_program
*all times are expressed in CET
**all proceedings are moderated by Mateusz Marciniak
***the framework program can be downloaded as PDF
_day 1 (09.03.2021): 14:00-18:00
_CLICK HERE TO JOIN DAY 1 VIA MS TEAMS
_opening ceremony
14.00-14.05: Michal Klichowski
WELCOME FROM ORGANIZING COMMITTEE
14:05-14:15: Agnieszka Cybal-Michalska
WELCOME FROM ADAM MICKIEWICZ UNIVERSITY IN POZNAN
14:15-14:45: Grazia M.S. Mancini and Anna Jansen
FOUR YEARS NEURO-MIG: WHAT HAVE WE LEARNED
_keynote speakers – session 1a: HIGHLIGHT ON NEURONAL MIGRATION RESEARCH
14:45-15:15: Orly Reiner (Weizmann Institute of Science, Israel)
ESSENTIAL ROLES OF THE HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN U (HNRNPU) PROTEIN IN THE DEVELOPING BRAIN
+15 min discussion
HNRNPU encodes for the heterogeneous nuclear ribonucleoprotein U and is known to participate in RNA splicing and chromatin organization. Microdeletions in the 1q44 locus encompassing HNRNPU and other genes, and point mutations in HNRNPU result in multiple brain-related phenotypes including early-onset seizures, severe intellectual delay, and with a lower penetrance microcephaly, a thin corpus callosum, dysmorphic facial features and hypotonia. Our work has uncovered that the presence of the HNRNPU protein is required for the survival of neuronal progenitors. The expression and alternative splicing of multiple genes that participate in cell survival, cell motility and axonal path finding are affected following Hnrnpu’s conditional deletion. We identified pharmaceutical and genetic manipulations that can rescue neuronal stem cell death and genetics means to restore neuronal migration. Collectively, our studies point to previously unknown roles of HNRNPU during brain development.
15:30-16:00: Silvia Cappello (Max Planck Institute of Psychiatry, Germany)
MODELING NEURONAL HETEROTOPIA WITH MOUSE AND HUMAN CEREBRAL ORGANOIDS
+15 min discussion
Malformations of the human neocortex represent a major cause of developmental disabilities including severe epilepsy. However, mouse lines carrying mutations of genes so far identified in human patients with cortical malformations only partially recapitulate the expected cortical phenotypes and therefore do not provide fully reliable models to entirely understand the molecular and cellular mechanisms responsible for these disorders. Hence, we combine the in vivo mouse model and the in vitro human-derived neurons, cerebral organoids, and dorso-ventral assembloids in order to better comprehend the molecular and cellular mechanisms involved in progenitors’ proliferation and fate as well as migration and maturation of inhibitory and excitatory neurons during human brain development and tackle the causes of neurodevelopmental disorders. We particularly focus on mutations in genes influencing cell-cell contacts, extracellular matrix and secretion of vesicles and therefore study intrinsic and extrinsic mechanisms underlying the formation of neuronal heterotopia. Our data reveal an important contribution of cell non-autonomous mechanisms in the development of cortical malformations. Finally, we use these models to study patients’ neurons at the functional level. To this end, we established intracellular and extracellular recordings of human neurons in 2D and 3D and found that also functionally, neurons from patients are altered. Taken together, our results show that we can model human brain development and neuronal migration disorders using human 2D and 3D models contributing to open new avenues to bridge the gap of knowledge between human brain malformations and existing animal models.
_15 min coffee break
_keynote speakers – session 1b: HIGHLIGHT ON NEURONAL MIGRATION RESEARCH
16:30-17:00: Laurent Nguyen (University of Liege, Belgium) et R. Le Bail, C.G. Silva
IMPAIRED INTERNEURON MIGRATION ALTERS CEREBRAL CORTEX MORPHOGENESIS AND CONNECTIVITY
+15 min discussion
The cerebral cortex is an evolutionary advanced brain structure made of neuronal layers tangentially organized into areas that serve specialized functions. Corticogenesis implies a continuous rearrangement of a primordial structure that progresses through successive biological steps and whose disruption can lead to malformations of cortical development (MCDs). Our recent work showed that migrating cortical interneurons crosstalk with dorsal cortical progenitors to control their proliferation, thereby the output of projection neurons. We found that a precocious invasion of the cortex by interneurons knockout for CCP1 leads to an increased output and persistent accumulation of supernumerary projection neurons in upper layers of the frontal cortex. Importantly, the production of supernumerary projection neurons has previously been linked to social behavior deficits and ASD, in mice and humans. Here we will present some unpublished data suggesting that the early imapirment of interneuron migration into the developing cerebral cortex, alters its connectivity and primes social behavioral changes in mouse. This work, may ultimately shed new light on the ontogenic causes of some social behavior defects observed in neurodevelopmental disorders.
17:15-17:45: Katja Kobow (University Hospital Erlangen, Germany)
DNA METHYLATION-BASED CLASSIFICATION OF MALFORMATIONS OF CORTICAL DEVELOPMENT
+15 min discussion and day 1 summary
Accurate histopathological diagnosis is crucial for optimal management of patients with epilepsy. For the broad spectrum of malformations of cortical development (MCD), standardization of the diagnostic process has been shown to be particularly challenging with substantial inter-observer variability in the histopathological diagnosis of many MCD. Some molecular grouping has been introduced including genetic marks, but only for selected entities. Diagnostic discordance and uncertainty may confound assignment of genetic variants to disease entities and compromise decision-making in clinical practice as well as the interpretation and validity of clinical trial results. Thus, other objective molecular markers should be considered. It has been convincingly shown that particularly DNA methylation profiling is highly robust and reproducible even from small samples and formalin-fixed paraffin embedded tissue, and such profiles have been widely used to classify CNS tumours. In line with these findings, we recently provided first evidence that epilepsy-associated structural brain lesions can be classified based on DNA methylation. Here we analyzed genome-wide DNA methylation patterns of almost 300 MCD patients and no seizure controls using Illumina 850K arrays to identify distinct methylation classes of MCD applying three different approaches, i.e., pairwise comparisons, machine learning and deep learning. Our data clearly show the suitability of DNA methylation-based MCD classification across all major histopathological entities amenable to epilepsy surgery and age groups, and its potential application in a routine diagnostic setting. Thus, the availability of this method may have a substantial impact on diagnostic precision compared to standard methods. Our results provide a blueprint for the generation of machine-learning-based disease classifiers across other epilepsy-associated structural brain lesions, with the potential to fundamentally transform epilepsy neuropathology.
_day 2 (10.03.2021): 12:00-19:30
_CLICK HERE TO JOIN DAY 2 VIA MS TEAMS
_keynote speakers – session 2a: CLINIC AND NEUROIMAGING OF BRAIN MALFORMATIONS
12:00-12:30: Nataliya Di Donato (Dresden University of Technology, Germany)
LISSENCEPHALY: IS IT STILL A CLASSICAL NEURONAL MIGRATION DISORDER?
