Neuronal activity promotes secretory autophagy for the extracellular release of α-synuclein.

Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.

Effects of selegiline on neuronal autophagy involving α-synuclein secretion.

Cell-to-cell transmission of α-synuclein (α-syn) pathology underlies the spread of neurodegeneration in Parkinson's disease. α-Syn secretion is an important factor in the transmission of α-syn pathology. However, it is unclear how α-syn secretion is therapeutically modulated. Here, we investigated effects of monoamine oxidase (MAO)-B inhibitor selegiline on α-syn secretion. Treatment with selegiline promoted α-syn secretion in mouse primary cortical neuron cultures, and this increase was kept under glial cell-eliminated condition by Ara-C. Selegiline-induced α-syn secretion was blocked by cytosolic Ca2+ chelator BAPTA-AM in primary neurons. Selegiline-induced α-syn secretion was retained in MAOA siRNA knockdown, whereas it was abrogated by ATG5 knockdown in SH-SY5Y cells. Selegiline increased LC3-II generation with a reduction in intracellular p62/SQSTM1 levels in primary neurons. The increase in LC3-II generation was blocked by co-treatment with BAPTA-AM in primary neurons. Additionally, fractionation experiments showed that selegiline-induced α-syn secretion occurred in non-extracellular vesicle fractions of primary neurons and SH-SY5Y cells. Collectively, these findings show that selegiline promotes neuronal autophagy involving secretion of non-exosomal α-syn via a change of cytosolic Ca2+ levels.

Monoamine Oxidase-B Inhibition Facilitates α-Synuclein Secretion In Vitro and Delays Its Aggregation in rAAV-Based Rat Models of Parkinson's Disease.

Cell-to-cell transmission of α-synuclein (α-syn) pathology is considered to underlie the spread of neurodegeneration in Parkinson's disease (PD). Previous studies have demonstrated that α-syn is secreted under physiological conditions in neuronal cell lines and primary neurons. However, the molecular mechanisms that regulate extracellular α-syn secretion remain unclear. In this study, we found that inhibition of monoamine oxidase-B (MAO-B) enzymatic activity facilitated α-syn secretion in human neuroblastoma SH-SY5Y cells. Both inhibition of MAO-B by selegiline or rasagiline and siRNA-mediated knock-down of MAO-B facilitated α-syn secretion. However, TVP-1022, the S-isomer of rasagiline that is 1000 times less active, failed to facilitate α-syn secretion. Additionally, the MAO-B inhibition-induced increase in α-syn secretion was unaffected by brefeldin A, which inhibits endoplasmic reticulum (ER)/Golgi transport, but was blocked by probenecid and glyburide, which inhibit ATP-binding cassette (ABC) transporter function. MAO-B inhibition preferentially facilitated the secretion of detergent-insoluble α-syn protein and decreased its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Moreover, in a rat model (male Sprague Dawley rats) generated by injecting recombinant adeno-associated virus (rAAV)-A53T α-syn, subcutaneous administration of selegiline delayed the striatal formation of Ser129-phosphorylated α-syn aggregates, and mitigated loss of nigrostriatal dopaminergic neurons. Selegiline also delayed α-syn aggregation and dopaminergic neuronal loss in a cell-to-cell transmission rat model (male Sprague Dawley rats) generated by injecting rAAV-wild-type α-syn and externally inoculating α-syn fibrils into the striatum. These findings suggest that MAO-B inhibition modulates the intracellular clearance of detergent-insoluble α-syn via the ABC transporter-mediated non-classical secretion pathway, and temporarily suppresses the formation and transmission of α-syn aggregates.SIGNIFICANCE STATEMENT The identification of a neuroprotective agent that slows or stops the progression of motor impairments is required to treat Parkinson's disease (PD). The process of α-synuclein (α-syn) aggregation is thought to underlie neurodegeneration in PD. Here, we demonstrated that pharmacological inhibition or knock-down of monoamine oxidase-B (MAO-B) in SH-SY5Y cells facilitated α-syn secretion via a non-classical pathway involving an ATP-binding cassette (ABC) transporter. MAO-B inhibition preferentially facilitated secretion of detergent-insoluble α-syn protein and reduced its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Additionally, MAO-B inhibition by selegiline protected A53T α-syn-induced nigrostriatal dopaminergic neuronal loss and suppressed the formation and cell-to-cell transmission of α-syn aggregates in rat models. We therefore propose a new function of MAO-B inhibition that modulates α-syn secretion and aggregation.

