Lipoic acid modulates inflammatory responses of monocytes and monocyte-derived macrophages from healthy and relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a disabling neuroinflammatory disease. Its etiology is unknown, but both oxidative stress and inflammation appear to be involved in disease pathology. Macrophages are the predominant cell type in acute inflammatory brain lesions in MS. Macrophages produce proinflammatory and toxic molecules that promote demyelination and are key players in phagocytosis/degradation of myelin sheathes. Lipoic acid (LA) is an inexpensive, endogenously produced small molecule that exhibits antioxidant and anti-inflammatory effects. Treatment with LA is protective in MS and other inflammatory diseases. To examine the mechanism(s) by which LA may attenuate inflammatory lesion activity in MS, we used healthy control and MS cells to evaluate the effects of LA on levels of inflammatory cytokines, phagocytosis and the immunomodulator cyclic adenosine monophosphate (cAMP) in monocytes and monocyte-derived macrophages (MDMs). LA treatment resulted in a generally less inflammatory phenotype of monocytes and MDMs from healthy controls, and (to a lesser degree) MS donors. LA inhibited monocyte secretion of cytokines relevant to MS in monocytes, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß; LA effects on secretion of these cytokines in MDMs were mixed with inhibition of TNF-α and IL-6, but stimulation of IL-1ß, the latter perhaps as a result of altered macrophage polarization. LA inhibited phagocytosis in both monocytes and MDMs, and increased cAMP levels in monocytes. LA may modulate inflammatory cytokine secretion and phagocytosis via a cAMP-mediated mechanism.

Lipoic Acid Stimulates cAMP Production in Healthy Control and Secondary Progressive MS Subjects.

Lipoic acid (LA) exhibits antioxidant and anti-inflammatory properties; supplementation reduces disease severity and T lymphocyte migration into the central nervous system in a murine model of multiple sclerosis (MS), and administration in secondary progressive MS (SPMS) subjects reduces brain atrophy compared to placebo. The mechanism of action (MOA) of LA's efficacy in suppression of MS pathology is incompletely understood. LA stimulates production of the immunomodulator cyclic AMP (cAMP) in vitro. To determine whether cAMP could be involved in the MOA of LA in vivo, we performed a clinical trial to examine whether LA stimulates cAMP production in healthy control and MS subjects, and whether there are differences in the bioavailability of LA between groups. We administered 1200 mg of oral LA to healthy control, relapsing remitting MS (RRMS) and SPMS subjects, and measured plasma LA and cAMP levels in peripheral blood mononuclear cells (PBMCs). There were no significant differences between the groups in pharmacokinetic (PK) parameters. Healthy and SPMS subjects had increased cAMP at 2 and 4 h post-LA treatment compared to baseline, while RRMS subjects showed decreases in cAMP. Additionally, plasma concentrations of prostaglandin E2 (PGE2, a known cAMP stimulator) were significantly lower in female RRMS subjects compared to female HC and SPMS subjects 4 h after LA ingestion. These data indicate that cAMP could be part of the MOA of LA in SPMS, and that there is a divergent response to LA in RRMS subjects that may have implications in the efficacy of immunomodulatory drugs. This clinical trial, "Defining the Anti-inflammatory Role of Lipoic Acid in Multiple Sclerosis," NCT00997438, is registered at https://clinicaltrials.gov/ct2/show/record/NCT00997438 .

Donor variability may mask dimethyl fumarate's effects on nuclear factor E2-related factor 2 in human peripheral blood mononuclear cells.

OBJECTIVE: Dimethyl fumarate (DMF) is an anti-inflammatory and antioxidant drug used to treat multiple sclerosis, but its mechanism(s) of action are not fully understood. In central nervous system (CNS) cells, DMF activates nuclear factor E2-related factor 2 (Nrf2), perhaps ameliorating oxidative stress-induced damage. However, it is not known whether DMF also activates Nrf2 in peripheral immune cells, which are known to participate in CNS demyelination. We conducted a single observation study to determine whether DMF can activate Nrf2 in peripheral immune cells in vitro. RESULTS: We performed enzyme-linked immunosorbent assays to measure Nrf2 activation in nuclear extracts of human peripheral blood mononuclear cells treated with DMF at time points from 0 to 6 h, initially determining that DMF did not activate Nrf2, and that the mechanism(s) of action of DMF may thus differ in the periphery compared to the CNS. However, further analyses of our data suggest that high Tmax variability is masking Nrf2 activation in individual donors. Additionally, there may be sub-populations of responders, perhaps related to genetic polymorphisms in Nrf2.

Analysis of IL-6, IL-1ß and TNF-α production in monocytes isolated from multiple sclerosis patients treated with disease modifying drugs.