+15 min discussion
Lissencephaly represents a heterogeneous group of cortical malformations characterized by the reduced or absent gyri, shallow sulci and abnormally thick cortex. Histologically lissencephaly is characterized by abnormal cortical layering. Clinically we distinguish more than 20 morphological subtypes and 31 disease genes have been associated with lissencephaly. The current classification of malformations of cortical development defines lissencephaly as a neuronal migration disorder and places it between disorders of cellular proliferation and post migration organization. However, the recent discoveries in the genetic spectrum and cellular mechanisms of lissencephaly questions this probably over-simplified definition. 25 from 31 lissencephaly-associated genes encode for structural or regulatory cytoskeletal proteins. However, the remaining genes represent transcriptional factors, regulators of apoptosis and cell cycle demonstrating that other developmental mechanism rather than impaired migration can lead to lissencephaly as well. Moreover, the recent data from 3D neuronal cultures – cerebral organoids modelling lissencephaly clearly demonstrates an abnormal differentiation and disturbed proliferation of the neural progenitor cells. These phenomena were seen in PAFAH1B1-, KATNB1-, as well as ACTB- and ACTG1-associated models. This shows that even lissencephaly types associated with the imparted cytoskeletal structure and function have a complex disease mechanisms going far beyond the abnormal neuronal migration. This lecture focuses on the current genetic landscape of lissencephaly and novel insights from the animal and cellular disease models.
12:45-13:15: Renske Oegema (Utrecht University, Netherlands)
A CLINICAL APPROACH TO HETEROTOPIA
+15 min discussion
Neuronal heterotopia form a group of brain malformations caused by a failure of neuronal migration. It is considered one of the most frequent malformations of cortical development. Heterotopia are associated with epilepsy and developmental delay, but can also occur as an incidental finding. Periventricular nodular heterotopia (PVNH), with nodules along the ventricular walls, are the most common subtype, but other more rare subtypes exist. The etiologic spectrum is broad encompassing chromosomal copy number variants, single gene mutations and non-genetic causes. Establishing a diagnosis can be challenging to the clinician, but can have important implications for patient management and genetic counseling. In this presentation the classification of heterotopia and its etiology will be discussed, with examples of common and more rare syndromes and associations.
13:30-14:00: Maarten Lequin (Utrecht University, Netherlands)
IS IMAGING STILL USEFUL IN MALFORMATIONS OF CORTICAL DEVELOPMENT? AN UPDATE
+15 min discussion
Malformations of cortical development (MCD) comprise a large, heterogeneous group of disorders related to disruption of the cortex formation caused by various genetic, infectious or vascular etiologies. They are characterized by an abnormal structure (micro- or macroscopic) of the cortex. The first classification based on imaging was introduced by Barkovich 1996 and in the following years up-dated. All these imaging-based classifications are focused on the concept that the development of the cortex is divided into three major stages, keeping in mind that each of these stages is composed of many different, and important, processes that overlap in time: Cell proliferation and apoptosis, cell migration and post-migrational development. These different processes take place at different times in different parts of the brain, so many parts of different processes actually occur at the same time. This lecture will focus on new insights of genetic pathways important for normal brain development, resulting in the phenotypic imaging features, seen in patients with MCD.
_15 min coffee break
_keynote speakers – session 2b: CLINIC AND NEUROIMAGING OF BRAIN MALFORMATIONS
14:30-15:00: Nadia Bahi-Buisson (Necker–Enfants Malades Hospita, France) et S. Farcy, C. Maillard, F. Francis, A. Baffet
FURTHER ADVANCES IN ELUCIDATING MOLECULAR AND CELLULAR MECHANISMS UNDERLYING DYNEINOPATHIES
+15 min discussion
Neocortical development requires tightly regulated processes, and perturbations lead to malformations of cortical development (MCDs). Our groups have demonstrated the importance of the cytoskeleton during these key developmental steps. Recently, Cytoplasmic dynein 1, heavy chain 1 (DYNC1H1) mutations were identified in MCDs, as well as in spinal muscular atrophy (SMA-LED), a motor neuron degeneration disorder, referred to as « Dyneinopathies ». This protein is a minus-end directed molecular motor, mainly involved in retro-transport. The wide spectrum of these disorders together with dynein’s pleomorphic cellular functions, raise fundamental questions about the effect of mutations on different cellular partners and processes. In this project, we united molecular and cellular neurobiologists, with clinicians and geneticists to question how distinct mutations perturb dynein’s behaviour in patient-derived cells, first in patients’ fibroblasts, and then on cerebral organoids. Individual genotype-phenotype correlations will advance comprehension of these severe and variable disorders. We were able to collect a cohort of 66 new patients with pathogenic mutation in DYNC1H1 through the COST Network. Our data allowed us to refine the radiological hallmarks of brain dyneinopathy which consist of frontal dysgyria, parieto-occipital agyria without pontocerebellar hypoplasia and basal ganglia dysmorphism which distinguish it from tubulinopathies. Electroneuromyography (ENMG) studies allowed us to identify two distinct motorneuron disease profile, the first non progressive SMALED and the second suggestive of progressive motor axonal neuropathy. Mutated organoids for MCD-causing mutations and SMA-LED-causing mutations showed a significant decrease in size of the mutated organoids for brain malformations. There is no significant difference in size with the organoids mutated for the SMA-LED disorder, which is in agreement with the patient’s phenotypes. We now have all the tools to study dynein-dependant functions in details in those cerebral organoids: mitotic spindle integrity and orientation, NSC proliferation rate, neuronal migration. In conclusion, the wide spectrum of dyneinopathies reflects different dysfonctions of this large protein. Our data highlights that at least the organoids models are powerful to study the neurobiological dysfunctions at the level of the brain development.
15:15-15:45: Andrew Fry (Cardiff University, United Kingdom)
DELINEATING NMDAR-ASSOCIATED MALFORMATIONS OF CORTICAL DEVELOPMENT
+15 min discussion
The N-methyl-D-aspartate receptor (NMDAR) is a key ligand-gated glutamate receptor expressed on synaptic membranes. The NMDAR is a heteromeric protein typically composed of two mandatory GluN1 subunits (encoded by the GRIN1 gene) and two GluN2 subunits (encoded by GRIN2A, GRIN2B, GRIN2C or GRIN2D) which have variable temporal and spatial expression. The GluN1/GluN2B form of the NMDAR is widely expressed in the fetal brain. Pathogenic variants in NMDAR subunits have been described in patients with a spectrum of neurological phenotypes include developmental delay, intellectual disability, epilepsy, cortical visual impairment, neurobehavioral problems and movement disorders. A subset of patients with pathogenic missense variants in GRIN1 and GRIN2B have been found to have malformations of cortical development (MCDs). We reviewed the clinical, molecular and radiological features of 25 individuals with GRIN1- or GRIN2B-related MCDs. The phenotypes associated with both genes were very similar. Common clinical features in the individuals were severe developmental delay and medication-resistant seizures. Affected individuals had diffuse bilateral dysgyria often with occipital sparing. The MRI appearance of the dysgyria was consistent with polymicrogyria. Histopathology from two affected fetuses confirmed the presence of diffuse and poorly laminated polymicrogyria. Other MRI findings include thinning of the corpus callosum, enlarged ventricles, reduced white matter, dysmorphic basal ganglia and abnormal hippocampi. MCD-associated NMDA receptor mutations tend to cluster in the M3 (gating mechanism) and S2 (ligand binding) domains of GluN1/2B suggesting a genotype-phenotype correlation. Based on evidence from electrophysiological analysis and animal models it has been proposed that NMDAR -associated dysgyria results from excitotoxic damage to the fetal cortex. NMDAR-mediated excitotoxicity has also been linked to other causes of polymicrogyria (metabolic, infectious and hypoxic-ischaemic insults).