Assessment of clinical factors affecting outcome of myasthenia gravis.

INTRODUCTION/AIMS: In myasthenia gravis (MG) therapy, achieving Myasthenia Gravis Foundation of America minimal manifestation (MM) or better status is proposed as a desirable target. However, this level of control is often not achieved and clinical factors affecting prognosis remain unclear. METHODS: Participants were 104 consecutive patients with MG who visited Osaka Medical College Hospital. We retrospectively assessed the association of clinical and laboratory features at baseline with prognosis. Eighty patients who achieved MM or better status were classified as the good outcome group and the remaining 24 patients were classified as the poor outcome group. RESULTS: The rate of dysphagia at baseline was significantly higher in the poor outcome group than in the good outcome group (P = .002). The levels of serum total protein and albumin at baseline were both significantly lower in the poor outcome group than in the good outcome group (P = .036 and P = .014, respectively). In addition, Controlling Nutritional Status scores at baseline were significantly higher in the poor outcome group than in the good outcome group (P = .043). Multivariate analysis using a Cox proportional hazards model showed that dysphagia (hazard ratio [HR], 6.92; 95% confidence interval [CI], 1.49-40.31) and hypoalbuminemia (HR, 2.57; 95% CI, 1.04-6.57) at baseline were risk factors that predicted prognosis. DISCUSSION: These findings suggest that dysphagia and hypoalbuminemia at baseline are associated with outcomes and are predictive risk factors for poorer outcomes in patients with MG.

Exploration of pathomechanism using comprehensive analysis of serum cytokines in polymyositis/dermatomyositis-interstitial lung disease.

OBJECTIVES: To elucidate the serum cytokine profile and address the pathomechanism of interstitial lung disease (ILD) complicated with PM/DM. METHODS: Forty patients with PM/DM-ILD were enrolled, and principal components analysis and cluster analysis were performed to classify patients into subgroups. Additionally, we compared cytokine profiles between the survivors and dead patients and between anti-melanoma differentiation-associated gene 5 antibody- and anti-aminoacyl tRNA synthetase antibody-positive ILD patients. We also examined the association of various cytokines with disease activity indicators and prognosis of ILD. RESULTS: The principal components analysis data allowed classification of the cytokine profile into three groups: group 1, neutrophilic and M1-macrophage-driven cytokines; group 2, type 1 Th cell-driven and M2-macrophage-induced cytokines; and group 3, M2-macrophage-driven cytokines. Cluster analysis showed the presence of PM/DM-ILD patient groups with high or low levels of total cytokines. Ninety percent of patients who died of ILD were included in clusters with high cytokine levels. Serum cytokine levels of all groups were significantly higher in the anti-melanoma differentiation-associated gene 5 antibody-positive patients than in the anti-aminoacyl tRNA synthetase antibody-positive patients. Groups 1 and 2 significantly correlated with known factors for poor prognosis, such as serum ferritin levels and alveolar-arterial oxygen difference. Serum cytokine levels of patients in group 1 were significantly higher initially and at 2 and 4 weeks in those who died. CONCLUSION: These findings suggested that the activation of monocytes, macrophages and type 1 Th cells, and neutrophils play roles in the pathomechanism of PM/DM-ILD, and group 1 cytokines could be useful biomarkers for predicting prognosis of PM/DM-ILD.

Clinical features of Guillain-Barré syndrome patients with elevated serum creatine kinase levels.

