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Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece-principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa., Abbreviations: GALC: galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyde.

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of β-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Spermatogenesis is a complex developmental program in which interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis. The Twitcher mouse is a model of Krabbe disease, characterised by the deficiency of galactosylceramidase, the enzyme that hydrolyses galactosylceramide and galactosylsphingosine. Galactosyl-alkyl-acyl-glycerol, a precursor of seminolipid, the most abundant glycolipid in spermatozoa, is also a substrate for galactosylceramidase. Altered sphingolipid metabolism has been suggested to be the cause of the morphological abnormalities reported previously in the spermatogenesis of Twitcher. However, given the frequency of infertility associated with neurological impairment, we hypothesised that an unbalanced hormonal profile could contribute to male infertility in this mutant. In order to clarify this issue, we investigated potential variations in the expression of hormones and hormone receptors involved in the regulation of spermatogenesis. Our data show that, in the brain of Twitcher mouse, gonadotrophin-releasing hormone (GnRH), LH and FSH gene expression is decreased, whereas expression of androgen receptor (AR) and inhibin ?A (INH?A) is increased. The changes in gene expression for the LH and FSH receptors and AR in the testes support the hypothesis that altered sphingolipid metabolism is not the only cause of Twitcher infertility.

Globoid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the galactosylceramidase gene. The objective of the study was to present information about the fatty acid (FA) composition of the brain and serum of twitcher mice, a mouse model of Krabbe disease, compared to wild type, in order to identify biomarker of disease progression. We defined the FA profiles by identifying the main components present in serum and brain using GC-EI-MS analysis. The FA percentage composition was measured and data were analyzed considering the disease and the mouse age as experimental factors. Significant correlations were established, both in brain and in serum, in the fatty acid percentage composition of twitcher compared to wild type mice. The most abundant saturated fatty acid in brain was the palmitic acid (C16:0) with mean values significantly increased in twitcher mouse (p = 0.0142); moreover, three monounsaturated, three polyunsaturated (PUFA) and a plasmalogen were significantly correlated to disease. In the serum highly significant differences were observed between the two groups for three polyunsaturated fatty acids. In fact, the docosahexaenoic acid (C22:6n3c) content was significantly increased (p = 0.0116), while the C20 PUFA (C20:3n6c and C20:5n3c) were significantly decreased in twitcher serum samples. Our study shows a specific FA profile that may help to define a possible pattern that could distinguish between twitcher and wild type; these data are likely to provide insight in the identification of new biomarkers to monitor the disease progression and thereby permit the critical analysis of therapeutic approaches.

Krabbe disease or globoid cell leukodystrophy is a degenerative, lysosomal storage disease resulting from the deficiency of β-galactocerebrosidase activity. This enzyme catalyzes the lysosomal hydrolysis of galactocerebroside and psychosine. Krabbe disease is inherited as an autosomal recessive trait, and many of the 70 disease-causing mutations identified in the GALC gene are associated with protein misfolding. Recent studies have shown that enzyme inhibitors can sometimes translocate misfolded polypeptides to their appropriate target organelle bypassing the normal cellular quality control machinery and resulting in enhanced activity. In search for pharmacological chaperones that could rescue the β-galactocerebrosidase activity, we investigated the effect of α-Lobeline or 3',4',7-trihydroxyisoflavone on several patient-derived fibroblast cell lines carrying missense mutations, rather than on transduced cell lines. Incubation of these cell lines with α-lobeline or 3',4',7-trihydroxyisoflavone leads to an increase of β-galacocerebrosidase activity in p.G553R + p.G553R, in p.E130K + p.N295T and in p.G57S + p.G57S mutant forms over the critical threshold. The low but sustained expression of β-galactocerebrosidase induced by these compounds is a promising result; in fact, it is known that residual enzyme activity of only 15-20% is sufficient for clinical efficacy. The molecular interaction of the two chaperones with β-galactocerebrosidase is also supported by in silico analysis. Collectively, our combined in silico-in vitro approach indicate α-lobeline and 3',4',7-trihydroxyisoflavone as two potential pharmacological chaperones for the treatment or improvement of quality of life in selected Krabbe disease patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mammalian spermatogenesis is a complex hormone-dependent developmental program where interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis. The Twitcher mouse is a model for the Krabbe disease, characterized by the deficiency of galactosylceramidase (GALC), a lysosomal enzyme that hydrolyzes the terminal galactose from galactosylceramide, a typical component of the myelin membrane. In addition, GALC catalyzes the hydrolysis of the terminal galactose from galactosyl-alkyl-acyl-glycerol, precursor of seminolipids, specifically expressed on the membrane of germ cells. Previous data reported by our group demonstrated that glycolipids play an important role in sperm maturation and differentiation. Moreover, we hypothesized that the severe impairment of the central nervous system that affects the Twitcher mouse could interfere with the hypothalamus-pituitary-gonadal axis function, contributing to infertility. To highlight this hypothesis we have determined, at molecular level, the potential variation in expression pattern of brain hormones involved in spermatogenesis regulation.

