Pathophysiological role of MLC1, a protein involved in megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), is a rare congenital and incurable leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation. The majority of MLC patients carry mutations in the MLC1 gene encoding a membrane protein named MLC1 that is highly expressed in brain astrocytes contacting blood vessels, ependyma and meninges. Although the neuropathological features of MLC disease, the molecular structure and the cellular localization of MLC1 suggest a possible involvement of this protein in astrocyte-mediated osmoregulatory processes, the function of MLC1 is still unknown. Understanding the function of MLC1 protein whose mutations are the main cause of MLC is an essential step toward identification of disease mechanisms and development of effective therapies. During the course of this thesis project we generated new data on MLC1 expression, distribution and functional associated pathways in astrocytes that are deregulated by pathological mutations, paving the way for the identification of the specific MLC1 function. We found that: i) endogenous MLC1 protein is expressed in cultured astrocytes, particularly in the plasma membrane where it interacts with caveolin-1 and proteins of the dystrophin/dystroglycan complex (DCG), and also in intracellular organelles and endoplasmic reticulum (Lanciotti et al., 2010); ii) MLC1 undergoes endolysosomal trafficking and, most of the missense mutations found in patients hamper MLC1 intracellular trafficking and localization at the plasma membrane (Lanciotti et al., 2010, 2012); iii) MLC1 directly binds the beta-1 subunit of the Na, K-ATPase enzyme and is part of a multiprotein complex that includes the inward rectifying potassium channel 4.1 (Kir4.1), caveolin-1 and syntrophin, and is involved in astrocyte response to hyposmotic stress (Brignone et al., 2011). Moreover, we generated a human pathological model based on astrocytoma cell lines overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. Using this new MLC disease model we found that WT, but not mutated MLC1, functionally interacts with the transient receptor potential cation channel-4 (TRPV4) to activate swelling-induced calcium influx in astrocytes during hyposmotic stress (Lanciotti et al, 2012). These findings, together with a recent study showing defects in a chloride current in patient-derived lymphoblast cell lines subjected to hyposmosis (Ridder et al, 2011), represent the first evidence that the MLC1 protein is involved in the molecular pathways regulating astrocyte response to osmotic changes

Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy. Supplementary information accompanies this paper in the files section.peer-reviewe

Pathophysiological role of MLC1, a protein involved in megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), is a rare congenital and incurable leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation. The majority of MLC patients carry mutations in the MLC1 gene encoding a membrane protein named MLC1 that is highly expressed in brain astrocytes contacting blood vessels, ependyma and meninges. Although the neuropathological features of MLC disease, the molecular structure and the cellular localization of MLC1 suggest a possible involvement of this protein in astrocyte-mediated osmoregulatory processes, the function of MLC1 is still unknown. Understanding the function of MLC1 protein whose mutations are the main cause of MLC is an essential step toward identification of disease mechanisms and development of effective therapies. During the course of this thesis project we generated new data on MLC1 expression, distribution and functional associated pathways in astrocytes that are deregulated by pathological mutations, paving the way for the identification of the specific MLC1 function. We found that: i) endogenous MLC1 protein is expressed in cultured astrocytes, particularly in the plasma membrane where it interacts with caveolin-1 and proteins of the dystrophin/dystroglycan complex (DCG), and also in intracellular organelles and endoplasmic reticulum (Lanciotti et al., 2010); ii) MLC1 undergoes endolysosomal trafficking and, most of the missense mutations found in patients hamper MLC1 intracellular trafficking and localization at the plasma membrane (Lanciotti et al., 2010, 2012); iii) MLC1 directly binds the beta-1 subunit of the Na, K-ATPase enzyme and is part of a multiprotein complex that includes the inward rectifying potassium channel 4.1 (Kir4.1), caveolin-1 and syntrophin, and is involved in astrocyte response to hyposmotic stress (Brignone et al., 2011). Moreover, we generated a human pathological model based on astrocytoma cell lines overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. Using this new MLC disease model we found that WT, but not mutated MLC1, functionally interacts with the transient receptor potential cation channel-4 (TRPV4) to activate swelling-induced calcium influx in astrocytes during hyposmotic stress (Lanciotti et al, 2012). These findings, together with a recent study showing defects in a chloride current in patient-derived lymphoblast cell lines subjected to hyposmosis (Ridder et al, 2011), represent the first evidence that the MLC1 protein is involved in the molecular pathways regulating astrocyte response to osmotic changes

Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies

Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions

Megalencephalic Leukoencephalopathy with Subcortical Cysts Disease-Linked MLC1 Protein Favors Gap-Junction Intercellular Communication by Regulating Connexin 43 Trafficking in Astrocytes

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions

NRF2 and PPAR-γ Pathways in Oligodendrocyte Progenitors: Focus on ROS Protection, Mitochondrial Biogenesis and Promotion of Cell Differentiation

An adequate protection from oxidative and inflammatory reactions, together with the promotion of oligodendrocyte progenitor (OP) differentiation, is needed to recover from myelin damage in demyelinating diseases. Mitochondria are targets of inflammatory and oxidative insults and are essential in oligodendrocyte differentiation. It is known that nuclear factor-erythroid 2-related factor/antioxidant responsive element (NRF2/ARE) and peroxisome proliferator-activated receptor gamma/PPAR-γ response element (PPAR-γ/PPRE) pathways control inflammation and overcome mitochondrial impairment. In this study, we analyzed the effects of activators of these pathways on mitochondrial features, protection from inflammatory/mitochondrial insults and cell differentiation in OP cultures, to depict the specificities and similarities of their actions. We used dimethyl-fumarate (DMF) and pioglitazone (pio) as agents activating NRF2 and PPAR-γ, respectively, and two synthetic hybrids acting differently on the NRF2/ARE pathway. Only DMF and compound 1 caused early effects on the mitochondria. Both DMF and pio induced mitochondrial biogenesis but different antioxidant repertoires. Moreover, pio induced OP differentiation more efficiently than DMF. Finally, DMF, pio and compound 1 protected from tumor necrosis factor-alpha (TNF-α) insult, with pio showing faster kinetics of action and compound 1 a higher activity than DMF. In conclusion, NRF2 and PPAR-γ by inducing partially overlapping pathways accomplish complementary functions aimed at the preservation of mitochondrial function, the defense against oxidative stress and the promotion of OP differentiation

The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1′s proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema

Multimorbidity and polypharmacy in the elderly: Lessons from REPOSI

The dramatic demographic changes that are occurring in the third millennium are modifying the mission of generalist professionals such as primary care physicians and internists. Multiple chronic diseases and the related prescription of multiple medications are becoming typical problems and present many challenges. Unfortunately, the available evidence regarding the efficacy of medications has been generated by clinical trials involving patients completely different from those currently admitted to internal medicine: much younger, affected by a single disease and managed in a highly controlled research environment. Because only registries can provide information on drug effectiveness in real-life conditions, REPOSI started in 2008 with the goal of acquiring data on elderly people acutely admitted to medical or geriatric hospital wards in Italy. The main goals of the registry were to evaluate drug prescription appropriateness, the relationship between multimorbidity/polypharmacy and such cogent outcomes as hospital mortality and re-hospitalization, and the identification of disease clusters that most often concomitantly occur in the elderly. The findings of 3-yearly REPOSI runs (2008, 2010, 2012) suggest the following pertinent tasks for the internist in order to optimally handle their elderly patients: the management of multiple medications, the need to become acquainted with geriatric multidimensional tools, the promotion and implementation of a multidisciplinary team approach to patient health and care and the corresponding involvement of patients and their relatives and caregivers. There is also a need for more research, tailored to the peculiar features of the multimorbid elderly patient