BACKGROUND: It is not well defined whether Guillain-Barré syndrome (GBS) patients with elevated serum creatine kinase (CK) levels have characteristic clinical features and are related to the subgroups of GBS. METHODS: We retrospectively studied 51 consecutive patients with GBS, who visited our hospital, and compared clinical, laboratory and electrophysiological findings between patients with and without elevated CK levels. RESULTS: Of 51 patients, 14 patients (27%) showed an elevation of serum CK levels. When compared with patients with the normal CK levels, the ratios of male, antecedent infections, and anti-GM1 antibody positivity were significantly higher in patients with elevated CK levels. The ratios of hypoesthesia, cranial nerve involvement, and urinary retention were significantly less in patients with elevated CK levels. There were no significant differences in disability at peak between two groups. In the electrophysiological examination, sensory nerve abnormalities were not observed. Although some patients with elevated CK levels showed prolongation of distal motor latencies (DMLs) and increase of durations in the initial examination, development of the prolongation of DMLs and increase of durations was not observed in the follow-up examinations. The findings were consistent with acute motor axonal neuropathy (AMAN) with reversible conduction failure (RCF) but not acute inflammatory demyelinating polyneuropathy (AIDP). CONCLUSIONS: The results suggest that the GBS patients with elevated CK levels represent not a group of AIDP but a group of AMAN with axonal degeneration or RCF even though the initial electrophysiological examination shows AIDP pattern.

Fulminant Guillain-Barré syndrome showing severe pharyngeal-cervical-brachial weakness in the recovery phase: a case report.

BACKGROUND: Fulminant Guillain-Barré syndrome (GBS) is characterized clinically by rapid progression of severe symptoms, such as the absence of brainstem reflexes, complete tetraplegia and respiratory arrest. The clinical course of fulminant GBS remains unclear. Here, we report a patient with fulminant GBS, who showed severe weakness of the pharyngeal-cervical-branchial (PCB) area in the recovery phase. CASE PRESENTATION: A 38-year-old man rapidly developed fulminant GBS. In blood examination, he was positive for a broad range of anti-ganglioside antibodies, including anti-GQ1b, GT1a, GT1b, GD1a, GD1b and GD3 IgG antibodies. We performed immunosuppressive therapies using intravenous immunoglobulin and intravenous methylprednisolone. Although disturbance of consciousness and weakness of the distal upper and lower limbs improved gradually, weakness of the oropharynx, neck, and proximal upper limbs were resistant to these therapies. Anti-GT1a IgG antibodies remained persistently positive. Consequently, mechanical ventilation and tube feeding were required for 7 and 10 months, respectively. Two years later, weakness of the proximal upper limbs and mild respiratory dysfunction remained as sequelae. CONCLUSION: Anti-GT1a IgG antibodies are known to be detected in patients with the PCB variant of GBS. In fulminant GBS, the persistent presence of anti-GT1a IgG antibodies may be associated with occurrence of severe PCB-like weakness in the recovery phase.

Clinical characteristics of long-term survival with noninvasive ventilation and factors affecting the transition to invasive ventilation in amyotrophic lateral sclerosis.

INTRODUCTION: We evaluated post-noninvasive ventilation survival and factors for the transition to tracheostomy in amyotrophic lateral sclerosis (ALS). METHODS: We analyzed 197 patients using a prospectively collected database with 114 patients since 2000. RESULTS: Among 114 patients, 59 patients underwent noninvasive ventilation (NIV), which prolonged the total median survival time to 43 months compared with 32 months without treatment. The best post-NIV survival was associated with a lack of bulbar symptoms, higher measured pulmonary function, and a slower rate of progression at diagnosis. The transition rate from NIV to tracheostomy gradually decreased over the years. Patients using NIV for more than 6 months were more likely to refuse tracheostomy and to be women. DISCUSSION: This study confirmed a positive survival effect with NIV, which was less effective in patients with bulbar dysfunction. Additional studies are required to determine the best timing for using NIV with ALS in patients with bulbar dysfunction. Muscle Nerve 58:770-776 2018.

Posterior reversible encephalopathy syndrome with extensive cytotoxic edema after blood transfusion: a case report and literature review.