Globoid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the galactosylceramidase (GALC) gene. Deficiency of GALC results in the accumulation of a highly cytotoxic metabolite galactosylsphingosine (psychosine). In the present study, we describe the development and validation of a sensitive and specific LC-ESI-tandem-MS method for the determination of psychosine in the serum of twitcher mice, the naturally occurring animal model of this disease. The method was validated in terms of accuracy, precision, specificity, linearity and sensitivity. Calibration plots were linear over the concentration range of 2.5-50ng/mL. Recovery of psychosine from serum was in the range 94.20-98.02%. The results of this study show that in the affected mice the concentration of psychosine (ranging from 2.53 to 33.27ng/mL) increased significantly with the progression of the disease. The maximum level (33.27ng/mL) was detected in the serum of one of the twitcher mice sacrificed at 40PND. Psychosine was not detected at significant levels in wild type mice. These results clearly demonstrate that this noninvasive, rapid, and highly sensitive LC-ESI-tandem-MS method is a very useful approach for the detection of psychosine in serum., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Krabbe disease or globoid cell leukodystrophy is an autosomal recessively inherited disorder caused by the deficiency of galactocerebrosidase, the lysosomal enzyme that catalyzes the hydrolysis of galactose from galactosylceramide and galactosylsphingosine (psychosine). Psychosine accumulation results in the loss of myelin and oligodendrocytes in the brain of Krabbe patients as well as twitcher mice (natural model of human Krabbe disease). The aim of the present research was to investigate in twitcher mice the potential role of a diet deficient in galactose enriched in soy isoflavones and a pool of antioxidants molecules, such as l-glutathione, coenzyme Q10, xanthophylls, in counteracting the toxic effects derived by psychosine accumulation. A second goal of this manuscript was to demonstrate suppression of the apoptotic effects of psychosine in cultured oligodendrocyte progenitor mice cells (OLP-II) with antioxidants. The affected twitcher mice began the milk-derivatives free diet on post-natal day 15 although they also received mother's milk until post-natal day 18. Nevertheless, average life span was increased 50%, from 32+/-2 to 48+/-3 days, onset of tremor was delayed 17 days (from 21 days in the untreated twitcher mice to 38 days in the treated affected mice) and the gait in the treated mice was normal until almost a week after the untreated animals died (38+/-1 days versus 32 days at death). Weight gain in the treated animals also progressed to 38 days compared with 22 days for the untreated affected twitcher mice. Protection of the OLP-II cells against psychosine was shown using the MTT test (the ability of the tetrazolium salt MTT to form a dark blue formazan product by mitochondrial dehydrogenase in viable cells) and assay of expression of p53 and TNF-related apoptosis-inducing ligand (TRAIL). The results showed a time-dependent and concentration-dependent decrease of OLP-II viability on exposure to psychosine and dose-dependent protection with the antioxidants xanthophylls and glutathione. They also demonstrated that psychosine-induced p53 induction of apoptosis and TNF-related apoptosis-inducing ligand receptors could be decreased by l-glutathione and xanthophylls. A dietary approach may constitute a promising clinical management of the late-infantile and juvenile forms of Krabbe leukodystrophy., (Published by Elsevier Inc.)

Krabbe disease or globoid cell leukodystrophy is an autosomal recessive disorder resulting from mutations in the galactocerebrosidase (GALC) gene. These mutations lead to deficient GALC activity, storage of substrates of the enzyme, including psychosine, death to oligodendrocytes, decreased myelination, production of globoid cells and eventually death to the individual. While most affected individuals are infants, late-onset forms are also recognized. In addition to human patients, several animal models have been well characterized, including the twitcher mouse. A spontaneously transformed progenitor cell line was isolated from an astrocyte-enriched fraction of normal mice, partially characterized and transduced with a retrovirus-containing mouse GALC cDNA to produce increased GALC activity (20-30-fold above baseline). These cells, called MAR-52, were injected into the brains of newborn affected twitcher mice. While there was only a modest increase in lifespan and body weight, there was clear evidence for the correction of the astrocytic gliosis, normal appearing oligodendrocytes and evidence for remyelination. We demonstrate that the exogenously supplied neural progenitor cells can donate GALC enzyme to oligodendrocytes in the brains of affected mice resulting in normal myelination in the area of donor cells. At this time, hematopoietic stem cell transplantation provides the best outcome in affected mice and is the only treatment available for human patients, but it does not result in a cure even when performed in asymptomatic newborns. Complete correction probably will require a combined approach to effectively treat patients with Krabbe disease. With developments in the isolation and characterization of stem cells, this approach may improve the outcome for individuals diagnosed in the future.

