Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E 2 (PGE 2 ). However, little is known about the molecular organization of the TRPV1 phosphorylationcomplexandspecificallyaboutscaffoldingproteinsthatpositiontheproteinkinaseA(PKA)holoenzymeproximaltoTRPV1foreffective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in ϳ80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-( p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE 2 decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE 2 effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (⌬36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in ⌬36 mice. The PGE 2 /PKA signaling defect in DRG neurons from ⌬36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE 2 -induced thermal hyperalgesia was significantly diminished in ⌬36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE 2 /PKA signaling pathway.

At least 99% of protein phosphorylation in eukaryotic cells occurs on Ser and Thr residues. The greater than 300 protein Ser/Thr kinases in the human genome are opposed by less than 30 protein Ser/Thr phosphatases, of which protein phosphatase 1 and protein phosphatase 2A (PP2A) 2 contribute the bulk of activity in most cell types. The notion of Ser/Thr phosphatases as promiscuous and constitutively active enzymes that simply provide the substrates for regulated kinase signaling has been challenged by the discovery of batteries of catalytic subunit-interacting proteins, which impart substrate specificity, subcellular localization, and responsiveness to phosphorylation (1, 2). In the case of PP2A, the predominant holoenzyme is formed by association of a core dimer of catalytic and scaffold subunits with one of a least 12 regulatory subunits in vertebrates. Since vertebrate A and C subunits are each encoded by two genes and since many regulatory subunits are diversified by alternative splicing, several dozen different PP2A heterotrimers are likely to exist in any given cell type. The three unrelated regulatory subunit gene families (B, BЈ, BЉ) are almost certain to utilize different mechanisms to control enzymatic activity and cellular localization of PP2A. The B-family (PR55) of regulatory subunits consists of predicted ␤-propellers with divergent N-terminal tails that act as subcellular targeting signals (3-5). In addition to interacting with phosphatase substrates (6 -12), BЈ-family (also referred to as B56, PR61) subunits are heavily phosphorylated, which may confer regulation by second messengers (13-16). Members of the BЉ-family of PP2A subunits (PR72/ 130, PR59, PR48) feature two calcium binding EF hands with presumed structural rather than regulatory functions (17). Striatin and SG2NA have been referred to as Bٞ regulatory subunits (18). It may be more appropriate to refer to them as PP2A dimer-associated proteins since their stability does not depend on association with the PP2A core enzyme (17, 19) and since they lack the A subunit binding consensus motif common to B, BЈ, and BЉ subunits (20).A growing body of evidence indicates that PP2A has complex inhibitory and stimulatory effects on hormone and growth factor signaling, in particular the extracellular-signal regulated (ERK)/mitogen-activated protein kinase (MAPK) cascade (21). PP2A substrates include G-protein-coupled receptors and receptor tyrosine kinases (22-24), receptorassociated proteins (25-28), and all three kinases of the ERK/MAPK cascade core module , MAPK/ERK kinase (MEK) (33)(34)(35) and ERK (36,37)).Here, we have begun to dissect the contribution of different PP2A holoenzymes to growth factor signaling in PC12 cells. The net effect of total PP2A silencing or inhibition was ERK and Akt hyperphosphorylation, most likely as a consequence of direct dephosphorylation of these kinases by PP2A. Cautioning against exclusive reliance on silencing approaches, protracted inhibition of PP2A by RNA interference (RNAi) resulted in a compensatory unco...

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

The molecular mechanism by which apolipoprotein E (apoE) suppresses inflammatory cytokine and NO production is unknown. Using an affinity purification approach, we found that peptide mimetics of apoE, derived from its receptor binding domain residues 130–150, bound to the SET protein, which is a potent physiological inhibitor of protein phosphatase 2A (PP2A). Both holo-apoE protein and apoE-mimetic peptides bound to the C-terminal region of SET, which is then associated with an increase in PP2A-mediated phosphatase activity. As physiological substrates for PP2A, the LPS-induced phosphorylation status of signaling MAPK and Akt kinase is reduced following treatment with apoE-mimetic peptides. On the basis of our previous report, in which apoE-mimetic peptides reduced I-κB kinase and NF-κB activation, we also demonstrate a mechanism for reduced production of inducible NO synthase protein and its NO product. These data provide evidence for a novel molecular mechanism by which apoE and apoE-mimetic peptides antagonize SET, thereby enhancing endogenous PP2A phosphatase activity, which reduces levels of phosphorylated kinases, signaling, and inflammatory response.