BACKGROUND: Posterior reversible encephalopathy syndrome (PRES) is described as a clinical-radiological disease entity with good prognosis. In brain MRI, PRES generally presents with vasogenic edema. Although PRES is induced by various causes, a small number of PRES cases have occurred after red cell blood transfusion. It is unclear whether there are characteristic features in PRES after blood transfusion. CASE PRESENTATION: Here, we report a case of 75-year-old Japanese woman who had acute exacerbation of subacute anemia by bleeding from gastric ulcer. After receiving a red cell blood transfusion, she showed disturbance of consciousness with extensive cytotoxic and small vasogenic edema in the occipitoparietal area on brain MRI. She was diagnosed as PRES and suffered irreversible impairments of visual acuity and fields in both eyes. We summarized and discussed clinical features of cases with PRES after blood transfusion. CONCLUSIONS: A total of 21 cases including the present one have been reported as PRES after blood transfusion. Of the cases, 20 of 21 were female, and 15 of 17 developed PRES in the course of chronic anemia lasting over 1 month. Anemia was severe in 15 of 20 cases, with hemoglobin levels < 3.5 g/dl. In 14 of 17 cases, hemoglobin levels increased to 5 g/dl by red cell blood transfusion until the onset of PRES. On brain MRI, 2 of 21 cases showed cytotoxic edema and 3 of 21 cases showed irreversible neurological disturbance. In this patient, the occurrence of PRES in subacute anemia and the presence of extensive cytotoxic brain edema with irreversible neurological deficits were characteristic points. When treating severe anemia, even with a subacute progression, we should consider a possibility that PRES occurs after blood transfusion with extensive cytotoxic brain edema and irreversible neurological changes.

Eosinophilic granulomatosis with polyangiitis complicated by subarachnoid hemorrhage and coronary vasculitis: a case report and review of the literature.

Eosinophilic granulomatosis with polyangiitis (EGPA) is characterized by necrotizing vasculitis of small-sized vessels with extravascular granulomas and eosinophilic infiltration. The case of a 48-year-old Japanese woman with EGPA, who presented concurrently with subarachnoid hemorrhage (SAH) and coronary vasculitis, is reported. She initially presented with bronchial asthma, and then 8 months later she developed various symptoms caused by systemic eosinophilic vasculitis and was admitted to our hospital. Three days after admission, she started oral corticosteroid therapy, and her 2009 Five-Factor Score (FFS) was 0. However, she developed an SAH, followed by coronary vasculitis 1 day later. With extensive treatment with a combination of betamethasone, cyclophosphamide, intravenous immunoglobulin, and rituximab, her systemic vasculitis improved dramatically. This seems to be the first case of EGPA with SAH and coronary vasculitis. In previous reports of EGPA with SAH, 4 of 11 cases developed SAH as an exacerbation of systemic vasculitis during remission induction therapy. The present patient also had SAH during remission induction therapy. However, the period between bronchial asthma and SAH was only 8 months. This is the shortest among case reports of EGPA with SAH. In addition, the present patient rapidly developed coronary vasculitis. These findings suggest that EGPA causes SAH and coronary vasculitis as early complications of systemic vasculitis. In EGPA, it is necessary to pay careful attention to rapid changes of disease activity, even when the FFS indicates a good prognosis.

Preventive Effect of Cilostazol on Pneumonia in Patients with Acute Cerebral Infarction.

BACKGROUND: The antiplatelet drug cilostazol decreases the risk of ischemic stroke recurrence in patients with chronic cerebral infarction. Additionally, cilostazol reduces the occurrence of pneumonia in these patients. The purpose of this study was to investigate whether cilostazol is effective for preventing pneumonia in patients with acute cerebral infarction. MATERIALS AND METHODS: A total of 199 consecutive Japanese patients with noncardioembolic acute cerebral infarction, who visited our hospital from January 2010 to April 2016, were retrospectively assessed by using medical records. We compared changes in the occurrence of pneumonia between cilostazol (n = 127) and noncilostazol (n = 72) groups. RESULTS: A total of 76% of patients in the cilostazol group were not administered other antiplatelet drugs. The median duration until cilostazol administration was 5 days (interquartile range = 2-8 days) after the onset of cerebral infarction. A total of 8.0% of the cohort was accompanied by pneumonia. The incidence of pneumonia in the cilostazol group was significantly lower than that in the noncilostazol group (4.7% versus 13.9%, P = .02). Within 30 days after acute cerebral infarction, the presence of neurological deterioration in the cilostazol group tended to be lower compared with the noncilostazol group, but this difference was not significant (5.5% versus 12.5%, P = .08). CONCLUSIONS: These findings suggest that cilostazol is effective for preventing pneumonia in patients with acute cerebral infarction.