Cystatin B (cystB) is an anti-protease implicated in EPM1, a degenerative disease of the central nervous system. This work analyzes the pattern of expression of cystB in developing and adult cerebellum, identifying the cystB positive cells by double immune-fluorescence microscopy using specific cell markers. In primary glial cells, cystB is found in progenitor and differentiated oligodendrocytes as well as in astrocytes. In the cerebellum, only oligodendrocyte progenitors express cystB. In myelin-producing cells, cystB synthesis is strongly down-regulated and the protein is not detectable. Astrocytes and Bergmann glia express cystB at all the developmental stages analyzed both in the cell body and in the fibers. Most neurons of developing and adult rat cerebellum do not express detectable amounts of cystB, with the exception of the Purkinje cells and of some cells of the differentiated molecular layer. In human cerebellum, cystB is present in Purkinje cells and Bergmann glial fibers only. cystB is also found in the cortical neurons of the dentate gyrus of the hippocampus. In rat cerebellum, cystB forms a complex with a number of proteins, two of which are specific to the nervous system. The cellular co-localization of cystB and its partners in developing and adult cerebellum is also shown.

The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A(+) mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A(+) mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288., (Copyright 2002 Wiley-Liss, Inc.)

Krabbe disease or globoid cell leukodystrophy is a disorder involving the white matter of the peripheral and central nervous systems. Mutations in the gene for the lysosomal enzyme galactocerebrosidase (GALC) result in low enzymatic activity and decreased ability to degrade galactolipids found almost exclusively in myelin. The pathological changes observed, including the presence of globoid cells and decreased myelin, appear to result from the toxic nature of psychosine and accumulation of galactosylceramide that cannot be degraded due to the GALC deficiency. Over 60 mutations have been identified in this gene. The great majority are disease-causing; however, a few are considered polymorphisms. While most patients present with symptoms within the first 6 months of life, others present later in life including adulthood. Even patients with the same genotype can have very different clinical presentations and course. The reason for this is not known. Treatment at this time is limited to hematopoietic stem cell transplantation that appears to slow the progression of the disease and improve the magnetic resonance images. Studies using stem cells and viral vectors to transduce transplantable cells are under way in model systems. In culture, oligodendrocytes from the twitcher mouse model can assume a normal appearance after differentiation if GALC activity is provided via viral transduction or uptake from donor cells. Therefore continued myelination and/or remyelination in patients will require supplying GALC activity by transplanted cells or viral vectors to still functional endogenous oligodendrocytes or transplantation of normal oligodendrocytes or stem cells that can differentiate into oligodendrocytes. Using the animal models these options can be explored., (Copyright 2000 Academic Press.)

The sphingomyelin pathway has been implicated in mediating the effect of several extracellular agents leading to important biochemical and cellular changes. The aim of this investigation is to study interleukin-1 beta (IL-1 beta) signaling in oligodendrocytes. For this purpose, the CG4 oligodendrocyte cells were differentiated and incubated with IL-1 beta. This treatment induced a time- and dose-dependent increase of the endocellular ceramide. To mimic the effect of the elevation of endogenous ceramide, the CG4 cells were treated with the ceramide analogue C2-ceramide. Cell survival, measured with the MTT assay, showed that, by increasing the concentration of ceramide, up to 40% of CG4 cells were dying within 6 h, similar data were obtained with the primary differentiated oligodendrocytes. Condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies indicated that apoptosis was the cause of death. Surprisingly, long-term exposure (72 h) to increasing concentrations of IL-1 beta, which increases intracellular ceramide, did not induce oligodendroglial cell death. These results show that an increase of intracellular ceramide is not sufficient to induce apoptosis in oligodendrocytes and that IL-1 beta signaling through the ceramide pathway in these cells can mediate functions other than programmed cell death.