Although mutations in the human doublecortin gene (DCX) cause profound defects in cortical neuronal migration, a genetic deletion of Dcx in mice produces a milder defect. A second locus, doublecortin-like kinase (Dclk), encodes a protein with similar "doublecortin domains" and microtubule stabilization properties that may compensate for Dcx. Here, we generate a mouse with a Dclk mutation that causes no obvious migrational abnormalities but show that mice mutant for both Dcx and Dclk demonstrate perinatal lethality, disorganized neocortical layering, and profound hippocampal cytoarchitectural disorganization. Surprisingly, Dcx(-/y);Dclk(-/-) mutants have widespread axonal defects, affecting the corpus callosum, anterior commissure, subcortical fiber tracts, and internal capsule. Dcx/Dclk-deficient dissociated neurons show abnormal axon outgrowth and dendritic structure, with defects in axonal transport of synaptic vesicle proteins. Dcx and Dclk may directly or indirectly regulate microtubule-based vesicle transport, a process critical to both neuronal migration and axon outgrowth.

SummaryDoublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.

Objective Recently, Christianson syndrome (CS) has been determined to be caused by mutations in the X-linked Na+/H+ Exchanger 6 (NHE6). We aimed to determine the diagnostic criteria and mutational spectrum for CS. Methods Twelve independent pedigrees (14 boys, ages 4 to 19) with mutations in NHE6 were administered standardized research assessments and mutations were characterized. Results The mutational spectrum was composed of 9 single nucleotide variants (SNVs), 2 indels and 1 CNV deletion. All mutations were protein-truncating or splicing mutations. We identified two recurrent mutations (c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were unique. In our study, seven of 12 mutations (58%) were de novo, in contrast to prior literature wherein mutations were largely inherited. We also report prominent neurological, medical and behavioral symptoms. All CS participants were non-verbal and had intellectual disability, epilepsy and ataxia. Many had prior diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included eye movement abnormalities (79%), postnatal microcephaly (92%) and MRI evidence of cerebellar atrophy (33%). Regression was noted in 50%, with recurrent presentations involving loss of words and/or the ability to walk. Medical symptoms, particularly gastrointestinal symptoms, were common. Height and body mass index measures were below normal ranges in most participants. Behavioral symptoms included hyperkinetic behavior (100%) and a majority exhibited high pain threshold. Interpretation This is the largest cohort of independent CS pedigrees reported. We propose diagnostic criteria for CS. CS represents a novel neurogenetic disorder with general relevance to autism, intellectual disability, Angelman syndrome, epilepsy and regression.

Cortical malformations associated with defects in neuronal migration result in severe developmental consequences including intractable epilepsy and intellectual disability. Genetic causes of migration defects have been identified with the advent and widespread use of high-resolution MRI and genetic techniques. Thus, the full phenotypic range of these genetic disorders is becoming apparent. Genes that cause lissencephaly, pachygyria, subcortical band heterotopia, and periventricular nodular heterotopias have been defined. Many of these genes are involved in cytoskeletal regulation including the function of microtubules (LIS1, TUBA1A,TUBB3, and DCX) and of actin (FilaminA). Thus, the molecular pathways regulating neuronal migration including the cytoskeletal pathways appear to be defined by human mutation syndromes. Basic science, including cell biology and animal models of these disorders, has informed our understanding of the pathogenesis of neuronal migration disorders and further progress depends on the continued integration of the clinical and basic sciences.

Type I lissencephaly, a genetic disease characterized by disorganized cortical layers and gyral abnormalities, is associated with severe cognitive impairment and epilepsy. Two genes, LIS1 and doublecortin (DCX), have been shown to be responsible for a large proportion of cases of type I lissencephaly. Both genes encode microtubule-associated proteins that have been shown to be important for radial migration of cortical pyramidal neurons. To investigate whether DCX also plays a role in cortical interneuron migration, we inactivated DCX in the ganglionic eminence of rat embryonic day 17 brain slices using short hairpin RNA. We found that, when DCX expression was blocked, the migration of interneurons from the ganglionic eminence to the cerebral cortex was slowed but not absent, similar to what had previously been reported for radial neuronal migration. In addition, the processes of DCX-deficient migrating interneurons were more branched than their counterparts in control experiments. These effects were rescued by DCX overexpression, confirming the specificity to DCX inactivation. A similar delay in interneuron migration was observed when Doublecortin-like kinase (DCLK), a microtubuleassociated protein related to DCX, was inactivated, although the morphology of the cells was not affected. The importance of these genes in interneuron migration was confirmed by our finding that the cortices of Dcx, Dclk, and Dcx/Dclk mutant mice contained a reduced number of such cells in the cortex and their distribution was different compared with wild-type controls. However, the defect was different for each group of mutant animals, suggesting that DCX and DCLK have distinct roles in cortical interneuron migration.

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system.

SUMMARY Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clockflox/flox and PV-Cre; Clockflox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clockflox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clockflox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clockflox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory post-synaptic currents. Finally, video-EEG of Emx-Cre; Clockflox/flox mice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule (MT) binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semi-quantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, wide spread differences are found in actin-associated proteins as compared with wild type axons. Decreases in actin-binding proteins, α-actinin-1, α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued, by full length Dcx, and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C terminal S/P rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin Like Kinase 1 (Dclk1) deficiency is consistent with actin defect as these axons are selectively deficient in axon guidance, but not elongation.