The β1 subunit of the Na,K-ATPase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 (MLC1) in brain astrocytes: new insights into MLC pathogenesis

Brignone, Maria Stefania; Lanciotti, Angela; Macioce, Pompeo; Macchia, Gianfranco; Gaetani, Matteo; Aloisi, Francesca; Petrucci, Tamara C.; Ambrosini, Elena

doi:10.1093/hmg/ddq435

Cited by 52 publications

(71 citation statements)

References 80 publications

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“…Altogether, these data confirm the hypothesis that MLC1 is involved in the regulation of organelle acidity, possibly by limiting vesicle acidification. A similar behavior has been described for the Na, K-ATPase which exerts an essential role in the maintenance of the slightly acidic pH typical of early endosomes in which it is localized (Cain and Murphy, 1988; Cain et al, 1989; Feldmann et al, 2007; Grabe and Oster, 2001) and whose direct interaction with MLC1 has been demonstrated by our group (Brignone et al, 2011). Future experiments will aim to clarify the exact mechanisms through which MLC1 can influence endosomal pH and the molecular and functional relationships between MLC1 and V-ATPase.…”

Section: Discussionsupporting

confidence: 82%

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

Brignone

Lanciotti

Visentin

et al. 2014

Neurobiology of Disease

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Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1+, Rab5+) and recycling (Rab11+) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

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Section: Discussionsupporting

confidence: 82%

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

Brignone

Lanciotti

Visentin

et al. 2014

Neurobiology of Disease

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“…Expression of proteins allegedly associated or interacting with MLC113, 30, 31, 32, 33, 34 was evaluated by immunohistochemistry in 7‐month‐old mice. Compared to wild‐type animals, expression of the water channel aquaporin4 was redistributed along the cell bodies and processes of Glialcam ‐null astrocytes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Most previous studies on MLC1 function are based on patient leukocytes, artificial cell systems and scarcely available patient brain tissue obtained from biopsies and one autopsy 5, 6, 8, 9, 10, 15, 21, 30, 31, 32, 33, 34, 37. To allow research on MLC1 dysfunction in the intact brain, we previously developed an Mlc1 ‐null mouse that models the human disease 18.…”

Section: Discussionmentioning

confidence: 99%

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

Bugiani

Dubey

Breur

et al. 2017

Ann Clin Transl Neurol

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ObjectiveMegalencephalic leukoencephalopathy with cysts (MLC) is a genetic infantile‐onset disease characterized by macrocephaly and white matter edema due to loss of MLC1 function. Recessive mutations in either MLC1 or GLIALCAM cause the disease. MLC1 is involved in astrocytic volume regulation; GlialCAM ensures the correct membrane localization of MLC1. Their exact role in brain ion‐water homeostasis is only partly defined. We characterized Glialcam‐null mice for further studies.MethodsWe investigated the consequences of loss of GlialCAM in Glialcam‐null mice and compared GlialCAM developmental expression in mice and men.Results Glialcam‐null mice had early‐onset megalencephaly and increased brain water content. From 3 weeks, astrocytes were abnormal with swollen processes abutting blood vessels. Concomitantly, progressive white matter vacuolization developed due to intramyelinic edema. Glialcam‐null astrocytes showed abolished expression of MLC1, reduced expression of the chloride channel ClC‐2 and increased expression and redistribution of the water channel aquaporin4. Expression of other MLC1‐interacting proteins and the volume regulated anion channel LRRC8A was unchanged. In mice, GlialCAM expression increased until 3 weeks and then stabilized. In humans, GlialCAM expression was highest in the first 3 years to then decrease and stabilize from approximately 5 years.Interpretation Glialcam‐null mice replicate the early stages of the human disease with early‐onset intramyelinic edema. The earliest change is astrocytic swelling, further substantiating that a defect in astrocytic volume regulation is the primary cellular defect in MLC. GlialCAM expression affects expression of MLC1, ClC‐2 and aquaporin4, indicating that abnormal interplay between these proteins is a disease mechanism in megalencephalic leukoencephalopathy with cysts.

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“…K + homeostasis is tightly linked to the homeostasis of other ions, and MLC1 and GlialCAM colocalize and/or interact with several proteins involved in ion and water homeostasis. This includes the dystrophin glycoprotein complex,5, 45 Na + /K + ‐ATPase,46 the swelling‐sensitive cation channel, TRPV4,47 and the gap‐junction protein, connexin 43 20. MLC1 and GlialCAM may therefore play a central role in organizing these components necessary for astrocyte ion and water homeostasis.…”

Section: Discussionmentioning

confidence: 99%

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts

Dubey

Brouwers

Hamilton

et al. 2018

Annals of Neurology

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ObjectiveLoss of function of the astrocyte‐specific protein MLC1 leads to the childhood‐onset leukodystrophy “megalencephalic leukoencephalopathy with subcortical cysts” (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking.MethodsWe analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole‐cell patch‐clamp recordings and K+‐sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC.ResultsAn early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+ dynamics and neuronal network activity are abnormal in MLC mice.InterpretationDisturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636–649

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The β1 subunit of the Na,K-ATPase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 (MLC1) in brain astrocytes: new insights into MLC pathogenesis

Cited by 52 publications

References 80 publications

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts

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