Efficacy and safety of combination therapy with prednisolone and oral tacrolimus for progressive interstitial pneumonia with systemic sclerosis: A retrospective study.

OBJECTIVES: We retrospectively investigated efficacy and safety of combination therapy with prednisolone (PSL) and tacrolimus (TAC) for progressive interstitial pneumonitis with systemic sclerosis (SSc-PIP). METHODS: We studied 11 patients with SSc-PIP who received combination therapy with PSL (0.5 mg/kg/d) and TAC (3 mg/d). RESULTS: Baseline Hugh-Jones grades were I, II, III, and IV in 2, 6, 2, and 1 patients, respectively. Krebs von den Lungen-6 (KL-6) values were elevated to 914 (range 300-2614) U/mL. % Diffusing capacity of carbon monoxide (%DLco) remarkably decreased to 47.4 (range 9.7-64.4) %. All patients were alive at 1 year after therapy. In response to treatment, interstitial pneumonia (IP) improved in three patients, stable in seven patients, and deteriorated in one patient. Total ground-glass opacity (GGO) score improved (p = .005). No significant changes occurred in values of KL-6, % forced vital capacity (%FVC), and %DLco. Presently, all seven patients who could be followed up were alive. IP improved in three patients and stable in four patients. Total GGO score improved (p = .016). KL-6, %FVC, and %DLco did not change. Mild cytomegalovirus or herpes zoster infection occurred in two patients. Grade I renal injuries were observed in three and one patient at 1 year and present, respectively. CONCLUSION: Combination therapy with PSL and TAC appeared to be well tolerated and effective in suppressing the disease activity of SSc-PIP.

[Spinocerebellar ataxia 2 develop lower motor neuron involvement as an initial symptom: a case report].

A 36-year-old man has developed weakness of left thumb and atrophy of left thenar muscle and left first dorsal interosseous muscle without sensory disturbance for a year. A nerve conduction study revealed decreases in the amplitude of compound muscle action potentials and occurrence of F-waves on left medial nerve. Needle electromyography examination revealed positive sharp waves and later recruited motor units on left abductor pollicis brevis muscle. Brain MRI showed atrophy of bilateral cerebellar hemisphere. His grandmother and his two uncles have been diagnosed as spinocerebellar degeneration. After discharge, he developed bilateral lower limb ataxia. Genetic analysis showed heterozygous CAG repeat expansion (19/39) in ATXN2 gene, being diagnosed as spinocerebellar ataxia 2 (SCA2). A previous report has shown that motor neuron involvement is recognized as part of SCA2 in the same pedigree with full CAG repeat expansions in ATXN2 gene. We here report the patient with lower motor neuron involvement as an initial symptom of SCA2.

[A pedigree of myotonia congenita with a novel mutation p.F343C of the CLCN1 gene].

A Japanese woman experienced slowness of movement in her early teens and difficulty in opening her hands during pregnancy. On admission to our hospital at 42 years of age, she showed grip myotonia with warm-up phenomenon. However, she had neither muscle weakness, muscle atrophy, cold-induced symptomatic worsening nor episodes of transient weakness of the extremities. Needle electromyography of the first dorsal interosseous and anterior tibial muscles demonstrated myotonic discharges. Whole exome sequencing of the patient revealed a heterozygous single-base substitution in the CLCN1 gene (c.1028T>G, p.F343C). The same substitution was identified in affected members of her family (mother and brother) by Sanger sequencing, but not in healthy family members (father and a different brother). We diagnosed myotonia congenita (Thomsen disease) with a novel CLCN1 mutation in this pedigree. This mutation causes a single amino acid substitution in the I-J extracellular loop region of CLCN1. Amino acid changes in the I-J loop region are rare in an autosomal-dominantly inherited form of myotonia congenita. We think that this pedigree is precious to understand the pathogenesis of myotonia congenita.

