Toshihiko Shirafuji

Measurement of platelet-derived microparticle levels using an enzyme-linked immunosorbent assay in polymyositis and dermatomyositis patients

Platelet‐derived microparticle (PDMP) levels were measured using an enzyme‐linked immunosorbent assay (ELISA) to elucidate the role of platelet activation in patients with polymyositis or dermatomyositis (PM/DM). PDMP levels in active PM/DM patients (median 13.3 U/ml, interquartile range 9.9–20.7 U/ml, n = 16) and those in patients undergoing treatment (12.1 U/ml, 7.4–16.7 U/ml, n = 12) were significantly higher than in controls (6.5 U/ml, 5.0–8.4 U/ml, n = 26, vs. active, P = 0.0001; vs. treatment, P = 0.004). In a paired sampling study, PDMP decreased significantly after glucocorticoid treatment (P = 0.04). PDMP in the active PM/DM patients correlated significantly with serum C‐reactive protein levels (rs = 0.67, P = 0.01). These results suggest that platelets may play an important role in the inflammatory process, and that PDMP level could be a useful marker of inflammatory activity in PM/DM patients. Muscle Nerve 39: 586–590, 2009

Identification and characterization of PKCγ, a kinase associated with SCA14, as an amyloidogenic protein

Amyloid assemblies are associated with a wide range of human disorders, including Alzheimers and Parkinsons diseases. Here, we identify protein kinase C (PKC) γ, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKCγ in cultured cells, as well as in vitro incubation of PKCγ without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKCγ accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKCγ are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the C1 domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKCγ are highly toxic to neuronal cells. Based on these findings, we propose that PKCγ could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.

The Role of Pak-Interacting Exchange Factor-β Phosphorylation at Serines 340 and 583 by PKCγ in Dopamine Release

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-β (βPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates βPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated βPIX in vivo. Classical PKC inhibitors and βPIX knock-down (KD) significantly suppressed Ca2+-evoked dopamine release in PC12 cells. Wild-type βPIX, and not the βPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by βPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of βPIX at Ser340 and Ser583 has pivotal roles in Ca2+-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through β2PIX phosphorylation. The PKCγ-βPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

Negative charges in the flexible N-terminal domain of Rho GDP-dissociation inhibitors (RhoGDIs) regulate the targeting of the RhoGDI-Rac1 complex to membranes.

In its resting state, Rho GDP-dissociation inhibitor (RhoGDI) α forms a soluble cytoplasmic heterodimer with the GDP-bound form of Rac. Upon stimulation, the dissociation of RhoGDIα from the RhoGDIα–Rac complex is a mandatory step for Rac activation; however, this mechanism is poorly understood. In this study, we examined how the cytoplasm/membrane cycles of the RhoGDI–Rac complex are regulated, as well as where RhoGDI dissociates from the RhoGDI–Rac complex, during FcγR-mediated phagocytosis. The negatively charged and flexible N terminus (25 residues) of RhoGDIα, particularly its second negative amino acid cluster possessing five negatively charged amino acids, was a pivotal regulator in the cytoplasm/membrane cycles of the RhoGDI–Rac complex. We also found that RhoGDIα translocated to the phagosomes as a RhoGDIα–Rac1 complex, and this translocation was mediated by an interaction between the polybasic motif in the C terminus of Rac1 and anionic phospholipids produced on phagosomes, such as phosphatidic acid, that is, by a phagosome-targeting mechanism of Rac1. Thus, we demonstrated that the targeting/accumulation of the RhoGDIα–Rac1 complex to phagosomes is regulated by a balance between three factors: 1) the negatively charged and flexible N-terminal of RhoGDIα, 2) the binding affinity of RhoGDIα for Rac1, and 3) anionic phospholipids produced on phagosomes. Moreover, we demonstrated that the mechanism of targeting/accumulation of the RhoGDIα–Rac1 complex is also applicable for the RhoGDIβ-Rac1 complex.

The subcellular dynamics of the Gs-linked receptor GPR3 contribute to the local activation of PKA in cerebellar granular neurons

G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.

The Toll-like receptor 4-activated neuroprotective microglia subpopulation survives via granulocyte macrophage colony-stimulating factor and JAK2/STAT5 signaling.

