Deficiency of hyccin, a newly identified membrane protein, causes hypomyelination and congenital cataract.

We describe a new autosomal recessive white matter disorder ('hypomyelination and congenital cataract') characterized by hypomyelination of the central and peripheral nervous system, progressive neurological impairment and congenital cataract. We identified mutations in five affected families, resulting in a deficiency of hyccin, a newly identified 521-amino acid membrane protein. Our study highlights the essential role of hyccin in central and peripheral myelination.

CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes.

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25(hi) Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25(hi) Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro.

The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-ß (TGF-ß). Conversely, migration toward ABA, but not toward TGF-ß, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

Alteration of Th17 and Treg cell subpopulations co-exist in patients affected with systemic sclerosis.

Aim of the study has been to understand the relationship between TH17 and Treg cell subsets in patients affected with systemic sclerosis (SSc). Phenotypes and functions of Th17 and Treg cell subsets were analyzed in a series of 36 SSc patients. Th17 cell concentration in the peripheral blood was found to be increased in SSc patients with respect to healthy controls independently from type or stage of disease. After PBMC stimulation with a polyclonal stimulus or Candida albicans antigens the frequency of Th17 T cell clones was significantly higher in SSc patients with respect to controls suggesting the skewing of immune response in SSc patients toward Th17 cell generation/expansion. Concerning the Treg compartment, both CD4+CD25+ and CD8+CD28- Treg subsets showed quantitative and qualitative alteration in the peripheral blood of SSc patients. Collectively, these data highlight the existence of an imbalanced ratio between Th17 and Treg cell subsets in SSc patients.

Caveolin-3 T78M and T78K missense mutations lead to different phenotypes in vivo and in vitro.

Caveolins are the principal protein components of caveolae, invaginations of the plasma membrane involved in cell signaling and trafficking. Caveolin-3 (Cav-3) is the muscle-specific isoform of the caveolin family and mutations in the CAV3 gene lead to a large group of neuromuscular disorders. In unrelated patients, we identified two distinct CAV3 mutations involving the same codon 78. Patient 1, affected by dilated cardiomyopathy and limb girdle muscular dystrophy (LGMD)-1C, shows an autosomal recessive mutation converting threonine to methionine (T78M). Patient 2, affected by isolated familiar hyperCKemia, shows an autosomal dominant mutation converting threonine to lysine (T78K). Cav-3 wild type (WT) and Cav-3 mutations were transiently transfected into Cos-7 cells. Cav-3 WT and Cav-3 T78M mutant localized at the plasma membrane, whereas Cav-3 T78K was retained in a perinuclear compartment. Cav-3 T78K expression was decreased by 87% when compared with Cav-3 WT, whereas Cav-3 T78M protein levels were unchanged. To evaluate whether Cav-3 T78K and Cav-3 T78M mutants behaved with a dominant negative pattern, Cos-7 cells were cotransfected with green fluorescent protein (GFP)-Cav-3 WT in combination with either mutant or WT Cav-3. When cotransfected with Cav-3 WT or Cav-3 T78M, GFP-Cav-3 WT was localized at the plasma membrane, as expected. However, when cotransfected with Cav-3 T78K, GFP-Cav-3 WT was retained in a perinuclear compartment, and its protein levels were reduced by 60%, suggesting a dominant negative action. Accordingly, Cav-3 protein levels in muscles from a biopsy of patient 2 (T78K mutation) were reduced by 80%. In conclusion, CAV3 T78M and T78K mutations lead to distinct disorders showing different clinical features and inheritance, and displaying distinct phenotypes in vitro.

Mitochondrial DNA deletion in a child with mitochondrial encephalomyopathy, growth hormone deficiency, and hypoparathyroidism.

We report an 11-year-old boy with short stature, bilateral ptosis, sensorineural hearing loss, muscle weakness, and endocrine abnormalities. Brain magnetic resonance imaging (MRI) showed a bilateral abnormal signal in the globus pallidus and in the midbrain tegment. Muscle biopsy specimens showed ragged red and cytochrome c oxidase negative fibers, and biochemical analysis of muscle homogenate showed a partial defect of complex I and IV activities of the respiratory chain enzymes. Analysis of mitochondrial DNA by a polymerase chain reaction screening procedure and Southern blot revealed a novel heteroplasmic single mitochondrial DNA deletion of 7.8 kb in different tissues. This deletion was absent in the blood DNA of his mother and brother. This case further expands and confirms the wide clinical spectrum of mitochondrial disorders associated with single large-scale mitochondrial DNA deletions.

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies.

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2alpha-, 1beta-, 2gamma-, 1delta-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in alpha1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with alpha1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, alpha1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells. In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.

Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment.

In this report, we have developed a novel method to identify compounds that rescue the dystrophin-glycoprotein complex (DGC) in patients with Duchenne or Becker muscular dystrophy. Briefly, freshly isolated skeletal muscle biopsies (termed skeletal muscle explants) from patients with Duchenne or Becker muscular dystrophy were maintained under defined cell culture conditions for a 24-h period in the absence or presence of a specific candidate compound. Using this approach, we have demonstrated that treatment with a well-characterized proteasome inhibitor, MG-132, is sufficient to rescue the expression of dystrophin, beta-dystroglycan, and alpha-sarcoglycan in skeletal muscle explants from patients with Duchenne or Becker muscular dystrophy. These data are consistent with our previous findings regarding systemic treatment with MG-132 in a dystrophin-deficient mdx mouse model (Bonuccelli G, Sotgia F, Schubert W, Park D, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, and Lisanti MP. Am J Pathol 163: 1663-1675, 2003). Our present results may have important new implications for the possible pharmacological treatment of Duchenne or Becker muscular dystrophy in humans.