In vitro modeling of dendritic atrophy in Rett syndrome: determinants for phenotypic drug screening in neurodevelopmental disorders

Abstract

Dendritic atrophy, defined as the reduction in complexity of the neuronal arborization, is a hallmark of several neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, affecting 1:10,000 girls worldwide, is mainly caused by mutations in the MECP2 gene and has no cure. We describe here an in vitro model of dendritic atrophy in Mecp2 −/y mouse hippocampal primary cultures, suitable for phenotypic drug-screening. Using High-content imaging techniques, we systematically investigated the impact of culturing determinants on several parameters such as neuronal survival, total dendritic length, dendritic endpoints, soma size, cell clusterization, spontaneous activity. Determinants included cell-seeding density, glass or polystyrene substrates, coating with poly-ornithine with/without Matrigel and miniaturization from 24 to 96-half surface multiwell plates. We show that in all plate-sizes at densities below 320 cells/mm 2 , morphological parameters remained constant while spontaneous network activity decreased according to the cell-density. Mecp2 −/y neurons cultured at 160 cells/mm 2 density in 96 multiwell plates, displayed significant dendritic atrophy and showed a marked increase in dendritic length following treatment with Brain-derived neurotrophic factor (BDNF) or Mirtazapine. in conclusion, we have established a phenotypic assay suitable for fast screening of hundreds of compounds, which may be extended to other neurodevelopmental diseases with dendritic atrophy.Dendritic atrophy, defined as the reduction in branching complexity of neuronal dendritic processes, is a typical hallmark of several neurodevelopmental disorders 1-4 . With an incidence of 1:10,000 newborn girls, Rett syndrome (RTT) is one of the most common neurodevelopmental diseases with mental retardation in females 5 . In 90-95% of cases, the disease is caused by sporadic mutations in the MECP2 gene 6 . RTT is characterized by a period of early normal development followed by a regression phase, leading to loss of speech and acquired motor skills, presence of stereotypical hand movements, seizures and microcephaly 6 . Presently, there is no cure for Rett syndrome.Due to microcephaly, the brains of RTT patients show more closely packed neurons 7 and reduced dendritic complexity has been described in cerebral cortex, hypothalamus and hippocampus [8][9][10] . The dendritic atrophy observed in the cortex of RTT patients has been related to dysfunctions of neural networks and intellectual disability 9 , similarly to other neurodevelopmental disorders such as Fragile-X syndrome and Down syndrome 1,7 . Accordingly, modelling dendritic atrophy for these diseases is extremely important.Notably, in mouse models for Rett syndrome, reduced brain dimensions and dendritic atrophy has been found in the same brain regions as in humans 11,12 . We previously showed that in vivo treatment of Mecp2 −/y mice with the antidepressant Mirtazapine was able to rescue cortical thickness, neuronal soma area and apical dendrites diameter counteracting breathing abnorma...

Amyloid-β Impairs Dendritic Trafficking of Golgi-Like Organelles in the Early Phase Preceding Neurite Atrophy: Rescue by Mirtazapine

“…In Figure 7 , TDL data shown for the DMSO condition were different between Figures 7B,D which were carried out at different times. We previously reported that variations in the culture density can affect the TDL ( Nerli et al, 2020 ). However, irrespective of the actual value, the validity of each experiment is given by the comparison to its own control.…”

“…We also characterize the ability of mirtazapine, a noradrenergic and specific serotonergic tetracyclic antidepressant (NaSSA), to rescue Golgi trafficking. The neurotrophic effect of antidepressants is well known and demonstrated in several pathological models ( Castrén, 2004 ), and we have previously shown that mirtazapine can rescue dendritic atrophy and soma size shrinkage of cortical and hippocampal neurons in a mouse model of Rett syndrome ( Bittolo et al, 2016 ; Nerli et al, 2020 ).…”

“…An automated analysis approach was used to quantify the neurite retraction and recovery after 24 h of treatment. This automated analysis was performed using the open source bioinformatics toolkit NeuriteQuant, implemented in the free image analysis software ImageJ/Fiji ( Dehmelt et al, 2011 ; Nerli et al, 2020 ). Following a staining of neurites using an antibody against cytoskeletal proteins such as Tubulin-β-III or MAP-2, this tool allows for the measurement of total neuritic or dendritic length on primary neuronal cultures.…”

Oral Manifestations of Rett Syndrome—A Systematic Review

“…Conditions such as autistic spectrum disorder, Aspergers syndrome, attention deficit hyperactivity disorder, dyspraxia and Rett Syndrome are characterized as being neurodevelopment disorders [2]. Among these, Rett Syndrome has been exclusively linked with female predilection and mental retardation [3].…”

Implicações de variantes bialélicas de perda de função no gene TBCK, no transporte de vesículas de células progenitoras neurais derivadas de células-tronco pluripotentes induzidas

“…Drosophila melanogaster e células humanas sugeriram uma possível interação de TBCK com duas moléculas (Perini, 2012; A técnica de reprogramação de células somáticas adultas para um estado pluripotente, por meio da superexpressão de genes específicos (Takahashi et al, 2007;Yu et al, 2007) Em concordância com os achados em modelos animais da síndrome (Chao et al, 2007;Wang et al, 2013;Nerli et al, 2020), os neurônios diferenciados a partir de iPSC de pacientes apresentaram alterações morfológicas importantes, como diminuição do soma neuronal e alteração na densidade dos espinhos dendríticos, quando comparados aos controles, características que indicam menor maturidade e complexidade que neurônios saudáveis (Ardhanareeswaran et al, 2017;Nerli et al, 2020). Funcionalmente, esses neurônios derivados de iPSC apresentaram redução no número de sinapses excitatórias e defeitos na sinalização por cálcio, contribuindo para alterações de amplitude e frequência do potencial de ação.…”