Morphological damage in Sertoli, myoid and interstitial cells in a mouse model of mucopolysaccharidosis type I (MPS I).

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by a mutation in the IDUA gene, which codes α-L-iduronidase (IDUA), a lysosomal hydrolase that degrades two glycosaminoglycans (GAGs): heparan sulfate (HS) and dermatan sulfate (DS). GAGs are macromolecules found mainly in the extracellular matrix and have important signaling and structural roles which are essential to the maintenance of cell and tissue physiology. Nondegraded GAGs accumulate in various cell types, which characterizes MPS I as a multisystemic progressive disease. Many tissues and vital organs have been described in MPS I models, but there is a lack of studies focused on their effects on the reproductive tract. Our previous studies indicated lower sperm production and morphological damage in the epididymis and accessory glands in male MPS I mice, despite their ability to copulate and to impregnate females. Our aim was to improve the testicular characterization of the MPS I model, with a specific focus on ultrastructural observation of the different cell types that compose the seminiferous tubules and interstitium. We investigated the testicular morphology of 6-month-old male C57BL/6 wild-type (Idua+/+) and MPS I (Idua-/-) mice. We found vacuolated cells widely present in the interstitium and important signs of damage in myoid, Sertoli and Leydig cells. In the cytoplasmic region of Sertoli cells, we found an increased number of vesicles with substrates under digestion and a decreased number of electron-dense vesicles similar to lysosomes, suggesting an impaired flux of substrate degradation. Conclusions: Idua exerts an important role in the morphological maintenance of the seminiferous tubules and the testicular interstitium, which may influence the quality of spermatogenesis, having a greater effect with the progression of the disease.

Cathepsin B-associated Activation of Amyloidogenic Pathway in Murine Mucopolysaccharidosis Type I Brain Cortex.

Mucopolysaccharidosis type I (MPS I) is caused by genetic deficiency of α-l-iduronidase and impairment of lysosomal catabolism of heparan sulfate and dermatan sulfate. In the brain, these substrates accumulate in the lysosomes of neurons and glial cells, leading to neuroinflammation and neurodegeneration. Their storage also affects lysosomal homeostasis-inducing activity of several lysosomal proteases including cathepsin B (CATB). In the central nervous system, increased CATB activity has been associated with the deposition of amyloid plaques due to an alternative pro-amyloidogenic processing of the amyloid precursor protein (APP), suggesting a potential role of this enzyme in the neuropathology of MPS I. In this study, we report elevated levels of protein expression and activity of CATB in cortex tissues of 6-month-old MPS I (Idua -/- mice. Besides, increased CATB leakage from lysosomes to the cytoplasm of Idua -/- cortical pyramidal neurons was indicative of damaged lysosomal membranes. The increased CATB activity coincided with an elevated level of the 16-kDa C-terminal APP fragment, which together with unchanged levels of ß-secretase 1 was suggestive for the role of this enzyme in the amyloidogenic APP processing. Neuronal accumulation of Thioflavin-S-positive misfolded protein aggregates and drastically increased levels of neuroinflammatory glial fibrillary acidic protein (GFAP)-positive astrocytes and CD11b-positive activated microglia were observed in Idua -/- cortex by confocal fluorescent microscopy. Together, our results point to the existence of a novel CATB-associated alternative amyloidogenic pathway in MPS I brain induced by lysosomal storage and potentially leading to neurodegeneration.

Lipase-like 5 enzyme controls mitochondrial activity in response to starvation in Caenorhabditis elegans.

The C. elegans lipase-like 5 (lipl-5) gene is predicted to code for a lipase homologous to the human gastric acid lipase. Its expression was previously shown to be modulated by nutritional or immune cues, but nothing is known about its impact on the lipid landscape and ensuing functional consequences. In the present work, we used mutants lacking LIPL-5 protein and found that lipl-5 is important for normal lipidome composition as well as its remodeling in response to food deprivation. Particularly, lipids with signaling functions such as ceramides and mitochondrial lipids were affected by lipl-5 silencing. In comparison with wild type worms, animals lacking LIPL-5 were enriched in cardiolipins linked to polyunsaturated C20 fatty acids and coenzyme Q-9. Differences in mitochondrial lipid composition were accompanied by differences in mitochondrial activity as mitochondria from well-fed lipl-5 mutants were significantly more able to oxidize respiratory substrates when compared with mitochondria from well-fed wild type worms. Strikingly, starvation elicited important changes in mitochondrial activity in wild type worms, but not in lipl-5 worms. This indicates that this lipase is a determinant of mitochondrial functional remodeling in response to food withdrawal.