Spatial Fluctuation of Central Nervous System Lesions in X-linked Charcot-Marie-Tooth Disease with a Novel GJB1 Mutation.

X-linked Charcot-Marie-Tooth disease type 1 (CMTX1), the most common form of CMTX, is caused by gap-junction beta 1 (GJB1) mutations. We herein report a 25-year-old Japanese man with disorientation, right hemiparesis, and dysarthria. Brain magnetic resonance imaging (MRI) showed high signal intensities in the bilateral cerebral white matter on diffusion-weighted imaging. He had experienced 2 episodes of transient central nervous system symptoms (at 7 and 13 years old). A genetic analysis identified a novel GJB1 mutation, c.169C>T, p.Gln57*. MRI abnormalities shifted from the cerebral white matter to the corpus callosum and had disappeared at the five-month follow-up. Transient changes between these lesions may indicate CMTX1.

The role of Ser129 phosphorylation of α-synuclein in neurodegeneration of Parkinson's disease: a review of in vivo models.

Parkinson's disease is the most common neurodegenerative movement disorder. The motor impairments of Parkinson's disease are caused by the loss of dopaminergic neurons in the substantia nigra and associated with the appearance of fibrillar aggregates of α-synuclein (α-syn) called Lewy bodies. Approximately 90% of α-syn deposited in Lewy bodies is phosphorylated at serine 129 (Ser129). In contrast, only 4% or less of total α-syn is phosphorylated at this residue in the normal brain. This suggests that the accumulation of Ser129-phosphorylated α-syn leads to the formation of Lewy bodies and dopaminergic neurodegeneration in Parkinson's disease. Our laboratory and others have performed experiments using in vivo models of Parkinson's disease to elucidate the role of increased Ser129 phosphorylation in α-syn neurotoxicity. However, there has been a lack of consistency among these models. In this review, we summarize the main findings regarding the relationship between Ser129 phosphorylation and α-syn neurotoxicity, and examine the differences among models. We further discuss the role of Ser129 phosphorylation in α-syn aggregation and the future directions to test the potential of Ser129 phosphorylation as a therapeutic target for slowing the progression of Parkinson's disease.

[A case of anti-myelin oligodendrocyte glycoprotein antibody-positive multiphasic disseminated encephalomyelitis showing significant recovery after immunoadsorption plasmapheresis].

The patient is an 18-year-old female. She had a history of acute disseminated encephalomyelitis at the age of 6 and 7. She visited our hospital due to acute disturbance of consciousness, quadriplegia, and numbness of left upper and lower extremities. Brain MRI showed multiple DWI/FLAIR high-signal lesions in the bilateral cerebral hemispheres, cerebellum, and brainstem. Qualitative test indicated that serum anti-MOG antibodies was positive, and she was diagnosed with anti-MOG antibody-positive polyphasic disseminated encephalomyelitis. Intravenous mPSL pulse therapy was performed twice, but the symptoms worsened. As a second line treatment, plasma exchange was started. However, she developed transfusion related acute lung injury. Alternatively, she was treated with immunoadsorption plasmapheresis. Her symptoms were significantly improved. This case seems to be valuable because there are few reports showing effectiveness of immunoadsorption therapy on anti-MOG antibody-related diseases, especially for polyphasic disseminated encephalomyelitis.

GRK5-mediated inflammation and fibrosis exert cardioprotective effects during the acute phase of myocardial infarction.

During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G-protein-coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation-related genes through nuclear factor-κB activation, leading to an increase in the expression levels of fibrosis-related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5-mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild-type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.

Authentically phosphorylated α-synuclein at Ser129 accelerates neurodegeneration in a rat model of familial Parkinson's disease.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble α-synuclein (α-syn) called Lewy bodies (LBs). Approximately 90% of α-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total α-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated α-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in α-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T α-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated α-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T α-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated α-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated α-syn did not promote accumulation of detergent-insoluble α-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T α-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T α-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated α-syn accelerates A53T α-syn neurotoxicity without the formation of detergent-insoluble α-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates α-syn at Ser129.

Therapeutic effect of a novel curcumin derivative GT863 on a mouse model of amyotrophic lateral sclerosis.

The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H2O2- and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.