Cellular and Molecular Mechanisms Underly the Combined Treatment of Fasudil and Bone Marrow Derived-Neuronal Stem Cells in a Parkinson's Disease Mouse Model.

Bone marrow-derived neural stem cells (BM-NSCs) have shed light on novel therapeutic approaches for PD with the potential to halt or even reverse disease progression. Various strategies have been developed to promote therapeutic efficacy via optimizing implanted cells and the microenvironment of transplantation in the central nervous system (CNS). This current study further proved that the combination of fasudil, a Rho-kinase inhibitor, and BM-NSCs exhibited a synergetic effect on restoring neuron loss in the MPTP-PD mice model. It simultaneously unveiled cellular mechanisms underlying synergistic neuron-protection effects of fasudil and BM-NSCs, which included promoting the proliferation, and migration of endogenous NSCs, and contributing to microglia shift into the M2 phenotype. Corresponding molecular mechanisms were observed, including the inhibition of inflammatory responses, the elevation of neurotrophic factors, and the induction of WNT/ß-catenin and PI3K/Akt/mTOR signaling pathways. Our study provides evidence for the co-intervention of BM-NSCs and fasudil as a promising therapeutic method with enhanced efficacy in treating neurodegenerative diseases.

Neurofilament light is a novel biomarker for mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a complicated maternally inherited disorder lacking of sensitive and specific biomarkers. The objective of this study was to investigate the serum neurofilament light chain (NfL) as a novel biomarker of neurological dysfunction in MELAS. Patients with different status of MELAS were enrolled in this study. The Mini-Mental State Examination (MMSE) was given to the participants to evaluate cognition status. Multiple functional MRI was performed on the participants. Blood samples were collected and the serum NfL concentrations were determined by the single-molecule array technology (Simoa). This study enrolled 23 patients with MELAS, 15 people in the acute attack phase of MELAS and 10 people in the remission phase, including 2 patients in both acute attack and remission phase. Sixteen healthy controls (HCs) were also enrolled. Serum NfL level increased significantly in patients with MELAS. Serum NfL level in the acute attack group (146.73 [120.91-411.31] pg/ml, median [IQR]) was higher than in the remission group (40.31 [19.54-151.05] pg/ml, median [IQR]) and HCs group (7.70 [6.13-9.78] pg/ml, median [IQR]) (p < 0.05). The level of NfL in the remission phase group was higher than in HCs group (p < 0.05). A negative correlation was found between the serum NfL level and MMSE (p = 0.006, r = -0.650). The NfL concentration correlated positively with stroke-like lesion volume in the brain (r = 0.740, p < 0.001). Serum NfL may serve as a novel biomarker for the neurological dysfunction in MELAS patients.

Population Pharmacokinetic Analysis of Tacrolimus in Adult Chinese Patients with Myasthenia Gravis: A Prospective Study.

BACKGROUND AND OBJECTIVES: Tacrolimus is a widely used immunosuppressive agent with narrow therapeutic window. Nowadays, tacrolimus has gained acceptance as a therapeutic option in myasthenia gravis (MG) treament, however, little is known about its pharmacokinetic characteristics in MG population. In this study, we aimed to investigate the population pharmacokinetic (PopPK) of tacrolimus in patients with MG and to develop model-informed dosing regimens. METHODS: Trough concentrations of tacrolimus (267 measurements) and cytochrome P450 (CYP) genotypes were determined in 97 Chinese adults. The non-linear mixed-effects model was used for PopPK modeling. Monte Carlo simulations based on the established model were employed to design dosing regimens. RESULTS: The PopPK model was described using a one-compartment model with first-order absorption and elimination. The mean apparent clearance (CL/F) of tacrolimus was 17.1 L/h, with a between-subject variability of 20.1%. Covariate screening of demographic characteristics, blood test results, co-medications, and CYP3A5*3 or CYP3A4*1G polymorphisms showed that the CYP3A5*3 genotype and co-administration of a Wuzhi capsule significantly affected tacrolimus CL/F. CONCLUSIONS: For patients with the CYP3A5*3*3 allele, the required tacrolimus dose for 75% of subjects to achieve target trough concentrations of 4.8-15 ng/mL was 2 mg every 12 h (q12h). For patients with the CYP3A5*1*1 allele, the required dose was 2 mg tacrolimus q12h with a Wuzhi capsule, and for patients with the CYP3A5*1*3 allele, the required dose was 3 mg of tacrolimus q12h or 4 mg q24h co-administered with a Wuzhi capsule. This model could be employed to optimize individualized therapies for patients with MG.

Myasthenia gravis and specific immunotherapy: monoclonal antibodies.

