Granule Cell Dispersion in Human Temporal Lobe Epilepsy: Proteomics investigation of neurodevelopmental migratory pathways

Granule cell dispersion (GCD) is a common pathological feature observed in the hippocampus of patients with Mesial Temporal Lobe Epilepsy (MTLE). Pathomechanisms underlying GCD remain to be elucidated, but one hypothesis proposes aberrant reactivation of neurodevelopmental migratory pathways, possibly triggered by febrile seizures. This study aims to compare the proteomes of basal and dispersed granule cells in the hippocampus of eight MTLE patients with GCD to identify proteins that may mediate GCD in MTLE. Quantitative proteomics identified 1882 proteins, of which 29% were found in basal granule cells only, 17% in dispersed only and 54% in both samples. Bioinformatics analyses revealed upregulated proteins in dispersed samples were involved in developmental cellular migratory processes, including cytoskeletal remodelling, axon guidance and signalling by Ras homologous (Rho) family of GTPases (P<0.01). The expression of two Rho GTPases, RhoA and Rac1, was subsequently explored in immunohistochemical and in situ hybridisation studies involving eighteen MTLE cases with or without GCD, and three normal post mortem cases. In cases with GCD, most dispersed granule cells in the outer-granular and molecular layers have an elongated soma and bipolar processes, with intense RhoA immunolabelling at opposite poles of the cell soma, while most granule cells in the basal granule cell layer were devoid of RhoA. A higher density and percentage of cells expressing RhoA was observed in cases with GCD than without GCD (P<0.004). In GCD cases, the density and percentage of cells expressing RhoA was significantly higher in the inner molecular layer than granule cell layer (P<0.026), supporting proteomic findings. In situ hybridisation studies using probes against RHOA and RAC1 mRNAs revealed fine peri- and nuclear puncta in granule cells of all cases. The density of cells expressing RHOA mRNAs were significantly higher in the inner molecular layer of cases with GCD than without GCD(P=0.05). In summary, our study has found limited evidence for ongoing adult neurogenesis in the hippocampus of patients with MTLE, but evidence of differential dysmaturation between dispersed and basal granule cells has been demonstrated, and elevated expression of Rho GTPases in dispersed granule cells may contribute to the pathomechanisms underpinning GCD in MTLE

Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

A role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors.

神経細胞の配置メカニズムを解明－抑制性神経前駆細胞に特有の移動の機構が明らかに. 京都大学プレスリリース. 2012-1-16.In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration

Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2.

Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated "reciprocal knock-in mice"-mice that make lamin B2 from the Lmnb1 locus (Lmnb1(B2/B2)) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2(B1/B1)). Lmnb1(B2/B2) mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1(B2/B2) mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2(B1/B1) mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other

Early Postnatal Migration and Development of Layer II Pyramidal Neurons in the Rodent Cingulate/Retrosplenial Cortex

The cingulate and retrosplenial regions are major components of the dorsomedial (dm) limbic cortex and have been implicated in a range of cognitive functions such as emotion, attention, and spatial memory. While the structure and connectivity of these cortices are well characterized, little is known about their development. Notably, the timing and mode of migration that govern the appropriate positioning of late-born neurons remain unknown. Here, we analyzed migratory events during the early postnatal period from ventricular/subventricular zone (VZ/SVZ) to the cerebral cortex by transducing neuronal precursors in the VZ/SVZ of newborn rats/mice with Tomato/green fluorescent protein-encoding lentivectors. We have identified a pool of postmitotic pyramidal precursors in the dm part of the neonatal VZ/SVZ that migrate into the medial limbic cortex during the first postnatal week. Time-lapse imaging demonstrates that these cells migrate on radial glial fibers by locomotion and display morphological and behavioral changes as they travel through the white matter and enter into the cortical gray matter. In the granular retrosplenial cortex, these cells give rise to a Satb2+ pyramidal subtype and develop dendritic bundles in layer I. Our observations provide the first insight into the patterns and dynamics of cell migration into the medial limbic corte