16:00-16:30: Marije Meuwissen (University Hospital Antwerp, Belgium)
GENETIC TESTING IN CEREBRAL PALSY CONTRIBUTES SIGNIFICANTLY TO DIAGNOSIS: THE ANTWERP EXPERIENCE
+15 min discussion
Cerebral palsy (CP) is a clinical descriptive term defining a heterogeneous group of non-progressive, neurodevelopmental disorders of motor impairment, which co-occur with a wide range of medical conditions, including intellectual disability, speech and language deficits, autism, epilepsy and visual and/or hearing impairment. Historically, inadequate oxygen delivery to the brain during birth, was thought to be the leading cause of CP, but large population-based, controlled studies have shown that birth asphyxia is a rather uncommon cause and accounts for less than 10% of CP cases. This recent insight has led to an increased interest in genetic studies to elucidate its additional, mainly unknown causes. Recent studies confirm copy number variants and single gene mutations with large genetic heterogeneity as an etiology in children with CP. Whole exome sequencing studies in CP cases demonstrated an important contribution of genetic pathogenic variants, with a diagnostic yield of 14%. At the Antwerp University Hospital, approximately 550 children with CP are followed at the multidisciplinary cerebral palsy clinic. Genetic testing is offered to CP patients in the absence of risk factors such as documented peripartal asphyxia, prematurity <30 weeks or non-accidental head trauma. Testing involved SNP microarray to exclude pathogenic copy number variants followed by whole exome sequencing (trio-approach) with in silico filtering of variants in around 200 genes associated with CP or “CP-mimics”. The latter is defined as a neurodevelopmental disorder that initially presents with a CP phenotype, but with a differing natural history and prognosis. I will discuss the genetic findings in our CP cohort, underscoring the added value of genetic testing and confirming the heterogeneity of CP. This heterogeneity has triggered a discussion regarding the validity of CP as a clinical diagnosis. In our opinion, the clinical diagnosis of CP should not be altered, but it should warrant additional genetic testing to enable genetic subclassifications of CP, hereby improving genetic counselling of families e.g. regarding future family planning and contributing to an improved and overall more personalized treatment and follow-up of CP patients.
_15 min coffee break
_free abstract communications
17:00-17:15: Stephanie Baulac (Sorbonne University, France) et S. Baldassari, M. Chipaux, G. Dorfmüller
BRAIN MOSAICISM IN FOCAL CORTICAL DYSPLASIAS
+one-shot 5 min discussion
Molecular studies have revealed somatic mosaicism in focal cortical malformations, such as focal cortical dysplasia (FCD) and mild malformation of cortical development (MCD). Focal malformations result from somatic (post-zygotic) mutation in genes related to the mTORC1 or glycosylation pathways, occurring early neuroprogenitor cells and evolving into a mutated clonal cell population. The molecular signature of FCD2 is the presence of mosaic variants at low allele frequencies (usually <5%) in a set of genes belonging to the mTOR signaling cascade. Two types of mutational events may explain the occurrence of a dysplasia: single activating variants in MTOR or in genes encoding upstream activators of the pathway (AKT3, PIK3CA, RHEB), or ‘double-hit’ inactivating variants (SNV or loss-of-heterozygosity) in repressors of the pathway (DEPDC5, NPRL2/3, TSC1/2). All FCD2-associated variants lead to constitutive mTORC1 signaling activation in resected tissues. The size of the dysplastic lesion is directly correlated to the timing of occurrence of the post-zygotic somatic mutation, and therefore the variant load. The variant allele fraction correlates with the dysmorphic neuron density, and the highest variant load correlates with the seizure-onset zone. Furthermore, there is evidence from pools of laser-captured microdissected cells from FCD2 tissue that mTOR-activating variants are present only in dysmorphic neurons. Using targeted digital droplet PCR, we were able to detect the presence of somatic variants in CSF-derived circulating cell-free DNA samples in 5/12 epileptic patients. Our findings suggest potential opportunities for clinical use of CSF to establish a genetic diagnosis before surgery or in patients not eligible for surgery and use these patients’ genetic diagnoses to guide the use of targeted therapies.
17:20-17:35: Rossella Di Giaimo (Max Planck Institute of Psychiatry, Germany) et Z. Bekjarova, F. Di Matteo, F. Pipicelli, G. Maccarone, P. Kielkowski, S. Cappello
INSUFFICIENCY OF FUNCTIONAL CYSTATIN B IN HUMAN EPILEPSY IMPAIRS NEURONAL MIGRATION
+one-shot 5 min discussion
Progressive myoclonus epilepsy of Unverricht-Lundburg-type (EPM1) is an autosomal recessive neurodegenerative disorder that has the highest incidence among the PME worldwide. Loss-of-function mutations in the gene encoding CYSTATIN B (CSTB) are the primary genetic cause of EPM1. We showed that CSTB levels are critical for cortical development both, in vivo, in mouse embryonic brain cortex and, in vitro, in a 3D model of human brain development, human cerebral organoids (hCO) and patient-derived cerebral organoids. Increased amount of CSTB lead to accumulation of proliferating cells and affects the recruitment of interneurons. On the contrary, overexpression of the pathological CSTB mutant as well as low amount of functional CSTB in EPM1 organoids, results in a general decreased proliferation and significant reduction of interneurons. We decided to further inquire about the role of CSTB during neurogenesis by studying the impact of low amount of the protein in the cortical dorso-ventral patterning. To this aim, we generated patterned spheroids and we found out that ventral-patterned spheroids lost some of their identity in terms of progenitor cells and neurons, providing new important hints for the onset of the epileptic disorder. Moreover, proteomic analysis of EPM1 organoids suggested a defect in extracellular matrix organization and vesicle secretions. Thereby, we characterized the extracellular vesicles that are secreted by EPM1 hCO during their development in culture and compared the results with extracellular vesicles secreted by hCO generated by 2 different control cell lines. The dysregulated pathways in EPM1 patient derived vesicles are in line with the epileptic disease and strongly indicate that EPM1 phenotype depends at least in part by an altered mechanism of cell-cell communication mediated by extracellular vesicles.