Myasthenia gravis (MG) is an acquired autoimmune disease affecting the postsynaptic membrane of neuromuscular junctions and characterized by antibody-mediated T cell dependence and complement involvement. Cholinesterase inhibitors (e.g., pyridostigmine bromide), glucocorticoids, and azathioprine are currently recommended as first-line treatments for MG, though they have limitations, including potential toxicity and ineffectiveness in patients with refractory MG. In recent years, owing to an increasing understanding of MG pathogenesis the development and execution of clinical trials with novel biologics, including monoclonal antibodies (mAbs) that have demonstrated higher safety and more specificity, provide new opportunities for the treatment of MG. In this article, we review recent advances in MG pathogenesis and the mAbs that have been used for target-specific MG therapy.

Inhibition of ROCK activity regulates the balance of Th1, Th17 and Treg cells in myasthenia gravis.

Aberrant ROCK activation has been found in patients with several autoimmune diseases, but the role of ROCK in myasthenia gravis (MG) has not yet been clearly investigated. Here, we demonstrated that ROCK activity was significantly higher in peripheral blood mononuclear cells (PBMCs) from MG patients. ROCK inhibitor Fasudil down-regulated the proportions of Th1 and Th17 cells in PBMCs of MG patients in vitro. Intraperitoneal injection of Fasudil ameliorated the severity of experimental autoimmune myasthenia gravis (EAMG) rats and restored the balance of Th1/Th2/Th17/Treg subsets. Furthermore, Fasudil inhibited the proliferation of antigen-specific Th1 and Th17 cells, and inhibited CD4 + T cells differentiated into Th1 and Th17 through decreasing phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through increasing phosphorylated Stat5. We conclude that dysregulated ROCK activity may be involved in the pathogenic immune response of MG and inhibition of ROCK activity might serve as a novel treatment strategy for MG.

Immunosuppressive and monoclonal antibody treatment for myasthenia gravis: A network meta-analysis.

BACKGROUND: We intended to compare and rank all the immunotherapies including immunosuppressant agents or monoclonal antibodies for myasthenia gravis (MG). METHODS: A network meta-analysis was performed to synthesize the direct evidence and indirect evidence. Quantitative MG score (QMGS) was defined as the primary outcome. The secondary outcomes included the glucocorticoid reduction and hazard ratios (HR) from the counts of adverse events (AEs). RESULTS: We identified 14 studies including 808 MG patients. For the primary outcome, cyclosporine A (CsA) was hierarchically the best with statistical significances of -1.18 (-1.81, -0.59) vs placebo (PLA), -0.98 (-1.72, -0.23) vs mycophenolate mofetil, and -0.77 (-1.57, -0.032) vs tacrolimus (TAC). When the intervention periods were controlled, both eculizumab (ECZ) of -1.50 (-2.81, -0.18) and CsA of -1.23 (-1.81, -0.64) vs PLA reached a statistical significance. Belimumab and ECZ ranked the most tolerable therapies while CsA of 2.41 (0.58, 10.01) ranked the last vs PLA. CONCLUSION: These findings demonstrated that ECZ represented the most effective and tolerable therapeutic alternative to be recommended for refractory MG. TAC may be a beneficial therapy to treat MG extensively while the efficacy of CsA and cyclophosphamide may be limited by their multiple or severe AEs.

Rho kinase II interference by small hairpin RNA ameliorates 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine­induced parkinsonism in mice.

Novel therapeutic targets are required for the treatment of Parkinson's disease (PD). Previous studies suggest that the Rho/Rho­associated, coiled­coil­containing protein kinases (ROCKs) signaling pathway may be a promising therapeutic target in PD. To elucidate the importance of ROCKII in the pathogenesis of dopaminergic (DA) neuron loss and to investigate the efficacy of ROCK inhibitors in PD, ROCKII expression in the substantia nigra (SN) of mice was silenced through the injection of a lentivirus­based small hairpin RNA system. Empty lentivirus vectors served as controls. Mice were subsequently challenged with 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine (MPTP). The expression levels and activity of ROCKII were elevated in tyrosine hydroxylase­positive neurons and in cluster of differentiation (CD) 11b­positive microglia within the SN of MPTP­treated mice, which was accompanied by an increased level of expression of inducible nitric oxide synthase (iNOS) and activation of the Toll­like receptor (TLR)2/nuclear factor (NF)­κB signaling pathway in M1 microglia. ROCKII interference (RI) significantly improved movement disorder and attenuated DA neuron loss induced by MPTP. In addition, RI inhibited the activation of M1 microglia in the SN, exhibiting reduced activity of the TLR2/NF­κB signaling pathway and decreased expression levels of iNOS and inflammatory factors, including interleukin (IL)­1ß and IL­6. The results of the present study verify that ROCKII participates in the loss of DA neurons induced by MPTP and suggest that ROCKII inhibition may be a promising therapeutic target for PD.