Evidence that glycosaminoglycan storage and collagen deposition in the cauda epididymidis does not impair sperm viability in the Mucopolysaccharidosis type I mouse model.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by a deficiency of the lysosomal hydrolase, α-L-iduronidase (IDUA). IDUA degrades heparan and dermatan sulfates, two types of glycosaminoglycan (GAG), important signalling and structural molecules of the extracellular matrix. Because many cell types store GAGs, MPS I has been investigated in human and animal models. Enzyme replacement therapy is available for MPS I patients and has improved their life expectancy, allowing them to achieve reproductive age. The aim of this study was to evaluate epididymal and sperm morphology and function in a murine model of MPS I. We used C57BL Idua+/+ and Idua-/- adult male mice (6 months old) to investigate epididymal morphology, sperm ultrastructure, GAG characterisation and mating competence. Epithelial GAG storage, especially in the cauda epididymidis, was seen in Idua-/- mice. Regardless of the morphologic change and GAG storage found in the cauda epididymis, sperm morphology and motility were normal, similar to wild types. In the interstitium, vacuolated cells were found in addition to deposits of GAGs. Mating was not impaired in Idua-/- males and litter sizes were similar between groups. At the time point of the disease evaluated, the deficiency in IDUA affected the morphology of the epididymis in male Idua-/- mice, whereas sperm appearance and motility and the male's capacity to mate and impregnate females were preserved.

Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo.

Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.

Comparative in vitro study of photodynamic activity of hypericin and hypericinates in MCF-7 cells.

In this work we present a comparative in vitro study of photodynamic activity between hypericin (HYP) and some hypericinates (hypericin ionic pair with lysine or N-methylglucamine) in human mammary adenocarcinoma cells (MCF-7). The toxicity and phototoxicity of hypericin and hypericinates were compared, as well as their cellular uptake and localization and mutagenic, genotoxic and clonogenic capacity. Our results demonstrate that different cationic moieties promote differences in the hypericinate solubility in a biological environment, and can influence the cellular localization and the phototoxicity of the photosensitizer. It was verified that hypericinates have better efficiency to generate singlet oxygen than HYP, and a lower aggregation in biological medium. In vitro assays have shown that HYP and the hypericinates are able to permeate the MCF-7 cell membrane and accumulated in organelles near the nucleus. The difference in location, however, was not determinant to the cell death mechanism, and a higher prevalence of apoptosis for all studied compounds occurred. The photodynamic studies indicated that hypericinates were more effective than HYP and were able to inhibit the formation of cellular colonies, suggesting a possible ability to prevent the recurrence of tumors. It also appears that all compounds have relative safety for mutagenicity and genotoxicity, which opens up a further safe route for application in in vivo studies.

Altered Cellular Homeostasis in Murine MPS I Fibroblasts: Evidence of Cell-Specific Physiopathology.

Mucopolysaccharidosis type I (MPS I), a rare autosomal recessive disease, is caused by a deficiency of the lysosomal enzyme alfa-L-iduronidase. Impaired enzyme activity promotes glycosaminoglycans accumulation in several tissues and organs, leading to complex multisystemic complications. Several studies using animal models indicated different intracellular pathways involving MPS I physiopathology; however, the exact mechanisms underlying this syndrome are still not understood. Previous results from our group showed alterations in ionic homeostasis and cell viability of splenocytes and macrophages in Idua-/- mice. In the present study, we found altered intracellular ionic homeostasis in a different cell type (fibroblasts) from the same murine model. Idua-/- fibroblasts from 3-month-old mice presented higher cytoplasmatic and endoplasmic reticulum Ca2+ concentration, lower levels of mitochondrial Ca2+ and mitochondrial membrane potential and higher cytoplasmatic pH when compared to Idua+/+ animals. Also, Idua-/- fibroblasts were more resistant to the apoptotic induction with staurosporine, indicating a possible resistance to apoptotic induction in those cells. In addition, despite the intracellular ionic imbalance, no significant alterations were found in apoptosis and autophagy in Idua-/- fibroblasts, which implies that the ionic alterations did not activate those pathways. The investigation of mechanisms underlying the cellular physiopathology of lysosomal diseases is crucial for a better understanding about the progression of these diseases. Since splenocytes, macrophages, and fibroblasts have different embryonic origins and distinct structural and functional features, potentially altered signaling pathways found in a cell-specific manner in an alfa-L-iduronidase-deficient environment provide additional understanding of the clinical multisystemic presentation of this disease and provide new basis for improved therapeutic approaches.