17:40-17:55: Kristina Lanko (Erasmus University, Netherlands) et M.J.A. Weerts, T.S. Barakat
DELINEATING THE MOLECULAR AND PHENOTYPIC SPECTRUM OF THE SETD1B-RELATED SYNDROME
+one-shot 5 min discussion
SETD1B encodes a lysine-specific histone methyltransferase that methylates histone H3 at position lysine-4 (H3K4me1, H3K4me2, H3K4me3) and thereby is involved in the regulation of gene expression. Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay and seizures. To date, clinical features have been described for eleven patients with (likely) pathogenic SETD1B sequence variants. We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. By means of computational protein modelling we predict potential functional effect of SETD1B variants. Selected variants located in different functional domains of SETD1B were functionally tested using in vitro and genome-wide methylation assays, confirming in silico predictions. Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Interestingly, males are significantly overrepresented, and thus we speculate that sex-linked traits could affect susceptibility to clinical penetrance and the clinical spectrum of SETD1B variants. Together, this work expands the phenotypic and molecular spectrum associated with SETD1B variants, provides insightes into their functional effects and will ultimately facilitate the counseling regarding the clinical spectrum of newly diagnosed patients with the SETD1B-related syndrome.
18:00-18:15: Jordy Dekker (Erasmus University, Netherlands) et R. Schot, K.A. Aldinger, D.B. Everman, J.A. Sullivan, V. Shashi, M.S. Zaki, J.G. Gleeson, A. Vitobello, A.-S. Denomme-Pichon, A.-L. Mosca-Boidron, S. Nambot, L. Perrin, S.E. McKeown, F. D’Arco, R.J. Leventer, D. Doherty, W.B. Dobyns, G.M.S. Mancini, K.C. Slep
THE CLINICAL AND NEURORADIOLOGICAL SPECTRUM OF VARIANTS IN THE GAR DOMAIN OF MACF1
+one-shot 5 min discussion
Microtubule-actin crosslinking factor 1 (MACF1) is a member of the spectraplakin protein family, that cross-link different components of the cytoskeleton. The growth arrest specific 2 (Gas2)-related (GAR) domain of MACF1 interacts with microtubules and dominant variants affecting the GAR result in a brain malformation involving a predominant posterior lissencephaly and reduced or absent pontine crossing fibers resulting in a W-shaped hypoplastic brainstem. Here we describe six patients with a de novo variant in the GAR domain of MACF1, of which five have not been reported before. We also identified a variant in the most N-terminal of the four zinc-binding residues (c.15524G>A: p.Cys5175Tyr), where previously no other variants have been reported. All patients with a zinc-binding residue variant show on MRI a severe narrowing of the pons, cerebellar vermis hypoplasia and a variable lissencephaly severity. Patients present with global developmental delay, having impaired motor development and speech development ranging from no speech to using single words. Most patients develop epilepsy during their first year after birth. Furthermore, we identified one patient with a variant located between the two zinc-binding residue pairs (c.15575G>C: p.Arg5192Pro), likely to affect the β-sheet structure of the GAR-domain, whose MRI reveals only mild brainstem and cerebellar hypoplasia with cortical dysgyria, but no lissencephaly. These results show that variants in the zinc-binding residues of the MACF1 GAR domain result in a typical cortical malformation, where variants in the surrounding residues may be associated with a different phenotype.
18:20-18:35: Roel Quintens (Belgian Nuclear Research Centre [SCK•CEN], Belgium)
P53 ACTIVATES A DUAL TRANSCRIPTIONAL PROGRAM TO REGULATE BRAIN SIZE IN RESPONSE TO EMBRYONIC DNA DAMAGE
+one-shot 5 min discussion
p53 regulates the cellular DNA damage response (DDR). Hyperactivation of p53 during embryonic development, however, can lead to a range of developmental defects including microcephaly. Here, we induce microcephaly by acute irradiation (1 Gy of X-rays) of mouse fetuses at the onset of neurogenesis (E11). We used fluorescence microscopy and RNA sequencing to investigate radiation effects mostly at early time points after irradiation. Dorsal forebrain-specific Trp53 knock-out (cKO) mice were generated by crossing Trp53fl/fl mice to Emx1-Cre mice. Besides a classical DDR culminating in massive apoptosis, we observe ectopic neurons in the subventricular zone in the brains of irradiated mice, indicative of premature neuronal differentiation. A transcriptomic study indicates that p53 activates both DDR genes and differentiation-associated genes. In line with this, Trp53 cKO mice do not show this ectopic phenotype and partially restore brain size after irradiation. Irradiation furthermore induces an epithelial-to-mesenchymal transition-like process resembling the radiation-induced proneural-mesenchymal transition in glioma and glioma stem-like cells. Our results demonstrate a critical role for p53 beyond the DDR as a regulator of neural progenitor cell fate in response to DNA damage.
18:40-18:55: Kristina Lanko (Erasmus University, Netherlands) et L.E. Sanderson, T.S. Barakat
BI-ALLELIC VARIANTS IN HOPS COMPLEX SUBUNIT VPS41 CAUSE CEREBELLAR ATAXIA AND ABNORMAL MEMBRANE TRAFFICKING
+one-shot 5 min discussion
Membrane trafficking is a complex, essential process in eukaryotic cells responsible for protein transport and processing. Deficiencies in vacuolar protein sorting (VPS) proteins, key regulators of trafficking, cause abnormal intracellular segregation of macromolecules and organelles and are linked to human disease. VPS proteins function as part of complexes such as the homotypic fusion and vacuole protein sorting (HOPS) tethering complex, comprised of VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41. The HOPS-specific subunit VPS41 has been reported to promote viability of dopaminergic neurons in Parkinson’s disease but to date has not been linked to human disease. Here, we describe five unrelated families with nine affected individuals, all carrying homozygous variants in VPS41 which we show impact protein function. All affected individuals presented with a progressive neurodevelopmental disorder consisting of cognitive impairment, cerebellar atrophy/hypoplasia, motor dysfunction with ataxia and dystonia, and nystagmus. In silico analysis predicted that each variant could influence VPS41 stability and function. The knock-out of VPS41 in human ESC resulted in slightly altered cell morphology, change in the localization of transcription factor TFE3 and upregulation of lysosomal protein LAMP2. Re-introduction of mutant VPS41 could only partially restore these phenotypes, confirming in silico predictions. Zebrafish disease modelling supports the involvement of VPS41 dysfunction in the disorder, indicating lysosomal dysregulation throughout the brain and providing support for cerebellar and microglial abnormalities when vps41 was mutated. This provides the first example of human disease linked to the HOPS-specific subunit VPS41 and suggests the importance of HOPS complex activity for cerebellar function.