Multitarget intervention of Fasudil in the neuroprotection of dopaminergic neurons in MPTP-mouse model of Parkinson's disease.

Recent studies have demonstrated that activation of the Rho-associated kinase (ROCK) pathway participates in the dopaminergic neuron degeneration and possibly in Parkinson's disease (PD). In the current study, we tried to observe the therapeutic potential of ROCK inhibitor Fasudil against dopaminergic neuron injury in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mouse model of PD, and explore possible molecular mechanisms by enzyme-linked immunosorbent assay (ELISA), western blot and immunofluorescent assays. The results showed that MPTP-PD mice presented motor deficits, dopaminergic neuron loss, activation of inflammatory response and oxidative stress as well as ROCK and glycogen synthase kinase 3ß (GSK-3ß) signaling pathways. The administration of Fasudil exhibited neuroprotective effects against the dopaminergic neurons and improved the motor function recovery in the MPTP-PD mice, accompanied by the suppression of inflammatory responses (IL-1ß, TNF-α, NF-κB-p65 and TLR-2), and oxidative stress (iNOS and gp91Phox), which might be associated with the inhibition of ROCK and GSK-3ß activity. Simultaneously, the administration of Fasudil resulted in the shift from inflammatory M1 to anti-inflammatory/neurorepair M2 microglia. Additionally, Fasudil intervention enhanced the expression of anti-oxidative factors such as NF-E2-related factor 2 (Nrf2), Hmox as well as neurotrophic factor including GDNF. Our observations defined the neuroprotective effects of Fasudil in MPTP-PD mice, and we found a series of novel effector molecules and pathways for explaining the neuroprotective effects against dopaminergic neurons. However, a lot of investigations are warranted to further elucidate the crosstalk among Fasudil, oxidative stress, inflammatory response, GDNF and ROCK/NF-kB/Nrf2 pathways in the therapeutic potential of PD.

Novel LAMP2 mutations in Chinese patients with Danon disease cause varying degrees of clinical severity.

AIMS: Danon disease is an Xlinked dominant lysosomal glycogen storage disorder characterized by cardiomyopathy, skeletal myopathy, and mental retardation. This study described two Chinese cases of Danon disease in order to broaden the phenotypic and genetic spectrum. METHODS: Clinical data were collected and LAMP2 mutations were analyzed. RESULTS: Patient A had fluctuating limb weakness during 6 months follow-up and was diagnosed with drug-induced myopathy due to anti-hepatitis B therapy with lamivudine. However, the first muscle biopsy with large cytoplasmic vacuoles confused the diagnosis and led to the second biopsy that allowed for the final diagnosis. Patient B had severe cardiac disturbances leading to sudden death. Molecularly, patient A harbored a synonymous mutation adjacent to the exon 6-intron 6 junction; mRNA analysis provided evidence that totally abolished the donor site and caused skipping of exon 6. Patient B harbored a frame-shift deletion mutation in exon 3 (c.396delA) leading to a truncated protein. DISCUSSION: To our knowledge, this is the first report of Danon disease caused by a synonymous exon mutation that affected mRNA splicing, which indicates that a synonymous substitution may not be silent when it is in the exon sequences close to the splice sites. It is also the first description of Danon disease clinically presenting as druginduced myopathy at onset; the pathological changes might be the key point for making a differential diagnosis. *These two authors contributed equally to this work.

Novel mutations m.3959G>A and m.3995A>G in mitochondrial gene MT-ND1 associated with MELAS.

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are progressive neurodegenerative disorder associated with polygenetic, maternally inherited mutations in mitochondrial DNA. Approximately 80% of MELAS cases are caused by the mutation m.3243A>G of the mitochondrial tRNA(Leu (UUR)) gene (MT-TL1). We reported two probands with MELAS features. Muscle biopsy identified ragged-red fibers (RRF) in Gomori Trichrome staining. A respiratory chain function study showed decreased activity of mitochondrial respiratory chain complex I in both probands. Sequencing of the mitochondrial DNA revealed two novel MT-ND1 gene missense mutations, m.3959G>A and m.3995A>G, which are highly conserved among species. Protein secondary structure predictions demonstrated that these mutations may alter the peptide structure and may lead to decreased ND1 gene stability. Our findings suggest that these two novel mutations may contribute to the MELAS phenotypes of the patients in our study.

Genetic variability and clinical spectrum of Chinese patients with limb-girdle muscular dystrophy type 2A.

