Systemic infections in multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). It has been suggested that viral and bacterial infections contribute to the pathogenesis of MS. This review will give an overview about the influence of viral and bacterial infections on MS and experimental autoimmune encephalomyelitis (EAE). It will focus on bacterial infections and will also emphasise therapeutic consequences such as the impact of antibiotic treatment on the course of EAE. In summary, a growing body of evidence suggests that systemic infections are a risk factor for the initiation of autoimmune processes including the induction of acute events in MS. Experimental and clinical data strongly suggest early treatment of bacterial infections in MS patients to avoid aggravation and relapse.     

Synaptic plasticity in multiple sclerosis and in experimental autoimmune encephalomyelitis

Approximately half of all patients with multiple sclerosis (MS) experience cognitive dysfunction, including learning and memory impairment. Recent studies suggest that hippocampal pathology is involved, although the mechanisms underlying these deficits remain poorly understood. Evidence obtained from a mouse model of MS, the experimental autoimmune encephalomyelitis (EAE), suggests that in the hippocampus of EAE mice long-term potentiation (LTP) is favoured over long-term depression in response to repetitive synaptic activation, through a mechanism dependent on enhanced IL-1β released from infiltrating lymphocytes or activated microglia. Facilitated LTP during an immune-mediated attack might underlie functional recovery, but also cognitive deficits and excitotoxic neurodegeneration. Having identified that pro-inflammatory cytokines such as IL-1β can influence synaptic function and integrity in early MS, it is hoped that new treatments targeted towards preventing synaptic pathology can be developed.     

The LPS receptor, CD14, in experimental autoimmune encephalomyelitis and multiple sclerosis.

Innate immune receptors are crucial for defense against microorganisms. Recently, a cross-talk between innate and adaptive immunity has been considered. Here, we provide first evidence for a role of the key innate immune receptor, LPS receptor (CD14) in pathophysiology of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Indicating a functional importance in vivo, we show that CD14 deficiency increased clinical symptoms in active experimental autoimmune encephalomyelitis. Consistent with these observations, CD14 deficient mice exhibited a markedly enhanced infiltration of monocytes and neutrophils in brain and spinal cord. Moreover, we observed an increased immunoreactivity of CD14 in biopsy and post mortem brain tissues of multiple sclerosis patients compared to age-matched controls. Thus, the key innate immune receptor, CD14, may be of pathophysiological relevance in experimental autoimmune encephalomyelitis and multiple sclerosis.     

Clathrin box in G protein-coupled receptor kinase 2

beta(1)-Adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. However, beta(1)AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the beta(1)AR and GRK2 fusion protein (beta(1)AR/GRK2) is dependent on dynamin but independent of beta-arrestin and phosphorylation. The beta(1)AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of beta(1)AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as beta-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.     

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.     

Peptide substrates for G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X_1–3(S/T), (D/E)X_1–3(S/T)(D/E), or (D/E)X_0–2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (K_m, 33.9 μM; V_max, 0.35 pmol min⁻¹ mg⁻¹), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

Rapamycin combined with MCC950 to treat multiple sclerosis in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a highly disabling demyelinating disease, which mainly affects young adults and is difficult to cure. Activated microglia may be involved in the process of neuronal cell damage and release inflammatory cytokines to injure neurons. Rapamycin (RAPA), an immunosuppressant, can induce autophagy in microglia to delay the process of the disease. As an inhibitor of NLRP3, MCC950 (CP-456773) can regulate the activation of inflammasome. An experimental autoimmune encephalomyelitis model, a disease model of MS, was established to detect the role of activated microglia in the dynamic evolution of MS. Our research showed that RAPA and MCC950 could reduce both the clinical symptom and the release of cytokines in immune cells. MCC950 reduced interleukin-1β (IL-1β) production in vivo and enhanced the effect of RAPA. We hypothesized that inflammation and demyelination in the central nervous system can be reduced by inhibiting the immune response mediated by microglia. This study provides theoretical support to the therapeutic evaluation of RAPA and MCC950 to make the mammalian targets of RAPA and NLRP3 the therapeutic targets of MS.     

Chemical genetic engineering of G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.     

MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.     

Involvement of IL-1R/TLR signalling in experimental autoimmune encephalomyelitis and multiple sclerosis

Multiple sclerosis is a complex disease characterised by chronic inflammation, demyelination and axonal pathology resulting in progressive neurological disabilities. Multiple sclerosis is generally considered to be an autoimmune disease, even though the primary cause of the underlying autoimmune response is unknown. Epidemiological evidence suggests that both genetic and environmental factors play a key role in susceptibility to multiple sclerosis; however, the relative contributions of these factors in triggering the onset of the disease remain unclear. Several studies indicate that receptors belonging to the Interleukin-1 and Toll-like receptor families are crucially involved in the mechanisms underlying the development of experimental autoimmune encephalomyelitis, an animal model that mimics multiple sclerosis. Moreover, recent evidence highlights the importance of downstream signalling proteins in the Interleukin-1 and Toll-like receptor signalling pathways, namely, myeloid differentiation primary response protein 88 and Interleukin-1-receptor-associated kinase. This review summarises the current knowledge concerning the involvement of Interleukin-1/Toll-like receptor signalling in the development of experimental autoimmune encephalomyelitis and multiple sclerosis. A deeper understanding of the role of these important pathways in the pathogenesis of experimental autoimmune encephalomyelitis may eventually yield clinical benefits in the treatment of central nervous system-based inflammatory disorders.     

G protein-coupled receptor kinase 2 (GRK2) in migration and inflammation

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors and other plasma membrane receptors stimulated by chemotactic messengers. On top of that, GRK2 has been reported to interact with a variety of signal transduction proteins related to cell migration such as MEK, Akt, PI3Kgamma or GIT. Interestingly, the levels of expression and activity of this kinase are altered in a number of inflammatory disorders (as rheumatoid arthritis or multiple sclerosis), thus suggesting that it may play an important role in the onset or development of these pathologies. This review summarizes the mechanisms involved in the control of GRK2 expression and function and highlights novel functional interactions of this protein that might help to explain how altered GRK2 levels affects cell migration in different cell types and pathological settings.     

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.     

G protein-coupled receptors as therapeutic targets for multiple sclerosis

G protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants. They are considered as the most successful therapeutic targets for a broad spectrum of diseases. Multiple sclerosis (MS) is an inflammatory disease that is characterized by immune-mediated demyelination and degeneration of the central nervous system (CNS). It is the leading cause of non-traumatic disability in young adults. Great progress has been made over the past few decades in understanding the pathogenesis of MS. Numerous data from animal and clinical studies indicate that many GPCRs are critically involved in various aspects of MS pathogenesis, including antigen presentation, cytokine production, T-cell differentiation, T-cell proliferation, T-cell invasion, etc. In this review, we summarize the recent findings regarding the expression or functional changes of GPCRs in MS patients or animal models, and the influences of GPCRs on disease severity upon genetic or pharmacological manipulations. Hopefully some of these findings will lead to the development of novel therapies for MS in the near future.     

Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.     

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.     

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866Delta, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.