👤 O Bugiani

Volume-regulated anion channels (VRACs) are central to cell volume homeostasis. They mediate swelling-induced efflux of chloride and organic osmolytes to drive regulatory volume decrease. In the brain, VRACs have been proposed to play a key role in astrocytic volume regulation. Genetic defects in astrocytic VRAC modulating proteins (MLC1, GlialCAM, Aquaporin-4, GPRC5B) cause the leukodystrophy Megalencephalic leukoencephalopathy with subcortical cysts (MLC), characterized by chronic white matter edema and myelin vacuolization. Disrupted VRAC activity in MLC-patient-derived lymphoblasts and primary astrocytes from MLC mice further supports a pathogenic link between defective VRAC activity and MLC. Here, we studied the physiological and pathological consequences of astrocyte-specific removal of the essential VRAC subunit LRRC8A. In contrast to established MLC mouse models, astrocyte specific Lrrc8a knockout mice had normal brain water content, no myelin vacuolization, and preserved expression of MLC-related proteins. At a late age they developed a mildly ataxic gait and displayed increased severity of kainate-induced seizures. Two-photon imaging in acute brain slices revealed that astrocytes lacking LRRC8A show normal volume recovery and chloride dynamics upon high potassium-induced cell swelling. Together, these findings demonstrate that astrocyte LRRC8A is not essential for volume regulation in situ and that its loss alone is insufficient to cause the chronic white matter edema typical of MLC. The mild neurological deficits indicate a physiological role for astrocyte LRRC8A, but MLC pathology likely arises from broader dysregulation of the astrocytic protein complex coordinating ion and water homeostasis. Show less

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema. Show less

The majority of arthrogryposis multiplex congenita (AMC) and lethal forms of AMC such as foetal akinesia deformation sequence (FADS) cases are missed prenatally. We have demonstrated the additional value of foetal motor assessment and evaluation in a multidisciplinary team for the period 2007-2016. An applied care pathway was developed for foetuses presenting with joint contracture(s) in one anatomic region (e.g., talipes equinovarus [TEV]), more than one body part with non-progressive contractures and motility (AMC) and with deterioration over time (FADS). The multidisciplinary team of Amsterdam University Medical Centre Expertise Centre FADS and AMC developed the care pathway. Additional tools are provided including a motor assessment by ultrasound examination and a post-mortem assessment form. An eight-step care pathway is presented with a proposed timing for prenatal sonographic examination, genetic examinations, multidisciplinary meetings, prenatal and postnatal counselling of the parents by a specialist also treating after birth, and the follow-up of prenatal and postnatal findings with counselling for future pregnancies. The scheduled serial structural and motor sonograpahic assessment together with follow-up examinations and genetic analysis should be tailored per prenatal centre per available resources. The multidisciplinary care pathway may pave the way to increase the detection rate and diagnosis of isolated contracture(s), TEV with underlying genetic causes, and the rare phenotypes AMC/FADS and prompt treatment after birth within expertise teams. Show less

The diagnosis of fetal akinesia deformation sequence (FADS) is a challenge. Motor assessment is of additional value to advanced ultrasound examinations (AUE) for in utero FADS diagnosis before 24 weeks of gestation. All consecutive fetuses with greater than or equal to two contractures on the 20 week structural anomaly scan (2007-2016) were included. Findings at AUE, including motor assessment were analysed and related to outcome. Sixty-six fetuses fulfilled the inclusion criteria. On the basis of the first AUE, FADS was suspected in 13 of 66, arthrogryposis multiplex congenita (AMC) in 12 of 66, bilateral pes equinovares (BPEV) in 40 of 66, and Holt-Oram syndrome in one of 66. On the basis of the first motor assessment, the suspected diagnosis changed in 19 of 66, in 13 of 66 worsening to FADS, six of 66 amelioration from FADS, and confirmed FADS in seven of 13. The result was 20 FADS, seven AMC, and 38 BPEV. Second AUE in 44 fetuses showed additional contractures in two of eight FADS, and one intrauterine fetal death (IUFD). The second motor assessment changed the diagnosis in three of 43, one worsening from BPEV into FADS, two ameliorations from FADS, and confirmed FADS in seven by deterioration of motility. The result was nine FADS, six AMC, and 29 BPEV. The results suggest that motor assessment has additional value to distinguish between FADS, AMC, and BPEV. Show less

The neuronal ceroid lipofuscinoses (NCL) are progressive neurodegenerative diseases occurring in infancy and adulthood. Atypical forms of these diseases have been described and are particularly represented in the late-infantile and juvenile onset groups. Recent progress in biochemistry and molecular genetics has identified some of these variants as separate disease entities while disclosing the phenotypic variability of some classic forms. We report the results of a retrospective analysis performed on a series of 27 NCL patients, 15 of which were atypical as to clinical and/or pathological findings. Most of such patients, belonging to the late-infantile onset group and displaying homogeneous clinical-pathological features, were suggestive for CLN6. The two atypical juvenile NCL patients had features which resembled the "protracted form" of the disease. Given their relative frequency, strict clinical and pathological criteria are still the most useful tools for identifying and characterizing atypical forms and for defining phenotype-genotype correlations. Show less