Grant T. Corbett

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 Alzheimers disease.

Regulation of Cyclic AMP Response Element Binding and Hippocampal Plasticity-Related Genes by Peroxisome Proliferator-Activated Receptor α

Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that regulates genes involved in fatty acid catabolism. Here, we provide evidence that PPARα is constitutively expressed in nuclei of hippocampal neurons and, surprisingly, controls calcium influx and the expression of various plasticity-related genes via direct transcriptional regulation of cyclic AMP response element binding (CREB). Accordingly, Pparα-null, but not Pparβ-null, mice are deficient in CREB and memory-associated proteins and have decreased spatial learning and memory. Small hairpin RNA knockdown of PPARα in the hippocampus suppressed CREB and NR2A, rendering wild-type animals markedly poor in consolidating spatial memory, whereas introduction of PPARα to the hippocampus of Pparα-null mice increased hippocampal CREB and NR2A and improved spatial learning and memory. Through detailed analyses of CREB and NR2A activity, as well as spatial learning and memory in bone marrow chimeric animals lacking PPARα in the CNS, we uncover a mechanism for transcriptional control of Creb and associated plasticity genes by PPARα.

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.

Gemfibrozil, a Lipid-Lowering Drug, Upregulates IL-1 Receptor Antagonist in Mouse Cortical Neurons: Implications for Neuronal Self-Defense

Chronic inflammation is becoming a hallmark of several neurodegenerative disorders and accordingly, IL-1β, a proinflammatory cytokine, is implicated in the pathogenesis of neurodegenerative diseases. Although IL-1β binds to its high-affinity receptor, IL-1R, and upregulates proinflammatory signaling pathways, IL-1R antagonist (IL-1Ra) adheres to the same receptor and inhibits proinflammatory cell signaling. Therefore, upregulation of IL-1Ra is considered important in attenuating inflammation. The present study underlines a novel application of gemfibrozil (gem), a Food and Drug Administration-approved lipid-lowering drug, in increasing the expression of IL-1Ra in primary mouse and human neurons. Gem alone induced an early and pronounced increase in the expression of IL-1Ra in primary mouse cortical neurons. Activation of type IA p110α PI3K and Akt by gem and abrogation of gem-induced upregulation of IL-1Ra by inhibitors of PI3K and Akt indicate a role of the PI3K-Akt pathway in the upregulation of IL-1Ra. Gem also induced the activation of CREB via the PI3K-Akt pathway, and small interfering RNA attenuation of CREB abolished the gem-mediated increase in IL-1Ra. Furthermore, gem was able to protect neurons from IL-1β insult. However, small interfering RNA knockdown of neuronal IL-1Ra abrogated the protective effect of gem against IL-1β, suggesting that this drug increases the defense mechanism of cortical neurons via upregulation of IL-1Ra. Taken together, these results highlight the importance of the PI3K-Akt-CREB pathway in mediating gem-induced upregulation of IL-1Ra in neurons and suggest gem as a possible therapeutic treatment for propagating neuronal self-defense in neuroinflammatory and neurodegenerative disorders.

Activation of peroxisome proliferator-activated receptor α stimulates ADAM10-mediated proteolysis of APP

Significance Although β-amyloid (Aβ) peptides participate in the pathogenesis of Alzheimer’s disease (AD), the mechanisms that regulate Aβ production are poorly understood. Here, we demonstrate that activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) upregulates transcription of the “a disintegrin and metalloproteinase” 10 (Adam10) gene and shifts APP processing toward the α-secretase pathway. These findings suggest PPARα could be a therapeutic target for reducing Aβ burden in AD. Amyloid precursor protein (APP) derivative β-amyloid (Aβ) plays an important role in the pathogenesis of Alzheimer’s disease (AD). Sequential proteolysis of APP by β-secretase and γ-secretase generates Aβ. Conversely, the α-secretase “a disintegrin and metalloproteinase” 10 (ADAM10) cleaves APP within the eventual Aβ sequence and precludes Aβ generation. Therefore, up-regulation of ADAM10 represents a plausible therapeutic strategy to combat overproduction of neurotoxic Aβ. Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that regulates genes involved in fatty acid metabolism. Here, we determined that the Adam10 promoter harbors PPAR response elements; that knockdown of PPARα, but not PPARβ or PPARγ, decreases the expression of Adam10; and that lentiviral overexpression of PPARα restored ADAM10 expression in Ppara−/− neurons. Gemfibrozil, an agonist of PPARα, induced the recruitment of PPARα:retinoid x receptor α, but not PPARγ coactivator 1α (PGC1α), to the Adam10 promoter in wild-type mouse hippocampal neurons and shifted APP processing toward the α-secretase, as determined by augmented soluble APPα and decreased Aβ production. Accordingly, Ppara−/− mice displayed elevated SDS-stable, endogenous Aβ and Aβ1–42 relative to wild-type littermates, whereas 5XFAD mice null for PPARα (5X/α−/−) exhibited greater cerebral Aβ load relative to 5XFAD littermates. These results identify PPARα as an important factor regulating neuronal ADAM10 expression and, thus, α-secretase proteolysis of APP.

