Open Access
Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects
(IRL Press at Oxford University Press, 2008-08-30) Duarri, Anna; Teijido Hermida, Óscar; López-Hernández, Tania; Scheper, Gert C.; Barriere, Herve; Boor, Ilja; Aguado, Fernando; Zorzano, Antonio; Palacín, Manuel; Martínez, Albert; Lukacs, Gergely L.; Van der Knaap, Marjo S.; Nunes, Virginia; Estévez, Raúl; Ciencias de la Salud; Osasun Zientziak
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.
Open Access
Vacuolating megalencephalic leukoencephalopathy with subcortical cysts: functional studies of novel variants in MLC1
(Wiley, 2006-02-08) Montagna, Giorgia; Teijido Hermida, Óscar; Eymard-Pierre, Eleonore; Muraki, Koutarou; Cohen, Bruce; Loizzo, Annalivia; Grosso, Pietro; Tedeschi, Gioacchino; Palacín, Manuel; Boespflug-Tanguy, Odile; Bertini, Enrico; Santorelli, Filippo M.; Estévez, Raúl; Ciencias de la Salud; Osasun Zientziak
Nine new unrelated patients presenting vacuolating myelinopathy with subcortical cysts were identified and analyzed for variations in the MLC1 gene. We detected 12 mutations (p.Leu37fs, p.Met80Val, p.Leu83Phe, p.Pro92Ser, p.Ser93Leu, p.Ile108fs, p.Gly130Arg, p.Cys171fs, p.Glu202Lys, p.Ser269Tyr, p.Ala275Asn, and p.Leu310_311insLeu) of which nine were novel. In one patient we did not detect mutations. Using a heterologous system, three new missense variants (p.Glu202Lys, p.Ser269Tyr, and p.Ala275Asn) and a single leucine insertion (p.Leu310insLeu) - lying in a stretch of seven leucines - were functionally assayed by determining total protein levels and mutant protein expression at the plasma membrane. No correlation was observed between mutation, clinical features, and plasma membrane expression of mutant protein.
Open Access
Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel
(American Society for Cell Biology, 2005-03-16) Dejean, Laurent M.; Martínez-Caballero, Sonia; Guo, Liang; Hughes, Cynthia; Teijido Hermida, Óscar; Ducret, Thomas; Ichas, François; Korsmeyer, Stanley J.; Antonsson, Bruno; Jonas, Elizabeth A.; Kinnally, Kathleen W.; Ciencias de la Salud; Osasun Zientziak
Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.
Open Access
Distribution of thyrotropin-releasing hormone immunoreactivity in the brain of the dogfish Scyliorhinus canicula
(Wiley, 2002-10-30) Teijido Hermida, Óscar; Manso, María Jesús; Anadón, Ramón; Ciencias de la Salud; Osasun Zientziak
To improve knowledge of the peptidergic systems of elasmobranch brains, the distribution of thyrotropin-releasing hormone-immunoreactive (TRHir) neurons and fibers was studied in the brain of the small-spotted dogfish (Scyliorhinus canicula L.). In the olfactory bulbs, small granule neurons richly innervated the olfactory glomeruli. In the telencephalic hemispheres, small TRHir neurons were observed in the superficial dorsal pallium, whereas TRHir fibers were widely distributed in pallial and subpallial regions. In the preoptic region, TRHir neurons formed a caudal ventrolateral group in the preoptic nucleus. In the hypothalamus, the most conspicuous TRHir populations were associated with the lateral hypothalamic recess, but small TRHir populations were found in the posterior tubercle and ventral wall of the posterior recess. The preoptic region and hypothalamus exhibited rich innervation by TRHir fibers. TRHir fibers were observed coursing to the neurohypophysis and the neuroepithelium of the saccus vasculosus, but not to the neurohemal region of the median eminence. Some stellate-like TRHir cells were observed in a few cell cords of the neurointermediate lobe of the hypophysis. The thalamus, pretectum, and midbrain lacked TRHir neurons. Further TRHir neuronal populations were observed in the central gray and superior raphe nucleus of the isthmus, and a few TRHir cells were located in the nucleus of the trigeminal descending tract at the level of the rostral spinal cord. In the brainstem, the central gray, interpeduncular nucleus, secondary visceral region of the isthmus, rhombencephalic raphe, inferior olive, vagal lobe, and Cajal's commissural nucleus were all richly TRHir-innervated. Comparison of the distribution of TRHir neurons observed in the dogfish brain with that observed in teleosts and tetrapods reveals strong resemblance but also interesting differences, indicating the presence of both a conserved basic vertebrate pattern and a number of derived characters.
Open Access
Localization and functional analyses of the MLC1 protein involved in megalencephalic leukoencephalopathy with subcortical cysts
(IRL Press at Oxford University Press, 2004-09-14) Teijido Hermida, Óscar; Martínez, Albert; Pusch, Michael; Zorzano, Antonio; Soriano, Eduardo; Río, José Antonio del; Palacín, Manuel; Estévez, Raúl; Ciencias de la Salud; Osasun Zientziak
Mutations in the MLC1 gene are responsible for one form of the neurological disorder megalencephalic leukoencephalopathy with subcortical cysts (MLC). The disease is a type of vacuolating myelinopathy. The biochemical properties and the function of the MLC1 protein are unknown. To characterize MLC1, we generated polyclonal antibodies. The MLC1 protein was detected in the brain, assembled into higher molecular complexes, as assessed by assembly-dependent trafficking assays. In situ hybridization and immunohistochemistry were used to determine MLC1 localization within the adult mouse brain. MLC1 was expressed in neurons, detected preferentially in particular axonal tracts. This expression pattern correlates with the major phenotype observed in the disease. In addition, it was expressed in some astrocytes, concentrating in Bergmann glia, the astrocyte end-feet membranes adjacent to blood vessels and in astrocyte¿astrocyte membrane contact regions. Other neuronal barriers, such as the ependyma and the pia mater, were also positive for MLC1 expression. MLC1 was detected in vivo and in heterologous systems at the plasma membrane. MLC mutations impaired folding, and the defect was corrected in vitro by addition of curcumin, a Ca2+-ATPase inhibitor. In summary, this study provides an explanation as to why mutations in MLC1 provoke the disease and points to a possible therapy for some patients.