Automated four-dimensional long term imaging enables single cell tracking within organotypic brain slices to study neurodevelopment and degeneration.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes

DNA Methylation program in normal and alcohol-induced thinning cortex

While cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7–16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis

Microtubule plus-end binding protein CLASP2 in neural development

Normal brain function is dependent on the correct positioning and connectivity of neurons established during development. The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to lipoprotein receptors and inducing tyrosine phosphorylation of the intracellular adaptor protein Dab1. Mutations in genes of the Reelin signaling pathway lead to profound defects in neuronal positioning during brain development in both mice and humans. However, the molecular mechanisms by which Reelin controls neuronal morphology and migration are unknown. We have used a systems analysis approach to identify genes perturbed in the Reelin signaling pathway and identified microtubule stabilizing CLIP-associated protein 2 (CLASP2) as a key cytoskeletal modifier of Reelin mutant phenotypes. Currently, little is known about the role of CLASP2 in the developing brain. We propose that CLASP2 is a key effector in the Reelin signaling pathway controlling basic aspects of cortical layering, neuronal morphology, and function. CLASP2 is a plus-end tracking protein and this localization places CLASP2 in a strategic position to control neurite outgrowth, directionality, and responsiveness to extracellular cues. Our results demonstrate that CLASP2 expression correlates with neurite length and synaptic activity in primary neuron cultures; however, the role of CLASP2 during brain development was unknown. In this dissertation, we have characterized the role of CLASP2 during cortical development by in utero electroporation of shRNA plasmids and found that silencing CLASP2 in migrating neurons leads to mislocalized cells at deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. In addition, we found that GSK3β-mediated phosphorylation of CLASP2 controls Dab1 binding and is required for regulating CLASP2 effects on neuron morphology and migration. This dissertation provides the first steps in gaining insight into how Reelin signaling affects cytoskeletal reorganization to regulate fundamental features of neuronal migration, positioning and morphogenesis

Insights From Pretzel Syndrome: The Role of STRADA in Neuronal Migration and Cortical Development

Pretzel Syndrome (also Polyhydramnios, Megalencephaly, and Symptomatic Epilepsy syndrome; PMSE) is a recently described rare neurodevelopmental disorder occurring in the Old Order Mennonite pediatric population, and characterized by intractable infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter, suggestive of failed neuronal migration. PMSE is caused by a homozygous deletion of exons 9-13 of LYK5/STRADA, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin (mTOR). Therefore, we hypothesize that STRADA plays a critical role in neuronal migration through modulating mTOR (specifically mTOR complex 1, mTORC1) signaling, and that therapeutic mTORC1 inhibition can ameliorate features of the PMSE disease phenotype. To test this hypothesis, we model PMSE in vitro using stable shRNA knockdown of STRADA (STRADA KD) in mouse neural progenitor cells (mNPCs). In vivo, we use in utero electroporation to create focal STRADA KD in the developing mouse brain. We show that STRADA depletion disrupts pathfinding and polarization in migrating mNPCs in vitro, and this effect can be rescued by inhibition of mTORC1 with rapamycin or of its downstream effector p70S6kinase (p70S6K) with PF-4708671 (p70S6Ki), indicating an mTORC1-specific dependence. We then define a pathway for this effect downstream of mTORC1, through insulin receptor substrate 1 (IRS1) signaling to cofilin, and finally modulating actin dynamics. In vivo, we demonstrate that STRADA KD causes a cortical lamination defect in the mouse, which can be rescued with rapamycin treatment, confirming the dependence of STRADA\u27s effect on mTORC1 signaling and suggesting an important target for patient therapy. To correlate our mouse model with PMSE, we demonstrate congruent mTORC1 and downstream signaling and rescue of migration deficit with rapamycin and p70S6Ki in PMSE patient fibroblasts. Finally, we report reduction of seizure frequency with rapamycin treatment in previously intractable PMSE patients. Our findings define a novel role for STRADA in neuronal migration, demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition can serve as an effective therapeutic bio-target in PMSE as well as other devastating mTOR-associated neurodevelopmental disorders