19:00-19:15: Daphne Smits (Erasmus University, Netherlands) et R. Schot, P. Magini, L. Vandervore, M. Columbaro, E. Kasteleijn, F. Palombo, L. Iommarini, M. Lequin, A.M. Porcelli, P. Govaert, M. Dremmen, M.C.Y. de Wit, M. Severino, M.T. Divizia, N. Ordonez-Herrera, A. Alhashem, A. Al Fares, M. Al Ghamdi, A. Rolfs, P. Bauer, J. Demmers, F. Verheijen, M. Wilke, M. van Slegtenhorst, P.J. van der Spek, A. Jansen, R. Stottmann, R. Hufnagel, R. Hopkin, D. Aljeaid, W. Wiszniewski, P. Gawlinski, F. Alkuraya, V. Stanley, A. Bertoli-Avella, G. Mirzaa, W.B. Dobyns, M. Seri, T.Pippucci, M.Fornerod, G.M.S. Mancini
STRESS: CAN NEURONAL PROGENITORS HANDLE IT
+one-shot 5 min discussion and day 2 summary
Primary microcephaly is a genetically heterogeneous disorder often caused by rare variants in genes coding for proteins regulating the cell cycle. Many microcephaly subjects present with simplified gyration of the cerebral cortex, without overt structural anomalies, non-progressive intellectual disability, and normal life expectancy. On the other hand, a group of children with congenital microcephaly is affected by severe and progressive encephalopathy and early demise. We performed cell biological studies to elucidate the pathogenic mechanisms contributing to disease in children with progressive primary microcephaly caused by SMPD4 variants. Children from 12 unrelated families presented with progressive primary microcephaly, a simplified gyral pattern, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for neutral sphingomyelinase-3. Overexpression of human SMPD4 showed localization to the outer nuclear envelope and the ER, and additionally revealed interactions with several nuclear pore complex proteins. Fibroblasts from affected individuals as well as SMPD4 depleted neural stem cells show a prolonged mitosis and defects in nuclear pore complex assembly. In addition, the absence of SMPD4 leads to alterations in the assembly of the new nuclear envelope following mitosis. Interestingly, mutant fibroblasts also show ER cisternae abnormalities, increased autophagy and higher susceptibility to apoptosis, all signs of ER-stress. Loss of SMPD4 causes a severe neurodevelopmental disorder including progressive primary microcephaly. Our results show the contribution of cell cycle related alterations, such as nuclear pore assembly, nuclear envelope dynamics and mitotic duration to disease pathogenesis. Additionally, SMPD4 seems to be involved in ER-stress regulation, thereby adding a second component to disease mechanism. Over the last years, variants in several genes coding for proteins involved in the ER-stress response have been reported to cause progressive microcephaly, suggesting a relation between this pathway and a more severe clinical course (e.g. IER3IP1, TMX2, PYCR2, EIF2AK3, EIF2S3, RNF13). Many of these are ER membrane proteins. Our study provides evidence for a direct influence of an ER-membrane protein on regulation of mitosis in neural progenitors, a mechanism distinct from apoptosis regulation. Studying the relevance of this mechanism in neurogenesis and its potential to predict clinical outcome in this group of microcephaly will improve clinical management and provide tools for intervention on disease progression in the coming years.
_day 3 (11.03.2021): 14:00-19:30
_CLICK HERE TO JOIN DAY 3 VIA MS TEAMS
_flash-talk session
14:00-14:05: Jean-Christophe Vermoyal (Mediterranean Institute of Neurobiology [INMED], France) et L. Goirand-Lopez, A. Vinck, A. Fortoul, F. Watrin, F. Francis, S. Capello, T. Marissal, J.-B. Manent
COMPARATIVE ASSESSMENT OF NETWORK-LEVEL DEFECTS IN RODENT MODELS OF SUBCORTICAL BAND HETEROTOPIA AND PERIVENTRICULAR HETEROTOPIA
The mammalian neocortex is a highly organized structure composed of six layers of pyramidal neurons. During corticogenesis neuronal progenitors proliferate and give birth to young neurons that will migrate to their appropriate layers, and then integrate into mature functional networks. If one of these three developmental steps (proliferation, migration and maturation) is impaired, malformations of cortical development (MCDs) may occur. Most MCDs have a genetic origin and are associated with epileptic seizures consisting in an abnormal, excessive and hypersynchronous discharge of a population of cortical neurons. Subcortical band heterotopia and periventricular nodular heterotopia (SBH and PVNH) are neuronal migration disorders characterized by the presence of misplaced neurons forming neuronal heterotopia. SBH describes a band of grey matter separated from the cortex and lateral ventricles by zones of white matter, whereas PVNH describes nodular masses of grey matter located along the ventricular walls. Although epilepsy is one of the most common presenting features, the precise location and properties of epileptogenic neuronal networks remain unclear in patients with these two forms of grey matter heterotopia. By utilizing two genetic rodents models of SBH and PVNH (Eml1 cKO and RhoA cKO mice, respectively), we sought to gain further access to the putative location and properties of these defective networks by looking at neuronal activity inside the heterotopia and normotopic cortex. For this purpose, we used wide-field two-photon calcium imaging to record the activity of large groups of neurons in acute slices covering both the heterotopia and the overlying normotopic cortex. This method allowed us to determine if spatially restricted groups of co-active neurons (clusters) are present in both structures. We found that clusters were usually shared between normotopic and heterotopic cortices, while one of the two fields was most often identified as a leader.
14:05-14:10: Aurelien Fortoul (Mediterranean Institute of Neurobiology [INMED], France) et F. Francis, S. Capello, J.-B. Manent, F. Watrin
IMPACT OF HETEROTOPIA ON PRIMARY SOMATOSENSORY CORTEX DEVELOPEMENT AND THALAMOCORTICAL CONNECTIVITY IN SUBCORTICAL BAND HETEROTOPIA AND PERIVENTRICULAR HETEROTOPIA MOUSE MODELS
Heterotopias are a group of cortical malformations characterized by abnormal neuronal positioning. In subcortical band heterotopias (SBH), newborn neurons exit the ventricular germinal zone but fail to migrate correctly into the cortex. In periventricular heterotopia (PVNH), newborn neurons fail to exit the ventricular zone and accumulate in clusters along the ventricles. Patients with SBH or PVNH often display intractable epilepsy and intellectual disability, owing most likely to a disorganized cortical connectivity. How these heterotopic neuronal clusters, in or below the white matter, impact cortical development and connectivity is not well understood. In order to progress in this understanding, we have analyzed two mouse models, representative of either SBH or PVNH , focusing on primary somatosensory cortex development and thalamocortical connectivity. The SBH model results from the conditional inactivation of the Eml1 gene (Eml1 cKO; F. Francis), coding for a microtubule associated protein. Mice develop massive bilateral heterotopias surrounded by white matter, as a consequence of altered distribution and abnormal cycling of apical progenitors. The PVNH model results from the conditional inactivation of the RhoA gene (RkoA cKO), affecting the actin and microtubule cytoskeletons. These mice also develop massive bilateral heterotopias under the white matter, as a consequence of apical progenitors defaults (abnormal adherent junctions, loss of apical anchoring). In both models, we studied the laminar organization of the normocortex and the neuronal composition of the heterotopias by performing immunohistochemistry with Cux1, RORb and CTIP2 antibodies. We have analyzed the barrel field organization and the thalamocortical connectivity by performing immunocytochemistry with V-Glut2, RORb and Netrin antibodies and viral tracing. Finally, we have investigated the subplate organization, this transient structure playing an important role in thalamocortical circuit assembly, by performing immunohistochemistry with a Complexin 3 antibody. This comparative analysis shows that each model display very specific features in their cortical and heterotopia organization and thalamocortical connectivity. Interestingly, a functional “barrel field-like” structure seems to be present in the Eml1 cKO heterotopia, implying a shared or duplicated somatotopy in this SBH model.
