Transgenic modeling of Ndr2 gene amplification reveals disturbance of hippocampus circuitry and function.

Duplications and deletions of short chromosomal fragments are increasingly recognized as the cause for rare neurodevelopmental conditions and disorders. The NDR2 gene encodes a protein kinase important for neuronal development and is part of a microduplication region on chromosome 12 that is associated with intellectual disabilities, autism, and epilepsy. We developed a conditional transgenic mouse with increased Ndr2 expression in postmigratory forebrain neurons to study the consequences of an increased gene dosage of this Hippo pathway kinase on brain circuitry and cognitive functions. Our analysis reveals reduced terminal fields and synaptic transmission of hippocampal mossy fibers, altered hippocampal network activity, and deficits in mossy fiber-dependent behaviors. Reduced doublecortin expression and protein interactome analysis indicate that transgenic Ndr2 disturbs the maturation of granule cells in the dentate gyrus. Together, our data suggest that increased expression of Ndr2 may critically contribute to the development of intellectual disabilities upon gene amplification.

Ndr2 Kinase Controls Neurite Outgrowth and Dendritic Branching Through α1 Integrin Expression.

The serine/threonine kinase Ndr2 has been shown to control the inside-out activation of the ß1subunit of integrins and the formation of neurites in both primary neurons and neurally differentiated pheochromacytoma (PC12) cells. In this study, we demonstrate that Ndr2 kinase furthermore determines the substrate specificity of neurite extension in PC12 cells via expression of α1ß1 integrins. We show that stable overexpression of Ndr2 in PC12 cells increases neurite growth and extension on poly-D-lysine substrate, likely involving an increased expression of active ß1 integrin in the growth tips of these cells. By contrast, the Ndr2 overexpressing cells do not show the α1ß1 integrin-mediated enhancement of neurite growth on collagen IV substrate that can be seen in control cells. Moreover, they entirely fail to increase in response to activation of α1ß1 integrins via a soluble KTS ligand and show a diminished accumulation of α1 integrin in neurite tips, although the expression of this subunit is induced during differentiation to comparable levels as in control cells. Finally, we demonstrate that Ndr2 overexpression similarly inhibits the α1ß1 integrin-dependent dendritic growth of primary hippocampal neurons on laminin 111 substrate. By contrast, lack of Ndr2 impairs the dendritic growth regardless of the substrate. Together, these results suggest that Ndr2 regulates α1 integrin trafficking in addition to ß1 integrin subunit activation and thereby controls the neurite growth on different extracellular matrix (ECM) substrates.

Mutations in MICAL-1cause autosomal-dominant lateral temporal epilepsy.

OBJECTIVE: Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic focal epilepsy characterized by auditory symptoms. Two genes, LGI1 and RELN, encoding secreted proteins, are implicated in the etiology of ADLTE, but half of the affected families remain genetically unsolved, and the underlying molecular mechanisms are yet to be clarified. We aimed to identify additional genes causing ADLTE to better understand the genetic basis and molecular pathway underlying this epileptic disorder. METHODS: A cohort of Italian ADLTE families was examined by whole exome sequencing combined with genome-wide single-nucleotide polymorphism-array linkage analysis. RESULTS: We identified two ADLTE-causing variants in the MICAL-1 gene: a p.Gly150Ser substitution occurring in the enzymatically active monooxygenase (MO) domain and a p.Ala1065fs frameshift indel in the C-terminal domain, which inhibits the oxidoreductase activity of the MO domain. Each variant segregated with ADLTE in a single family. Examination of candidate variants in additional genes excluded their implication in ADLTE. In cell-based assays, both variants significantly increased MICAL-1 oxidoreductase activity and induced cell contraction in COS7 cells, which likely resulted from deregulation of F-actin dynamics. INTERPRETATION: MICAL-1 oxidoreductase activity induces disassembly of actin filaments, thereby regulating the organization of the actin cytoskeleton in developing and adult neurons and in other cell types. This suggests that dysregulation of the actin cytoskeleton dynamics is a likely mechanism by which MICAL-1 pathogenic variants lead to ADLTE. Ann Neurol 2018;83:483-493.