Sodium Phenylbutyrate Enhances Astrocytic Neurotrophin Synthesis via Protein Kinase C (PKC)-mediated Activation of cAMP-response Element-binding Protein (CREB): IMPLICATIONS FOR ALZHEIMER DISEASE THERAPY*

Background: Increase in neurotrophic factors in the brain is a possible therapeutic approach for different neurodegenerative disorders. Results: Sodium phenylbutyrate, an FDA-approved drug for hyperammonemia, increases neurotrophic factors in brain cells via the PKC-CREB pathway. Conclusion: These results delineate a novel neurotrophic property of sodium phenylbutyrate. Significance: Sodium phenylbutyrate may be of therapeutic benefit in neurodegenerative disorders. Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser133) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

Gemfibrozil and Fenofibrate, Food and Drug Administration-approved Lipid-lowering Drugs, Up-regulate Tripeptidyl-peptidase 1 in Brain Cells via Peroxisome Proliferator-activated Receptor α IMPLICATIONS FOR LATE INFANTILE BATTEN DISEASE THERAPY

Background: Increase in tripeptidyl-peptidase 1 (TPP1) is a possible therapeutic approach for late infantile Batten disease or neuronal ceroid lipofuscinosis (LINCL). Results: Gemfibrozil and fenofibrate, FDA-approved drugs for hyperlipidemia, stimulate TPP1 in brain cells via the PPARα/RXRα pathway. Conclusion: These results delineate a novel TPP1 up-regulating property of gemfibrozil and fenofibrate. Significance: Gemfibrozil and fenofibrate may be of therapeutic benefit in Batten disease. The classical late infantile neuronal ceroid lipofuscinosis (LINCLs) is an autosomal recessive disease, where the defective gene is Cln2, encoding tripeptidyl-peptidase I (TPP1). At the molecular level, LINCL is caused by accumulation of autofluorescent storage materials in neurons and other cell types. Currently, there is no established treatment for this fatal disease. This study reveals a novel use of gemfibrozil and fenofibrate, Food and Drug Administration-approved lipid-lowering drugs, in up-regulating TPP1 in brain cells. Both gemfibrozil and fenofibrate up-regulated mRNA, protein, and enzymatic activity of TPP1 in primary mouse neurons and astrocytes as well as human astrocytes and neuronal cells. Because gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor-α (PPARα), the role of PPARα in gemfibrozil- and fenofibrate-mediated up-regulation of TPP1 was investigated revealing that both drugs up-regulated TPP1 mRNA, protein, and enzymatic activity both in vitro and in vivo in wild type (WT) and PPARβ−/−, but not PPARα−/−, mice. In an attempt to delineate the mechanism of TPP1 up-regulation, it was found that the effects of the fibrate drugs were abrogated in the absence of retinoid X receptor-α (RXRα), a molecule known to form a heterodimer with PPARα. Accordingly, all-trans-retinoic acid, alone or together with gemfibrozil, up-regulated TPP1. Co-immunoprecipitation and ChIP studies revealed the formation of a PPARα/RXRα heterodimer and binding of the heterodimer to an RXR-binding site on the Cln2 promoter. Together, this study demonstrates a unique mechanism for the up-regulation of TPP1 by fibrate drugs via PPARα/RXRα pathway.

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

Alzheimers 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 β (IKKβ) 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 Aβ 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.

Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo

Summary A major challenge for clinical application of pluripotent stem cell therapy for Parkinsons disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

Detection of Aggregation-Competent Tau in Neuron-Derived Extracellular Vesicles

Progressive cerebral accumulation of tau aggregates is a defining feature of Alzheimer’s disease (AD). A popular theory that seeks to explain the apparent spread of neurofibrillary tangle pathology proposes that aggregated tau is passed from neuron to neuron. Such a templated seeding process requires that the transferred tau contains the microtubule binding repeat domains that are necessary for aggregation. While it is not clear how a protein such as tau can move from cell to cell, previous reports have suggested that this may involve extracellular vesicles (EVs). Thus, measurement of tau in EVs may both provide insights on the molecular pathology of AD and facilitate biomarker development. Here, we report the use of sensitive immunoassays specific for full-length (FL) tau and mid-region tau, which we applied to analyze EVs from human induced pluripotent stem cell (iPSC)-derived neuron (iN) conditioned media, cerebrospinal fluid (CSF), and plasma. In each case, most tau was free-floating with a small component inside EVs. The majority of free-floating tau detected by the mid-region assay was not detected by our FL assays, indicating that most free-floating tau is truncated. Inside EVs, the mid-region assay also detected more tau than the FL assay, but the ratio of FL-positive to mid-region-positive tau was higher inside exosomes than in free solution. These studies demonstrate the presence of minute amounts of free-floating and exosome-contained FL tau in human biofluids. Given the potential for FL tau to aggregate, we conclude that further investigation of these pools of extracellular tau and how they change during disease is merited.