14:10-14:15: Donia Zaidi (National Institute for Health and Medical Research [INSERM], France) et J. Ferent, S.R. Puiggros, B.N. Yigit, N. Ozlu, D. Jabaudon, F. Francis
INVESTIGATION OF CELL CYCLE DYSFUNCTIONS IN THE MURINE EML1 CONDITIONAL KNOCKOUT MODEL OF HETEROTOPIA
Subcortical band heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of misplaced (heterotopic) neurons within the white matter. Eml1 codes for a microtubule-binding protein and is currently the only gene consistently found mutated in both human and rodent heterotopia. We generated a forebrain-specific conditional knock-out model to investigate Eml1’s role in cortical development (Eml1 cKO). Mammalian cortical development depends on radial glial cells (RG), which undergo regulated divisions at the apical side of the ventricular zone (VZ). We have previously shown that Eml1 loss-of-function aberrantly positions RG outside the VZ in association with centrosome and primary cilia defects. Since the centrosome is also essential to cell division, we aim now to decipher the link between cilia/centrosomal defects and potential proliferation alterations in Eml1 cKO RG. First, we show that Eml1 and Cep170, a specific mother centriole protein, interact, and this interaction is reduced when Eml1 carries a human heterotopia mutation. Since cilia formation and centrosome dynamics are dependent on cell cycle phases, we investigated cell cycle dynamics in the Eml1 cKO model. We found increased RG cells in S phase present at the cKO ventricular surface, as well as mislocalized mitotic cells. In addition, S phase length appeared longer in ectopic RG outside the VZ. Looking at different developmental stages we now further aim to understand the RG defects leading to detachment across corticogenesis. Finally, we performed a single cell RNA-seq analysis of cortical cells. Gene expression clustering identified a unique cell population in the Eml1 cKO which do not fit within the cell types found in the control dataset. These cycling cells may represent the detached progenitors causing the formation of the heterotopia.
14:15-14:20: Richard Belvindrah (Sorbonne University and National Institute for Health and Medical Research [INSERM], France) et M. Penisson, F. Francis
LIS1 MUTATION PREVENTS BASAL RADIAL GLIA-LIKE CELL PRODUCTION IN THE MOUSE
Human cortical malformations are associated with progenitor proliferation and neuronal migration abnormalities. Progenitor cells include apical radial glia, intermediate progenitors and basal (or outer) radial glia (bRGs or oRGs). The LIS1 gene coding for a dynein regulator, is mutated in human lissencephaly, associated also in some cases with microcephaly. LIS1 was shown to be important during cell division and neuronal migration. bRGs being few in number in lissencephalic species (e.g. the mouse) but abundant in gyrencephalic brains, we first generated bRG-like cells in the mouse embryonic brain, in order to investigate the role of Lis1 in their formation. This was achieved by in utero electroporation of a hominoid-specific gene TBC1D3 (coding for a RAB-GAP protein) at mouse embryonic day (E) 14.5. We first confirmed that TBC1D3 expression in wild-type (WT) brain generates numerous Pax6+ bRG-like cells that are basally localized. Second, using the same approach, we assessed the formation of these cells in heterozygote Lis1 mutant brains. Our results show that Lis1 depletion in the forebrain from E9.5 prevented subsequent TBC1D3-induced bRG-like cell amplification. Indeed, we observe disruption of the ventricular zone (VZ) in the mutant. Lis1 depletion altered adhesion proteins and mitotic spindle orientations at the ventricular surface and increased the proportion of abventricular mitoses. We conclude that perturbation of Lis1/LIS1 dosage is likely to be detrimental for appropriate progenitor number and position, contributing to lissencephaly pathogenesis.
14:20-14:25: Stefanie Brock (Free University of Brussels [VUB], Belgium) et F. Cools, A. Jansen
NEUROPATHOLOGY OF GENETICALLY DEFINED MALFORMATIONS OF CORTICAL DEVELOPMENT – A SYSTEMATIC LITERATURE REVIEW
Malformations of cortical development (MCD) include a heterogeneous spectrum of clinical, imaging, molecular and histopathological entities. While the understanding of genetic causes of MCD has improved with the availability of next-generation sequencing modalities, genotype-histopathological correlations remain limited. This is the first systematic review of molecular and neuropathological findings in patients with MCD to provide a comprehensive overview of the current literature. A systematic review was performed between November 2019 and February 2020. A MEDLINE search was conducted for 132 genes previously linked to MCD in order to identify studies reporting macroscopic and/or microscopic findings in patients with a confirmed genetic cause. 81 studies were included in this review reporting neuropathological features associated with pathogenic variants in 46 genes (46/132 genes, 34.8%). Four groups emerged, consisting of (1) 13 genes with well-defined histological-genotype correlations, (2) 27 genes for which neuropathological reports were limited, (3) 5 genes with conflicting neuropathological features, and (4) 87 genes for which no histological data were available. Lissencephaly and polymicrogyria were reported most frequently. Associated brain malformations were variably present, with abnormalities of the corpus callosum as most common associated feature. Neuropathological data in patients with MCD with a defined genetic cause is available only for a small number of genes. As each genetic cause might lead to unique histopathological features of MCD, standardized thorough neuropathological assessment and reporting should be encouraged. Histologic features can help improve the understanding of the pathogenesis of MCD and generate hypotheses with impact on further research directions.
14:25-14:30: Eva Medico Salsench (Erasmus University, Netherlands) et R. Maroofian, R. Deng, K. Lanko, B.P. Gonzalez, O. Sanchez-Lijarcio, S. Ibanez-Mico, A. Wojcik, M. Vargas, A. Schroeder, C.-T. Fong, A. Begtrup, I.H. Kaya, M. AlMuhaizea, D. Colak, H. Houlden, R.-J. Galjaard, N. Kaya, T.S. Barakat
EXPANDING THE MUTATIONAL LANDSCAPE AND CLINICAL PHENOTYPE OF THE YIF1B RELATED BRAIN DISORDER
Intracellular proteins involved in mediating vesicular trafficking in eukaryotic cells have been implicated in brain disorders, showing the relevance of the process for neuronal development in human. YIF1B is an essential protein involved in the anterograde trafficking from the endoplasmic reticulum to the cell membrane, and in Golgi apparatus architecture. We recently described a neurodevelopmental disorder caused by recessive variants in YIF1B, which has now been recognized by OMIM as Kaya-Barakat-Masson syndrome (KABAMAS, OMIM# 619125). So far, our study and that of Diaz et al. reported 16 affected individuals from 11 independent families. These individuals presented with a progressive encephalopathy with various degrees of movement disorders, microcephaly, and epilepsy. In all but one family, bi-allelic protein truncating variants were identified in YIF1B, with only a single bi-allelic missense mutation assumed to be causative. Here, we describe 6 additional individuals from 6 families harboring protein altering variants in YIF1B, 4 of which are homozygous or compound heterozygous missense variants. Interestingly, all YIF1B missense variants encountered localized in, or close to, the transmembrane domains, which were previously shown to be essential for YIF1B function. To investigate the function of these missense variants, we performed site-directed mutagenesis followed by expression and interaction studies, providing functional evidence from in vitro studies that these missense variants impact on YIF1B function. In addition, we compare the clinical phenotype between all currently known YIF1B cases to further delineate the mutational landscape and the clinical phenotype associated with this new disease entity.