OBJECTIVE AND DESIGN: The etiology of multiple sclerosis (MS) is unknown, but blood derived monocytes/macrophages are believed to be involved in the pathogenesis through phagocytosis of myelin and production of inflammatory mediators. The objective of this study is to examine inflammatory cytokines that are present at elevated levels in active MS lesions to determine whether there are differences between classically stimulated monocytes isolated from healthy control (HC) and relapsing-remitting MS (RRMS) subjects taking disease modifying drugs (DMDs), including dimethyl fumarate (DMF). SUBJECTS: Thirty-nine veterans of the US Armed Forces were enrolled, 21 health controls (HC), and 18 with relapsing-remitting MS (RRMS), all taking DMDs. METHODS: Use ELISAs to measure production of IL-6, IL-1ß and TNF-α by LPS-stimulated peripheral monocytes. RESULTS: Activation of monocytes from MS subjects produced significantly more IL-6 than healthy controls (49531 ± 20795 vs 10526 ± 4845), and IL-6 production trended higher in MS subjects taking DMF than those taking other DMDs (72186.9 ± 35156.2 vs 32585.8 ± 17135.4). There were no significant differences in IL-1ß or TNF-α secretion. CONCLUSIONS: Our data suggest that not all DMDs may provide disease modification by suppressing monocyte/macrophage production of pro-inflammatory mediators.

Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells.

Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and ß-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation.

Dimethyl fumarate activates the prostaglandin EP2 receptor and stimulates cAMP signaling in human peripheral blood mononuclear cells.

Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway.

Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity.

The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme. Its primary components are A-kinase anchoring proteins (AKAPs) 3 and 4, which suggests that the FS affects flagellar beating via the scaffolding of signaling pathways necessary for motility. Sperm proteins ROPN1 and ROPN1L bind AKAP3. To determine the role of ROPN1 and ROPN1L in sperm function, we created mice deficient in ROPN1 (RKO), mice deficient in ROPN1L (RLKO), and double knockout mice (DKO). All three strains of mice had normal testicular morphology and spermatogenesis. Only the DKOs had obvious defects in sperm morphology (thinning and shredding of the principal piece), which was accompanied by a reduction in AKAP3 levels. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. RKO mice had moderately impaired motility and increased levels of ROPN1L. DKO sperm were immotile. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. Together these data indicate that ROPN1L and ROPN1 compensate for each other in the absence of the opposing protein, possibly to maintain AKAP3 incorporation in the FS. Sperm from mice lacking ROPN1L exhibited reductions in both cAMP-dependent protein kinase (PKA) phosphorylation of a 270-kDa protein (perhaps FSCB), and in capacitation-induced tyrosine phosphorylation. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. These data demonstrate that mutations in ROPN1 and ROPN1L can cause defects in FS integrity, sperm motility, and PKA-dependent signaling processes, leading to male infertility.

PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells.

Controlled maturation of ovarian follicles is necessary for fertility. Follicles are restrained at an immature stage until stimulated by FSH secreted by pituitary gonadotropes. FSH acts on granulosa cells within the immature follicle to inhibit apoptosis, promote proliferation, stimulate production of steroid and protein hormones, and induce ligand receptors and signaling intermediates. The phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) pathway is a pivotal signaling corridor necessary for transducing the FSH signal. We report that protein kinase A (PKA) mediates the actions of FSH by signaling through multiple targets to activate PI3K/AKT. PKA uses a route that promotes phosphorylation of insulin receptor substrate-1 (IRS-1) on Tyr(989), a canonical binding site for the 85-kDa regulatory subunit of PI3K that allosterically activates the catalytic subunit. PI3K activation leads to activation of AKT through phosphorylation of AKT on Thr(308) and Ser(473). The adaptor growth factor receptor bound protein 2-associated binding protein 2 (GAB2) is present in a preformed complex with PI3K heterodimer and IRS-1, it is an A-kinase anchoring protein that binds the type I regulatory subunit of PKA, and it is phosphorylated by PKA on Ser(159). Overexpression of GAB2 enhances FSH-stimulated AKT phosphorylation. GAB2, thus, seems to coordinate signals from the FSH-stimulated rise in cAMP that leads to activation of PI3K/AKT. The ability of PKA to commandeer IRS-1 and GAB2, adaptors that normally integrate receptor/nonreceptor tyrosine kinase signaling into PI3K/AKT, reveals a previously unrecognized route for PKA to activate a pathway that promotes proliferation, inhibits apoptosis, enhances translation, and initiates differentiation of granulosa cells.

Loss of ASP but not ROPN1 reduces mammalian ciliary motility.

Protein kinase A (PKA) signaling is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins [AKAP-associated sperm protein (ASP), ropporin (ROPN1), sperm protein 17 (SP17) and calcium binding tyrosine-(Y)-phosphorylation regulated protein (CABYR)] share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease.

Myeloid translocation gene 16b is a dual A-kinase anchoring protein that interacts selectively with plexins in a phospho-regulated manner.

The myeloid translocation gene (MTG) homologue Nervy associates with PlexinA on the plasma membrane, where it functions as an A-kinase anchoring protein (AKAP) to modulate plexin-mediated semaphorin signaling in Drosophila. Mammalian MTG16b is an AKAP found in immune cells where plexin-mediated semaphorin signaling regulates immune responses. This study provides the first evidence that MTG16b is a dual AKAP capable of binding plexins. These interactions are selective (PlexinA1 and A3 bind MTG, while PlexinB1 does not) and can be regulated by PKA-phosphorylation. Collectively, these data suggest a possible mechanism for the targeting and integration of adenosine 3',5'-cyclic monophosphate (cAMP) and semaphorin signaling in immune cells.

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction.

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.

Identification and characterization of RHOA-interacting proteins in bovine spermatozoa.

In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro.

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa.

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.