Mis-splicing of Tau exon 10 in myotonic dystrophy type 1 is reproduced by overexpression of CELF2 but not by MBNL1 silencing.

Tau is the proteinaceous component of intraneuronal aggregates common to neurodegenerative diseases called Tauopathies, including myotonic dystrophy type 1. In myotonic dystrophy type 1, the presence of microtubule-associated protein Tau aggregates is associated with a mis-splicing of Tau. A toxic gain-of-function at the ribonucleic acid level is a major etiological factor responsible for the mis-splicing of several transcripts in myotonic dystrophy type 1. These are probably the consequence of a loss of muscleblind-like 1 (MBNL1) function or gain of CUGBP1 and ETR3-like factor 1 (CELF1) splicing function. Whether these two dysfunctions occur together or separately and whether all mis-splicing events in myotonic dystrophy type 1 brain result from one or both of these dysfunctions remains unknown. Here, we analyzed the splicing of Tau exons 2 and 10 in the brain of myotonic dystrophy type 1 patients. Two myotonic dystrophy type 1 patients showed a mis-splicing of exon 10 whereas exon 2-inclusion was reduced in all myotonic dystrophy type 1 patients. In order to determine the potential factors responsible for exon 10 mis-splicing, we studied the effect of the splicing factors muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1), CUGBP1 and ETR3-like factor 2 (CELF2), and CUGBP1 and ETR3-like factor 4 (CELF4) or a dominant-negative CUGBP1 and ETR-3 like factor (CELF) factor on Tau exon 10 splicing by ectopic expression or siRNA. Interestingly, the inclusion of Tau exon 10 is reduced by CUGBP1 and ETR3-like factor 2 (CELF2) whereas it is insensitive to the loss-of-function of muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1) gain-of-function, or a dominant-negative of CUGBP1 and ETR-3 like factor (CELF) factor. Moreover, we observed an increased expression of CUGBP1 and ETR3-like factor 2 (CELF2) only in the brain of myotonic dystrophy type 1 patients with a mis-splicing of exon 10. Taken together, our results indicate the occurrence of a mis-splicing event in myotonic dystrophy type 1 that is induced neither by a loss of muscleblind-like 1 (MBNL1) function nor by a gain of CUGBP1 and ETR3-like factor 1 (CELF1) function but is rather associated to CUGBP1 and ETR3-like factor 2 (CELF2) gain-of-function.

Immunohistochemical detection of glycoprotein hormone alpha subunit in somatoprolactinic and pure somatotroph adenomas.

Glycoprotein hormone alpha subunit (alpha SU) is expressed in nearly all thyreotroph adenomas and most gonadotrophinomas, but is less well documented in plurisecreting adenomas. We therefore examined the immunohistochemical (IHC) expression of alpha SU in a generally accepted model of plurisecreting adenomas (somatoprolactinic type) by comparison to a series of pure monosecreting somatotroph tumors. Fifty patients (32 females, 18 males) aged 15 to 68 years with clinical and/or biological acromegaly requiring adenomectomy were studied. Forty-five had clinical acromegaly and 5 had isolated amenorrhea and/or galactorrhea syndromes. Forty-eight of the 49 patients who had baseline assessments of plasma GH had a mean concentration of 5 ng/ml or more (normal value < 5). Fifteen of the 46 patients who had baseline measurements of plasma PRL had a prolactinemia value greater than 20 ng/ml (normal value < 20) but below 100 ng/ml, except for one patient. All the adenomas studied were positive by GH immunohistochemistry; 21 were immunostained by an antiPRL antibody and formed the "somatoprolactinic" (GH-PRL) group. Five of these 21 patients were male. The 12 female patients younger than 50 years had amenorrhea or galactorrhea, and one male patient complained of impotence. Eleven patients (9 females, 2 males) in this GH-PRL group had hyperprolactinemia. Sixteen of these GH-PRL adenomas were immunolabeled by alpha SU antiserum. The remaining 29 adenomas, which were immunonegative with the PRL antibody and formed the "somatotroph adenoma" (GH) group, were more frequent in male patients (13/29; 45%) compared to GH-PRL group. Eight amenorrhea or galactorrhea syndromes occurred among the 14 women younger than 50 years, 3 of whom had hyperprolactinemia. Thirteen of these 29 adenomas (45%) were immunopositive with alpha SU antibody. Compared to the GH group, the GH-PRL group had a significant higher frequency of amenorrhea and/or galactorrhea syndromes among women under 50 years (100% vs 57%; p < 0.01), as well as hyperprolactinemia (55% vs 15%; p < 0.01) and positive alpha SU immunoreactivity (76% vs 45%; p < 0.05). The frequency of extrasellar macroadenomas was not different according to PRL or alpha SU immunoreactivity. Thus, in this series of somatoprolactinic adenomas, alpha SU immunopositivity was slightly more frequent than in a control group of pure somatotroph adenomas. Moreover, hyperprolactinemia was more frequent in patients with GH-PRL adenomas, although the size of the pure and mixed adenomas was not different. These results suggest that hyperprolactinemia and/or alpha SU immunopositivity are more often associated with mixed GH-PRL adenomas.

Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation.

Treatment of diabetes mellitus by transplantation of isolated pancreatic islets could constitute an alternative to human pancreas allograft. Before transplantation, porcine islets are submitted to a procedure of isolation and purification. The quality of islets through these different steps may be assessed by morphological and functional studies. The aim of this work was the histological characterization of the four main cell types of porcine adult endocrine islets during the different steps of the isolation procedure using immunohistochemistry (IHC) applied in light (LM) and electron microscopy (EM). In fresh pancreas, islets were various sizes and shapes in LM. The number was not found different between the different portions of the pancreas. In IHC, insulin (Ins)-secreting cells accounted for the majority of the islet cells, while glucagon(Glu)-somatostatin (Som)- and polypeptide(PP)-immunoreactive cells, in decreasing number, were found in the mantle around the core of Ins-cells. In EM, B-cells contained poly-hedric granules with a dense central core and clear halo. Glu granules were spherical and very dense. D-cells and PP-cells were characterized by numerous granules, rather spherical and of inequal density for Som and more ellipsoidal for PP granules. After purification in Euroficoll, in EM, the four cellular types remained recognizable, but underwent vacuolization, mitochondrial swelling, and enlargement of intercellular spaces. After 3 days of culture on plastic dishes, as on Biopore membranes in a Millicell insert, microvilli appeared and vacuolization increased in EM. At the seventh day of culture, in EM, most of the cells were lysed in contrast to LM where at the same time, the four cell types were clearly identified by IHC but only in collagen matrix. Important discrepancies were noticed between LM and EM. This fact emphasizes the complementarity of morphological and functional studies in assessment of the quality of an islet isolation.