Neurofibromin haploinsufficiency results in altered spermatogenesis in a mouse model of neurofibromatosis type 1.

The fertility of men with neurofibromatosis 1 (NF1) is reduced. Despite this observation, gonadal function has not been examined in patients with NF1. In order to assess the role of reduced neurofibromin in the testes, we examined testicular morphology and function in an Nf1+/- mouse model. We found that although Nf1+/- male mice are able to reproduce, they have significantly fewer pups per litter than Nf1+/+ control males. Reduced fertility in Nf1+/- male mice is associated with disorganization of the seminiferous epithelium, with exfoliation of germ cells and immature spermatids into the tubule lumen. Morphometric analysis shows that these alterations are associated with decreased Leydig cell numbers and increased spermatid cell numbers. We hypothesized that hyper-activation of Ras in Nf1+/- males affects ectoplasmic specialization, a Sertoli-spermatid adherens junction involved in spermiation. Consistent with this idea, we found increased expression of phosphorylated ERK, a downstream effector of Ras that has been shown to alter ectoplasmic specialization, in Nf1+/- males in comparison to control Nf1+/+ littermates. These data demonstrate that neurofibromin haploinsufficiency impairs spermatogenesis and fertility in a mouse model of NF1.

Quantitative associations of scalp and body subcutaneous neurofibromas with internal plexiform tumors in neurofibromatosis 1.

Internal plexiform neurofibromas are a major cause of adverse outcomes in patients with neurofibromatosis 1 (NF1). We investigated the relationship of the numbers of subcutaneous neurofibromas of the scalp or body to internal plexiform tumor volume in 120 NF1 patients who had undergone whole body magnetic resonance imaging (MRI). We identified internal plexiform neurofibromas in 55% of patients, subcutaneous neurofibromas of the body in 75%, and subcutaneous neurofibromas of the scalp in 45%. The number of subcutaneous neurofibromas of the body and scalp were associated with each other (Spearman's Rho = 0.36; P < 0.001). The presence of internal tumors was associated with the presence (odds ratio [OR] = 4.38, 95% confidence interval [CI] 2.04-9.86, P < 0.001) and number (OR = 1.06 per neurofibroma, 95% CI 1.02-1.13, P < 0.001) of subcutaneous neurofibromas of the scalp. The total internal tumor volume was associated with the number of subcutaneous neurofibromas of the body (OR = 1.00086 per neurofibroma, 1.000089-1.0016, P = 0.029) and of the scalp (OR = 1.056 per neurofibroma, 1.029-1.083, P < 0.0001). Numbers of subcutaneous neurofibromas of the scalp and body are associated with internal plexiform tumor burden in NF1. Recognition of these associations may improve clinical management by helping to identify patients who will benefit most from whole body MRI and more intense clinical surveillance.

Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress.

Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long-term exposure to 10µM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport. However, we found that tunicamycin caused an immediate release of calcium from the endo/sarcoplasmic reticulum, which was sensitive to thapsigargin, and an influx of calcium through the plasma membrane, resulting in a significant increase in cytoplasmic calcium and depletion of endo/sarcoplasmic reticulum calcium. Furthermore, we observed that tunicamycin also induced contraction in intact vascular smooth muscle. By applying established procedures and antagonists, we established that tunicamycin did not directly activate physiological calcium channels, such as store-operated channels, voltage gated calcium channels, ryanodine receptors or inositol trisphosphate receptors. Instead, we found that its effects on cellular calcium fluxes closely resembled those of the known calcium ionophore, ionomycin. We have concluded that tunicamycin directly permeabilizes the plasma membrane and endo/sarcoplasmic reticulum to calcium, and is, therefore, inappropriate for studying the relationship between accumulation of unfolded proteins and endo/sarcoplasmic reticulum calcium dysregulation during the endo/sarcoplasmic reticulum stress response. In contrast, we also report that two other well-known endo/sarcoplasmic reticulum stress inducers, brefeldin A and dithiothreitol, did not exhibit similar increases in calcium permeability.