the formation of the stable unfolding intermediate, having higher surface hydrophobicity, may be one of the reasons for aggregation of MalZ and its recognition by chaperonin GroEL during the assisted folding pathway.