Impaired Hematopoiesis and Disrupted Monocyte/Macrophage Homeostasis in Mucopolysaccharidosis Type I Mice.

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disease caused by alpha-L-iduronidase deficiency in which heparan and dermatan sulfate degradation is compromised. Besides primary lysosomal glycosaminoglycan accumulation, further changes in cellular functions have also been described in several murine MPS models. Herein, we evaluated alterations in hematopoiesis and its implications on the production of mature progeny in a MPS I murine model. Despite the significant increase in hematopoietic stem cells, a reduction in common myeloid progenitors and granulocyte-macrophage progenitor cells was observed in Idua -/- mice bone marrow. Furthermore, no alterations in number, viability nor activation of cell death mechanisms were observed in Idua -/- mice mature macrophages but they presented higher sensitivity to apoptotic induction after staurosporine treatment. In addition, changes in Ca(2+) signaling and a reduction in phagocytosis ability were also found. In summary, our results revealed significant intracellular changes in mature Idua -/- macrophages related to alterations in Idua -/- mice hematopoiesis, revealing a disruption in cell homeostasis. These results provide new insights into physiopathology of MPS I.

The beneficial effects of strength exercise on hippocampal cell proliferation and apoptotic signaling is impaired by anabolic androgenic steroids.

Previous studies have shown that strength exercise improves memory and increases expression of a myriad of proteins involved on neuronal survival and synaptic plasticity in the hippocampus. Conversely, chronic exposure to supraphysiological levels of anabolic androgenic steroids (AAS) can induce psychiatric abnormalities, cognitive deficits, impair neurotransmission, alter the levels of neurotrophic factors, decrease cell proliferation and neurogenesis, and enhance neuronal cell death. In the present study, we investigated the effects of the AAS nandrolone decanoate (ND) administration during a strength exercise program on cell proliferation, apoptotic status and brain-derived neurotrophic factor (BDNF) expression in the rat hippocampus. Adult male Wistar rats were subjected to 4 weeks of progressive strength exercise in a vertical ladder apparatus with or without daily doses (5.0 mg/kg, SC) of ND. Immunohistochemistry analysis revealed that strength exercise increased significantly the number of Ki-67-positive cells (a cell proliferation marker) in dentate gyrus (DG) of hippocampus. However, this effect was abrogated when strength exercise was combined with ND. Although western blot analysis of whole hippocampus showed no significant differences in Bax and Bcl-2 protein expression among groups, the immunoreactivity of the pro-apoptotic protein Bax was significantly increased in DG, CA1 and CA3 hippocampal subfields of sedentary rats treated with ND. Moreover, the increase in the immunoreactivity of anti-apoptotic protein Bcl-2 (DG and CA3) induced by strength exercise was diminished by ND. There were no significant differences in BDNF expression among experimental groups. Therefore, the present findings suggest that the beneficial effects of strength exercise on hippocampal cell proliferation and apoptotic signaling are impaired by ND.

Visual Dysfunction of Type I and VI Mucopolysaccharidosis Patients Evaluated with Visual Evoked Cortical Potential.

PURPOSE: To evaluate the visual system of patients suffering from type I or VI mucopolysaccharidosis (MPS) by recording the visual evoked cortical potential (VECP). METHODS: Two patients with MPS VI and 2 patients with MPS I were tested before and after enzyme replacement therapy (ERT). A control group of 20 subjects was tested for statistical comparison. VECP was elicited by monocular stimulation with 1-Hz phase-reversal checkerboard patterns at 0.5 and 2 cycles per degree and with 16° of visual field. In all patients, both eyes were tested. VECP amplitude and latency were measured and compared with tolerance limits obtained from controls. RESULTS: MPS I and VI patients have a severe visual impairment that can be quantified by measuring VECPs. Even after several weeks of ERT, the visual impairment remained unaltered, indicating that the treatment had no significant influence on the visual conditions of MPS patients. Visual responses to high spatial frequencies were more deeply impaired than responses to low spatial frequencies. This can be explained by the kind of damage in the visual system that preferentially targets the eye optics. CONCLUSION: VECPs can be used to monitor the degree of visual impairment of MPS patients and to check ERT efficacy.

