Comparison of zebrafish and mice knockouts for Megalencephalic Leukoencephalopathy proteins indicates that GlialCAM/MLC1 forms a functional unit

Abstract

BackgroundMegalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by astrocyte and myelin vacuolization, epilepsy and early-onset macrocephaly. MLC is caused by mutations in MLC1 or GLIALCAM, coding for two membrane proteins with an unknown function that form a complex specifically expressed in astrocytes at cell-cell junctions. Recent studies in Mlc1−/− or Glialcam−/− mice and mlc1−/− zebrafish have shown that MLC1 regulates glial surface levels of GlialCAM in vivo and that GlialCAM is also required for MLC1 expression and localization at cell-cell junctions.MethodsWe have generated and analysed glialcama−/− zebrafish. We also generated zebrafish glialcama−/− mlc1−/− and mice double KO for both genes and performed magnetic resonance imaging, histological studies and biochemical analyses.Resultsglialcama−/− shows megalencephaly and increased fluid accumulation. In both zebrafish and mice, this phenotype is not aggravated by additional elimination of mlc1. Unlike mice, mlc1 protein expression and localization are unaltered in glialcama−/− zebrafish, possibly because there is an up-regulation of mlc1 mRNA. In line with these results, MLC1 overexpressed in Glialcam−/− mouse primary astrocytes is located at cell-cell junctions.ConclusionsThis work indicates that the two proteins involved in the pathogenesis of MLC, GlialCAM and MLC1, form a functional unit, and thus, that loss-of-function mutations in these genes cause leukodystrophy through a common pathway.

Proteome Profile of Myelin in the Zebrafish Brain

“…The zebrafish has become a frequently used model organism to study cellular and molecular mechanisms in the central nervous system, including myelination (Pogoda et al, 2006;Lyons and Talbot, 2015;Ackerman and Monk, 2016;Czopka, 2016) and oligodendrocyte differentiation (Ravanelli et al, 2018;Marisca et al, 2020). Notwithstanding the increasing relevance of zebrafish in assessing the pathomechanisms of human myelin-related diseases (Pérez-Rius et al, 2019;Keefe et al, 2020;Tsata et al, 2020), proteomic approaches to zebrafish myelin have fallen behind evolving technical standards. For example, no study is available thus far that systematically approaches the entire zebrafish CNS myelin proteome.…”

Megalencephalic Leukoencephalopathy: Insights Into Pathophysiology and Perspectives for Therapy

“…It will be interesting to use Mlc1 floxed mice to generate conditional KO specifically in astrocytes during development, or KI mice for Mlc1 containing missense mutations as well as to obtain tissue-specific knockouts for GlialCAM. In zebrafish, an mlc1 KO line has been generated (Sirisi et al, 2014), with a mutation that abolishes mlc1 expression in vitro and in vivo; a glialcama KO and a double KO for mlc1 and glialcama (Pérez-Rius et al, 2019).…”

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

“…These observations suggest that the comprehension of MLC molecular pathogenesis and the identification of possible molecular targets may help finding therapeutic strategies to correct the pathological defect or slow down disease progression. To this aim, several transgenic mouse models of MLC have been generated, mainly MLC1/GlialCAM KO [ 92 , 93 , 94 , 95 , 96 ], and double KO for both MLC1 and GlialCAM [ 97 ]. Moreover, zebrafish lines KO for MLC1/GlialCAM or both have been also developed [ 97 , 98 ] ( Figure 3 ).…”

“…To this aim, several transgenic mouse models of MLC have been generated, mainly MLC1/GlialCAM KO [ 92 , 93 , 94 , 95 , 96 ], and double KO for both MLC1 and GlialCAM [ 97 ]. Moreover, zebrafish lines KO for MLC1/GlialCAM or both have been also developed [ 97 , 98 ] ( Figure 3 ). In all these models, increased brain water content and cerebellar WM vacuolation have been reported, although at an advanced age (about 8 months) compared with the human disease.…”