Cortical Morphogenesis during Embryonic Development Is Regulated by miR-34c and miR-204.

The porcine brain closely resembles the human brain in aspects such as development and morphology. Temporal miRNA profiling in the developing embryonic porcine cortex revealed a distinct set of miRNAs, including miR-34c and miR-204, which exhibited a highly specific expression profile across the time of cortical folding. These miRNAs were found to target Doublecortin (DCX), known to be involved in neuron migration during cortical folding of gyrencephalic brains. In vivo modulation of miRNA expression in mouse embryos confirmed that miR-34c and miR-204 can control neuronal migration and cortical morphogenesis, presumably by posttranscriptional regulation of DCX.

Frizzled3 Controls Axonal Polarity and Intermediate Target Entry during Striatal Pathway Development.

The striatum is a large brain nucleus with an important role in the control of movement and emotions. Medium spiny neurons (MSNs) are striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts in the forebrain develop remains poorly understood. Here, we implicate the Wnt binding receptor Frizzled3 in several uncharacterized aspects of MSN pathway formation [i.e., anterior-posterior guidance of MSN axons in the striatum and their subsequent growth into the globus pallidus (GP), an important (intermediate) target]. In Frizzled3 knock-out mice, MSN axons fail to extend along the anterior-posterior axis of the striatum, and many do not reach the GP. Wnt5a acts as an attractant for MSN axons in vitro, is expressed in a posterior high, anterior low gradient in the striatum, and Wnt5a knock-out mice phenocopy striatal anterior-posterior defects observed in Frizzled3 mutants. This suggests that Wnt5a controls anterior-posterior guidance of MSN axons through Frizzled3. Axons that reach the GP in Frizzled3 knock-out mice fail to enter this structure. Surprisingly, entry of MSN axons into the GP non-cell-autonomously requires Frizzled3, and our data suggest that GP entry may be contingent on the correct positioning of "corridor" guidepost cells for thalamocortical axons by Frizzled3. Together, these data dissect MSN pathway development and reveal (non)cell-autonomous roles for Frizzled3 in MSN axon guidance. Further, they are the first to identify a gene that provides anterior-posterior axon guidance in a large brain nucleus and link Frizzled3 to corridor cell development. SIGNIFICANCE STATEMENT: Striatal axon pathways mediate complex physiological functions and are an important therapeutic target, underscoring the need to define how these connections are established. Remarkably, the molecular programs regulating striatal pathway development remain poorly characterized. Here, we determine the embryonic ontogeny of the two main striatal pathways (striatonigral and striatopallidal) and identify novel (non)cell-autonomous roles for the axon guidance receptor Frizzled3 in uncharacterized aspects of striatal pathway formation (i.e., anterior-posterior axon guidance in the striatum and axon entry into the globus pallidus). Further, our results link Frizzled3 to corridor guidepost cell development and suggest that an abnormal distribution of these cells has unexpected, widespread effects on the development of different axon tracts (i.e., striatal and thalamocortical axons).

The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth.

Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of ß1-integrin to stimulate the PKC- and CaMKII-dependent activation of ß1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated ß1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

Disruption of fear memory consolidation and reconsolidation by actin filament arrest in the basolateral amygdala.

The dynamic re-arrangement of actin filaments is an essential process in the plasticity of synaptic connections during memory formation. In this study, we determined in mice effects of actin filament arrest in the basolateral complex of the amygdala (BLA) at different time points after memory acquisition and re-activation, using the fungal cytotoxin phalloidin. Our data show a selective disruption of auditory cued but not contextual fear memory, when phalloidin was injected 6h after conditioning. In contrast, no effect was observed when phalloidin was applied after 24h, ruling out an interference with the retrieval or expression of conditioned fear. A comparable result was obtained after memory re-activation, hence suggesting similar actin-dependent mechanisms to be active during consolidation and reconsolidation of auditory fear memory. Biochemical analysis showed that phalloidin-mediated filament arrest leads to a transient increase of highly cross-linked actin filaments in the BLA, evident 2h after injection. Together, these observations indicate that dynamic re-arrangements of actin filaments in the BLA during a late phase of fear memory consolidation and reconsolidation are critical for fear memory storage.