Toll-like receptor (TLR) 4 mediates inflammation and is also known to trigger apoptosis in microglia. Our time-lapse observations showed that lipopolysaccharide (LPS) stimulation induced rapid death in primary cultures of rat microglia, while a portion of the microglia escaped from death and survived for much longer than 2 days, in which time, all of the control cells had died. However, it remains unclear how the LPS-stimulated microglia subpopulation could continue to survive in the absence of any supplied growth factors. In the present study, to clarify the mechanism underlying the LPS-stimulated survival, we investigated whether microglia could produce their own survival factors in response to LPS, focusing on macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-34, which are mainly supplied by astrocytes or neurons. The LPS-stimulated microglia drastically induced the expression of the GM-CSF mRNA and protein, while M-CSF and IL-34 levels were unchanged. The surviving microglia also significantly upregulated the expression of GM-CSF receptor (GM-CSFR) mRNA without affecting M-CSFR. As for the GM-CSFR downstream signal, LPS resulted in the phosphorylation of STAT5 and its translocation to the nucleus in the surviving microglia. Moreover, a specific JAK2 inhibitor, NVP-BSK805, suppressed STAT5 phosphorylation and microglia survival in response to LPS, indicating a critical role of the JAK2/STAT5 pathway in this survival mechanism. Together, these results suggest that a subpopulation of TLR4-activated microglia may survive by producing GM-CSF and up-regulating GM-CSFR. This autocrine GM-CSF pathway may activate the JAK2/STAT5 signaling pathway, which controls the transcription of survival-related genes. Finally, these surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia.

Validation of Anti-CSPα, SNAP25, Tyrosine Hydroxylase, Ubiquitin, Cleaved Caspase 3, and pSer PKC Motif Antibodies for Utilization in Western Blotting

There are many commercial antibodies with little information provided by their suppliers as to their reliability. Accordingly, commercial antibodies require proper validation before being used in scientific research. In this study, we validated several commercial antibodies, including anti-CSPα, SNAP25, tyrosine hydroxylase, ubiquitin, cleaved caspase 3, and pSer PKC motif. Anti-CSPα, SNAP25, and tyrosine hydroxylase antibodies could detect their endogenous target proteins with some degree of cross-reactivity. Furthermore, clear SNAP25 staining was observed with SNAP25 antibody. Antibodies directed against ubiquitin, cleaved caspase 3, and pSer PKC motif could detect poly-ubiquitination, apoptosis, and phosphorylation, respectively.

The Role of Cysteine String Protein α Phosphorylation at Serine 10 and 34 by Protein Kinase Cγ for Presynaptic Maintenance

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinsons disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ–CSPα–HSC70/HSP70–SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD. SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration.

The Development of Screening Methods to Identify Drugs to Limit ER Stress Using Wild-type and Mutant Serotonin Transporter.

The function of the serotonin transporter (SERT) is regulated by its membrane trafficking. Previously, we showed that the C-terminus-deleted mutant of SERT (SERTΔCT) exhibited an aberrant membrane trafficking and subsequent retention at the endoplasmic reticulum (ER). In addition, we found that proteasome inhibitor-induced ER stress resulted in the impairment of SERT membrane trafficking and retention of SERT at the ER, an impairment very similar to that of SERTΔCT. Based on the result that the chemical chaperone 4-phnylbutulic acid (4-PBA), which relieves ER stress, accelerated the membrane trafficking and upregulated SERT activity, we hypothesized that drugs that facilitate the membrane trafficking of SERT would have potential therapeutic effects on an ER stress-related disease. In this study, we aimed to develop simple screening methods for such drugs using SERT. We first validated the serotonin uptake assay using fluorescent substrates. This simple and reliable assay method was useful for screening for drugs that affected the wild-type SERT but not SERTΔCT. In addition, we verified an assay focusing on the formation of SERTΔCT aggregates. The drugs 4-PBA and SKF-10047 facilitated the trafficking of SERT to the membrane and reduced SERTΔCT aggregates, indicating that the drugs with such characters could be potential candidates for ER stress relief. For both assays, we clarified the usefulness of a high-content screening microscope. These results could pave the way for high-throughput screening for such drugs.