Gustavo Monteiro Viana

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.

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 β-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.

Mucopolysaccharidoses (MPS) are the group of lysosomal storage disorders caused by deficiencies of enzymes involved in the stepwise degradation of glycosaminoglycans. To identify brain pathology common for neurological MPS, we conducted a comprehensive analysis of brain cortex tissues from post-mortem autopsy materials of eight patients affected with MPS I, II, IIIA, IIIC, and IIID, and age-matched controls. Frozen brain tissues were analyzed for the abundance of glycosaminoglycans (heparan, dermatan, and keratan sulfates) by LC-MS/MS, glycosphingolipids by normal phase HPLC, and presence of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor superfamily member 10 (TNFSF10) by Western blotting. Fixed tissues were stained for the markers for microgliosis, astrogliosis, misfolded proteins, impaired autophagy, and GM2 ganglioside. Our results demonstrate that increase of heparan sulfate, decrease of keratan sulfate, and storage of simple monosialogangliosides 2 and 3 (GM2 and GM3) as well as the neutral glycosphingolipid, LacCer, together with neuroinflammation and neuronal accumulation of misfolded proteins are the hallmarks of brain pathology in MPS patients. These biomarkers are similar to those reported in the corresponding mouse models, suggesting that the pathological mechanism is common for all neurological MPS in humans and mice.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control or influence crucial biological processes. Attempts to describe its structure-function relationships with HS binding proteins in a classical ‘lock and key’ type manner, however, have been unsuccessful. HS chains are synthesized in a non-template driven process in the ER and Golgi apparatus, involving a large number of enzymes capable of fine-tuning structures. Changes in the localization of HS-modifying enzymes throughout the Golgi, rather than protein expression levels, were found to correlate with changes in the structure of HS. Following brefeldin A treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Further, shortly after treatment with heparin, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS trisulfated disaccharide content. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings highlight that the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking processes are critical prerequisite for the complete delineation of HS biosynthesis.

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.

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of ¹⁹F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing ¹⁹F NMR spectroscopy is followed. Furthermore, the ability of ¹⁹F labelled GAGs to be imaged using CARS microscopy is demonstrated. ¹⁹F labelled GAGs enable both ¹⁹F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.

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 Ca²⁺ 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. J. Cell. Physiol. 231: 698–707, 2016.

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.