14:30-14:35: Ece Sonmezler (Dokuz Eylul University, Turkey)
A NOVEL HOMOZYGOUS VARIANT IN THE TUBGCP2 GENE UNDERLYING SEVERE NEURODEVELOPMENTAL DISEASE ALTERS Γ-TURC RING COMPLEX
One of the three primary groups of cytoskeletal components is microtubules that are polymers of alpha/beta tubulin (α/β-tubulin) heterodimers. These long fibers are involved in many essential roles including maintenance of cell shape, cell movement, intracellular transport and cell division. Particularly in neuronal cells, microtubules contribute to establishing neuronal polarity, maintaining neuronal morphology, providing axonal transport and regulating signalling. Differently from microtubule components, α and β-tubulins, gamma-tubulin (γ-tubulin) and associated proteins nucleate microtubule assembly. Several components of the γ-tubulin ring complex (γ-TuRC) have been previously reported in human neurodevelopmental diseases. Here, our team describes a homozygous missense variant (NP_006650.1: c.931G>A; p.Glu311Lys) in Tubulin Gamma Complex Associated Protein 2 (TUBGCP2) gene encoding the γ-tubulin complex 2 (GCP2) protein in two siblings from a consanguineous Turkish family with dysmorphic features, developmental delay, brain malformation, and epilepsy. The electrostatic complementarity of GCP2 with GCP3 is predicted to be disrupted by this variant. In primary dermal fibroblasts carrying the variant, immunofluorescence studies suggested that the mutation in TUBGCP2 could perturb γ-TuRC localization during the cell cycle. However, levels of TUBGCP2 did not show any significant change. Through mass spectrometry, we observed dysregulation of multiple proteins involved in the assembly and organization of the cytoskeleton and the extracellular matrix. In summary, our functional and proteomic findings provide insight for the effects of TUBGCP2 mutations on nervous system development and its clinical manifestations.
14:35-14:40: Elena Perenthaler (Erasmus University, Netherlands) et S. Yousefi, R. Deng, A. Nikoncuk, T.S. Barakat
CHARACTERIZATION OF THE FUNCTIONAL ENHANCERS IN HUMAN NEURAL STEM CELLS
The development of the cerebral cortex is a complex and dynamic process. Alterations at any stage can result in a wide range of neurodevelopmental disorders (NDDs), among which malformations of cortical development (MCD), that are a common cause of developmental delay, intellectual disability, and epilepsy. Exome sequencing greatly increased the diagnostic yield of genetic forms of NDDs, allowing the identification of variations in hundreds of genes. Nevertheless, many cases remain genetically unexplained, hinting at variations in the non-coding genome. Among these non-coding regions are the understudied enhancers, cis-acting elements that control gene-expression in a temporal and tissue-specific manner during many key-developmental processes. Here, we combined analysis of transcription factor binding sites, histone modifications (ChIPseq) and open chromatin regions (ATACseq) with the massively parallel reporter assay ChIP-STARR-seq to identify the subset of functional enhancers in human neural stem cells, an in vitro model reflecting early brain development. This led to a genome-wide, quantitative map of enhancer activity that can be of relevance for MCDs and other neurodevelopmental disorders.
14:40-14:45: Julien Ferent (National Institute for Health and Medical Research [INSERM], France) et F. Francis
TRIDIMENSIONAL VISUALIZATION OF ECTOPIC AXONAL TRACTS WITHIN THE HETEROTOPIC CORTEX OF THE MURINE EML1 CONDITIONAL KNOCKOUT MODEL
Subcortical heterotopias are defined by the presence of misplaced neurons in the white matter. They can arise through abnormal neural progenitors and/or neuronal migration. EML1 shows mutations in several human patients presenting a ribbon-like subcortical heterotopia. We are using the Eml1 conditional knock-out (cKO) mouse model to investigate the molecular, cellular and organizational mechanisms involved in the development of this cortical malformation. In this study, we were interested in the morphological features which arise during the development of the malformation. Using the clarification protocol iDISCO, we show that the volume of the Eml1 cKO-induced heterotopia can be defined in 3D. We then set out to analyze the axonal projections of heterotopic neurons. Unexpectedly, by staining for the immunoglobulin TAG1 at E18.5, expressed along axons, we show large bundles of fasciculated axons in the cKO, extending from the surface of the cortex (normotopia), overlying the heterotopia. Similar projections were not seen in control brains. Moreover, TAG1+ axon bundles were also detected within the heterotopia itself. Investigating the brain at E15.5, we could already see the presence of ectopic axons within the normotopic cortex, associated with clusters of TAG1+ neurons, suggesting that this abnormal neuronal and axonal phenotype develops early on, even before the formation of the heterotopia itself. These results pave the way for further investigations of how the abnormal axon tracts form in this mouse model, and their impact on heterotopia development, as well as tractography analyses in human patients assessing fiber bundles above and within the heterotopia.
_15 min coffee break
_keynote speakers – session 3a: UNEXPLORED PATHS ON DISEASE MECHANISM AND THERAPY
15:00-15:30: Karina Krajden Haratz (Tel Aviv University, Israel)
WHAT NEXT: COST ACTION ON MIDBRAIN-HINDBRAIN MALFORMATIONS
+15 min discussion
The embryonic structures of the midbrain and hindbrain (MB-HB) and their differentiation process into the brainstem and cerebellum have been the focus of recent research in the fields of neurogenetics, developmental biology, prenatal and postnatal neuroimaging. However, the comprehension of MB-HB anomalies and their relationships with other malformations, either cerebral or systemic, still remains limited. For the last 5 years our multidisciplinary team has conducted a large multicenter project on prenatal features and diagnostic criteria of midbrain and hindbrain malformations, with a profitable collaboration between centers from all continents. The most important achievements of this work were: a) the development of optimal fetal imaging techniques for the MB-HB structures (either by US and fetal MRI); b) defining morphological diagnostic criteria for several anomalies in different gestational ages (when the presentation differs); c) identification of rare diseases never identified in fetuses until now; d) progresses in genetic evaluation of these cases. From the lessons we learned from the COST-MIG action, we consider very promising a similar project facing this specific group of anomalies, expanding our prenatal approach to a broader insight into the MBHB development and abnormal formation. This lecture aims to present our prenatal data and a new proposal for a future action.