INTRODUCTION: Previous studies of limb-girdle muscular dystrophy type 2A (LGMD2A) patients in many countries have suggested a heterogeneous genetic and clinical spectrum, but the genotypes and phenotypes of Chinese LGMD2A patients remain unclear. METHODS: We directly screened calpain-3 (CAPN3) in 18 Chinese Han subjects who exhibited severely reduced or completely absent calpain-3 expression, as determined by Western blot analysis. We subsequently analyzed genotype/phenotype correlations. RESULTS: Seventeen patients (94.4%) were identified who had at least 1 causative mutation. All 18 mutations were distributed along the entire gene, and 11 of the mutations were novel, including 4 missense mutations, 5 deletions, and 2 splicing mutations. The phenotypes of these Chinese LGMD2A patients varied from severe LGMD to distal myopathy, and even asymptomatic hyper-CK-emia. CONCLUSIONS: No evidential correlation was found between the genotypes and phenotypes of the patients assessed in this study. Western blot analysis is still a useful diagnostic method when genetic analysis is unavailable.

HLA-DQA1*03:02/DQB1*03:03:02 is strongly associated with susceptibility to childhood-onset ocular myasthenia gravis in Southern Han Chinese.

OBJECTIVE: Our aim was to investigate the correlation between onset age, clinical features and HLA-DQA1/DQB1 genetic variability in myasthenia gravis (MG) patients in Southern Han Chinese. METHODS: 205 MG patients and 100 controls were genotyped for HLA-DQA1 and -DQB1 using sequence-based typing (SBT) and analyzed for haplotype frequencies. Anti-acetylcholine receptor (AChR) autoantibodies were measured in all, and muscle-specific tyrosine kinase (MuSK) antibodies were tested in AChR antibody negative patients. RESULTS: HLA-DQA1/DQB1 haplotypes showed association only with childhood-onset MG. Haplotype DQA1*03:02/DQB1*03:03:02 (DQ9) was positively associated with the childhood-onset MG, while haplotype DQA1*02:01/DQB1*02:02 and DQA1*05:01:01/DQB1*02:01:01 (DQ2) were negatively associated with this group. Childhood-onset ocular MG patients had an extremely high phenotype frequency of DQ9 haplotype (90.1% of patients, 34.0% of controls, p≤0.0001, OR=17.8). CONCLUSIONS: The childhood-onset ocular MG in Southern Han Chinese may present a particular subgroup of distinct genetic background. Its correlation to the HLA haplotype DQA1*03:02/DQB1*03:03:02 might explain the phenotypic difference of MG between Han Chinese and Caucasians.

Clinical and pathological features in 15 Chinese patients with calpainopathy.

BACKGROUND: Calpainopathy is comprised of a group of myopathies caused by deficiency in calcium-activated, neutral protease (calpain-3). In this study we identify calpainopathy in a cohort of Chinese patients with unclassified myopathy and analyze its clinical and pathological features. METHODS: Sixty-six muscle biopsies were selected for combined Western blotting of dysferlin and calpain-3 after immunohistochemical staining. Clinical and pathological parameters of 15 confirmed calpainopathy cases were determined. RESULTS: The diagnosis of calpainopathy in 15 Chinese patients was confirmed by Western blot analysis. Fourteen subjects had progressive proximal muscle weakness; 1 presented with bilateral distal muscle atrophy of the lower extremities. Scapular winging was observed in 12 patients (80%), and joint contractures were found in 10 others (66.7%). Histopathological studies showed a high prevalence of lobulated fibers (66.7%). CONCLUSIONS: Chinese patients with calpainopathy share some common clinical and pathological features with the reported characteristics of non-Chinese patients.

Decrease of CD4(+)CD25(high)Foxp3(+) regulatory T cells and elevation of CD19(+)BAFF-R(+) B cells and soluble ICAM-1 in myasthenia gravis.

Myasthenia gravis (MG) is caused by T-cell-dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokine and chemokine sICAM-1 and to analyze the phenotypes of CD4(+) and CD8(+) regulatory cells as well as the expression of BAFF-R on CD19(+) B cells in peripheral blood from 75 MG patients and 50 healthy controls. There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-gamma, TNF-alpha, TGF-beta and sCTLA-4 in both sera and culture supernatants between MG patients and healthy controls. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Although the populations of CD8(+)CD28(-) and CD8(+)CD122(+) regulatory T cells were not different between MG patients and healthy controls, MG patients exhibited the decrease of CD4(+)CD25(high)Foxp3(+) regulatory T cells and the increase of CD19(+)BAFF-R(+) B cells, revealing that MG patients should display the dysfunction of T cell balance and the activation of B cell maturation.