Implantação de um protocolo laboratorial para o diagnóstico de Mucopolissacaridoses VI e VII / Evaluation of a laboratorial protocol for Mucopolysaccharidosis VI and VII

As Mucopolissacaridoses (MPS) correspondem a um grupo de doenças genéticas raras caracterizadas peladeficiência/ausência de enzimas lisossomais responsáveis pela degradação de glicosaminoglicanos (GAG). Uma vez não degradados,os GAG se acumulam em diversos tecidos do organismo, causando uma série de complicações patológicas, que iniciam desde o período fetal até a fase infantil. Foi realizada a implantação de um protocolo laboratorial para o diagnóstico de pacientes com MPS, através da mensuração da atividade de duas enzimas lisossomais: beta-glicuronidase e arilsulfatase B, deficientes nas MPS VII e VI, respectivamente. Os valores encontrados em indivíduos normais corresponderam aos valores de referência descritos para a doença. Umpaciente com suspeita clínica de MPS demonstrou níveis normais de ambas as enzimas, o que exclui a possibilidade do mesmo possuir MPS VI ou VII. A implantação de protocolos laboratoriais de mensuração enzimática na investigação de MPS permite a realização de diagnósticos mais rápidos e, dessa forma, pode contribuir para as condutas clínicas mais apropriadas.

Investigação de doença de Fabry em pacientes com insuficiência renal crônica. / Fabry disease investigation on chronic kidney failure patients

Objetivos: implantação de um protocolo clínico-laboratorial que permita o diagnóstico da doença de Fabry (DF). Método: estudo longitudinal retrospectivo e realizado no período de janeiro de 2007 a janeiro de 2008. Para a padronização da técnica foi coletado 10ml de sangue heparinizado de 50 indivíduos hígidos voluntários. O grupo de estudo contou com a participação de 11 pacientes do sexo masculino, maiores de 18 anos, com insuficiência renal crônica (IRC) de causa desconhecida e em processo de hemodiálise no Hospital de Clínicas Gaspar Vianna. Deste grupo, foi coletado 10ml de sangue heparinizado e aplicado um questionário clínico. Para todos os indivíduos foi feito o ensaio enzimático para a ?-Gal A. Os dados foram analisados no Software Bioestat 4.0. Resultados: o valor de referência variou de 9,72 a 41,81 nmoles/h/ml, não houve diferença estatística entre os gêneros. No grupo teste, os resultados variaram de 12,1 a 27,4 nmoles/h/ml e o achado clínico mais comum foi a hipertensão arterial sistêmica (HAS). Um indivíduo do grupo controle apresentou baixa atividade enzimática. Conclusões: a dosagem da ?-Gal A em plasma apresentou valores semelhantes aos encontrados na literatura (4 a 22 nmoles/h/mL) e foi capaz de identificar um indivíduo com baixa atividade enzimática. Não foi encontrado nenhum caso de DF no grupo com IRC.

Objectives: introdution a clinical and laboratorial protocol which allows Fabry disease (FD) diagnosis. Method: this is a retrospective longitudinal study and data were collected since january 2007 until january 2008. To technic standardize, 10ml of heparin blood sample from 50 healthy volunteers individuals were collected. The test group was composed by 11 male patients, older than 18 years old, with unknown chronic kidney failure (CKF) on hemodialysis treatment on Hospital de Clínicas Gaspar Vianna. From this group, it was also colected 10mL of heparin blood sample and it was applied a clinical questionary. For all individuals, the ?-Galactosidase A (?-Gal A) enzymatic assay was done. Data were analised on Bioestat 4.0 Software. Results: the reference values varied from 9,72 to 41,81 nmoles/h/mL, there was no statistical difference between genders. On the test group, the results varied from 12,1 to 27,4 nmoles/h/mL and the most common clinical manifestation was systemic arterial hypertension (SAH). One individual from control group presented low enzymatic activity. Conclusions: the plasma ?-Gal A dosage presented values similar to those found on literature (4 to 22 nmoles/h/mL) and it was capable of identify an individual with low enzymatic activity. In this study, no DF case was found on CKF group.