15:45-16:15: Gustavo Malinger (Tel Aviv University, Israel)
WHAT NEXT? MALFORMATIONS INVOLVING THE MIDBRAIN-HINDBRAIN. EARLY RECOGNITION BY TRANSVAGINAL HIGH RESOLUTION ULTRASOUND
+15 min discussion
We learned from the success of the Neuro-Mig Cost Action the importance of the recognition of malformations of cortical development during pregnancy. In our new proposal we want to evaluate the strengths of a similar approach to the diagnosis of Midbrain-Hindbrain (MBHB) malformations. From the beginning it is important to recognize that MBHB development follows a very specific and dynamic pattern and that the end result, the newborn HBHB, is very different from the one observed during the first and early second trimester. Before trying to diagnose specific pathologies it is of foremost importance to be knowledgeable with its normal appearance. We performed TVS examinations in 60 apparently normal fetuses between 14 and 19 weeks using a high resolution probes and were able to study and measure various structures in the median plane: the tectum, the tegmentum, the aqueduct of Sylvius, the pons, the Blake’s pouch, the 4th ventricle and the cisterna magna. In the axial planes: the tegmentum width, the pons, the cerebellum including the vermis, 4th ventricle and the cisterna magna. In the coronal plane the cerebellum and the vermis. We found that by 14 GW, the embryonic pontine and mesencephalic flexures are not observed, and the BS gained its final “mature” appearance. The growth of the different structures follows a linear pattern. During the study period we diagnosed 26 fetuses with anomalies involving the MBHB including aqueductal stenosis (11); kinked brainstem in the median plane (6) (2 diagnosed as Walker Warburg); cystic dilatations of the 4th ventricle (6); abnormal vermis (2) (one Joubert, one rhomboencephalosynapsis); Moebius syndrome (1). The mean gestational age at the time of the initial diagnosis was 15.6 weeks (range 14-18 weeks). We conclude that a complete visualization of the MBHB structures is possible early in pregnancy and that in some cases, probably the most severe of the spectrum, diagnosis of anomalies is possible at this stage enabling further investigation and genetic counseling. Since this field is still mostly unexplored the opportunities for a new COST action are to be considered.
16:30-17:00: Christopher A. Walsh (Harvard University, United States)
SOMATIC MUTATION AND GENOMIC DIVERSITY IN HUMAN BRAIN IN DEVELOPMENT, DISEASE, AND DEGENERATION
+15 min discussion
Although it had long been assumed that the genomes of all neurons are identical, recent work shows that every cell division causes mutations even during normal development, and that postmitotic neurons continue to accumulate mutations throughout life even in the absence of cell division. Recent studies implicate clonal somatic mutations in some brain malformations, other types of focal epilepsy, autism spectrum disorders, and schizophrenia. Reading out these developmental mutations reveals a forensic cell lineage map that records each cell division that generates each person. Sequencing the DNA genome from a single neuron reveals a universe of genomic diversity, with transposon insertion, copy number variants, and hundreds of point mutations distinguishing the genome of one neuron from another. Surprisingly, neurons accumulate a dozen of more mutations per year in human brain, with thousands of such mutations present in old age. SNV accumulate faster in rare genetic disorders, associated with precocious neurodegeneration and are likely to play a role in more common forms of degeneration as well. Supported by the NIMH, NINDS, and HHMI.
_15 min coffee break
_keynote speakers – session 3b: UNEXPLORED PATHS ON DISEASE MECHANISM AND THERAPY
17:30-18:00: Ghayda Mirzaa (University of Washington, United States)
MEGALENCEPHALY: ADVANCES FROM GENOMIC DISCOVERIES TO PRECISION-BASED MEDICINE
+15 min discussion
Megalencephaly disorders (MEG) constitute a growing spectrum of developmental disorders associated with early brain overgrowth, brain malformations including cortical dysplasia, and neurodevelopmental co-morbidities such as intractable epilepsy, autism and intellectual disability. Constitutional and mosaic mutations of multiple cell growth pathways, including the PI3K-AKT-MTOR and RAS pathways, have been identified in this spectrum; with promising therapeutic avenues on the horizon to ameliorate the associated neurological deficits (e.g. MTOR pathway inhibitors). However, significant molecular and clinical challenges remain. Widely used molecular diagnostic methods, such as standard-depth exome sequencing, lack the sensitivity to detect low-level mosaic mutations common in MEG. These methods are also not suitable for screening limited amounts of surgically resected brain tissues due to their high DNA input requirements. Ultimately, more optimal molecular diagnostic methods and high throughput pre-clinical models are necessary to identify and study the effects of MEG-related mutations on brain development. To address these challenges, we utilize a combination of ultra-deep targeted sequencing employing digital droplet PCR (ddPCR) as a new and sensitive tool for screening common mutations in the PI3K-AKT-MTOR pathway, and a quad based exome approach, sequencing lesional (affected) tissues at a higher depth (200-250X). To date, we performed genetic testing on a large cohort of MEG families and have identified both known and novel candidate genetic variants, including ultra-low-level mosaic mutations in the PI3K-AKT-MTOR pathway. Our data describe a gradient of mosaicism seen within the brain in hemimegalencephaly samples; with mutation-positive regions ranging in alternate allele frequencies (AAF) from 0.3% to 25.6% across several brain tissues. We subsequently aimed to model disease pathogenesis in vitro, selecting patient-derived fibroblast lines with mutations in key upstream (PIK3CA) and downstream (MTOR) nodes in the pathway. We generated induced pluripotent stem cells (iPSCs) carrying the common PIK3CAH1047R and MTORT1977I mutations to characterize these nodes. Our results suggest that targeted molecular analysis for the common PI3K-AKT-MTOR pathway mutations by ddPCR is an effective molecular diagnostic approach for MEG. Given the high sensitivity and low DNA input requirements for this assay, ddPCR may also be an effective molecular tool for other mosaic disorders with a narrow mutational spectrum. Our iPSC-derived lines provide a valuable model for dissecting the functional consequences of these mutations and future drug screens using PI3K-AKT-MTOR pathway inhibitors.
18:15-18:45: Carlos Cardoso (Mediterranean Institute of Neurobiology [INMED], France)
ABNORMAL DEVELOPMENT OF NEURONAL WIRING IN ANIMAL MODEL OF GREY MATTER HETEROTOPIA
+15 min discussion
Proper cerebral function relies on adequate activities of cortical neural circuits. This connectivity is the end-point of a long process of embryonic and postnatal development which depends on the proper generation and specification of dozens of neuronal subtypes, their migration to appropriate layers and the formation of synaptic connections between distinct neuronal subtypes. Alterations in any of these processes, due to genetic mutations and/or environmental insults (e.g. in utero infection, vascular injury…), lead to defined brain disorders collectively known as malformations of cortical development (MCD). The most common type of MCD, Grey Matter Heterotopia is characterized by the ectopic position of neurons along the ventricular walls or in the deep white matter. Most affected patients often have seizures and cognitive deficits. Mutations have been found in genes, including DCX and FLNA, that influence radial migration of postmitotic cells from the ventricular zone to the cortical plate. However, the sequence of events linking molecular disruption to clinical manifestation mostly remains undetermined. Our recent work revealed that abnormal neuronal positioning of excitatory neurons leads to defects in neurite outgrowth, synapse formation and transmission and longrange connectivity in Dcx and Flna animal models. Such alterations during development often lead to synaptic excitation/inhibition (E/I) imbalance in the mature brain, and this has been recognized as a mechanism at the basis of epilepsy and cognitive impairment.
_closing ceremony
19:00-19:25: Grazia M.S. Mancini, Anna Jansen and Carlos Cardoso
CLOSING REMARKS AND GENERAL DISCUSSION
19:25-19:30: Michal Klichowski
ACKNOWLEDGMENTS AND FAREWELL