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in ex vivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactive oxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.

We have previously shown that the botanical drug candidate PBI-05204, a supercritical CO 2 extract of Nerium oleander, provides neuroprotection in both in vitro and in vivo brain slice-based models for focal ischemia (Dunn et al., 2011). Intriguingly, plasma levels of the neurotrophin BDNF were increased in patients treated with PBI-05204 in a phase I clinical trial (Bidyasar et al., 2009). We thus tested the hypothesis that neuroprotection provided by PBI-05204 to rat brain slices damaged by oxygen-glucose deprivation (OGD) is mediated by BDNF. We found, in fact, that exogenous BDNF protein itself is sufficient to protect brain slices against OGD, whereas downstream activation of TrkB receptors for BDNF is necessary for neuroprotection provided by PBI-05204, using three independent methods. Finally, we provide evidence that oleandrin, the principal cardiac glycoside component of PBI-05204, can quantitatively account for regulation of BDNF at both the protein and transcriptional levels. Together, these findings support further investigation of cardiac glycosides in providing neuroprotection in the context of ischemic stroke.

Nerve growth factor (NGF) is critical for the differentiation and maintenance of neurons in the peripheral and central nervous system. Sustained autophosphorylation of the TrkA receptor tyrosine kinase and longlasting activation of downstream kinase cascades are hallmarks of NGF signaling, yet our knowledge of the molecular mechanisms underlying prolonged TrkA activity is incomplete. Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase composed of a scaffolding, catalytic, and regulatory subunit (B, B, and B؆ gene families). Here, we employ a combination of pharmacological inhibitors, regulatory subunit overexpression, PP2A scaffold subunit exchange, and RNA interference to show that PP2A containing B family regulatory subunits participates in sustained NGF signaling in PC12 cells. Specifically, two neuron-enriched regulatory subunits, B␤ and B␦, recruit PP2A into a complex with TrkA to dephosphorylate the NGF receptor on Ser/Thr residues and to potentiate its intrinsic Tyr kinase activity. Acting at the receptor level, PP2A/ B␤ and B␦ enhance NGF (but not epidermal growth factor or fibroblast growth factor) signaling through the Akt and Ras-mitogen-activated protein kinase cascades and promote neuritogenesis and differentiation of PC12 cells. Thus, select PP2A heterotrimers oppose desensitization of the TrkA receptor tyrosine kinase, perhaps through dephosphorylation of inhibitory Ser/Thr phosphorylation sites on the receptor itself, to maintain neurotrophinmediated developmental and survival signaling.

Neurons must maintain protein and mitochondrial quality control for optimal function, an energetically expensive process. The PPARs are ligand-activated transcription factors that promote mitochondrial biogenesis and oxidative metabolism. We recently determined that transcriptional dysregulation of PPARδ contributes to Huntington’s disease (HD), a progressive neurodegenerative disorder resulting from a CAG-polyglutamine repeat expansion in the huntingtin gene. We documented that the PPARδ agonist KD3010 is an effective therapy for HD in a mouse model. PPARδ forms a heterodimer with the retinoid X receptor (RXR), and RXR agonists are capable of promoting PPARδ activation. One compound with potent RXR agonist activity is the FDA-approved drug bexarotene. Here, we tested the therapeutic potential of bexarotene in HD, and found that bexarotene was neuroprotective in cellular models of HD, including medium spiny-like neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with HD. To evaluate bexarotene as a treatment for HD, we treated the N171-82Q mouse model with the drug and found that bexarotene improved motor function, reduced neurodegeneration, and increased survival. To determine the basis for PPARδ neuroprotection, we evaluated metabolic function and noted markedly impaired oxidative metabolism in HD neurons, which was rescued by bexarotene or KD3010. We examined mitochondrial and protein quality control in cellular models of HD, and observed that treatment with a PPARδ agonist promoted cellular quality control. By boosting cellular activities that are dysfunctional in HD, PPARδ activation may have therapeutic applications in HD and potentially other related neurodegenerative diseases.