Jilin Bai

RNAi reveals doublecortin is required for radial migration in rat neocortex

Mutations in the doublecortin gene (DCX) in humans cause malformation of the cerebral neocortex. Paradoxically, genetic deletion of Dcx in mice does not cause neocortical malformation. We used electroporation of plasmids encoding short hairpin RNA to create interference (RNAi) of DCX protein in utero, and we show that DCX is required for radial migration in developing rat neocortex. RNAi of DCX causes both cell-autonomous and non-cell autonomous disruptions in radial migration, and creates two disruptions in neocortical development. First, many neurons prematurely stop migrating to form subcortical band heterotopias within the intermediate zone and then white matter. Second, many neurons migrate into inappropriate neocortical lamina within normotopic cortex. In utero RNAi can therefore be effectively used to study the specific cellular roles of DCX in neocortical development and to produce an animal model of double cortex syndrome.

A Critical Function for β-Amyloid Precursor Protein in Neuronal Migration Revealed by In Utero RNA Interference

Physiological processing of the β-amyloid precursor protein (APP) generates amyloid β-protein, which can assemble into oligomers that mediate synaptic failure in Alzheimers disease. Two decades of research have led to human trials of compounds that chronically target this processing, and yet the normal function of APP in vivo remains unclear. We used the method of in utero electroporation of shRNA constructs into the developing cortex to acutely knock down APP in rodents. This approach revealed that neuronal precursor cells in embryonic cortex require APP to migrate correctly into the nascent cortical plate. cDNAs encoding human APP or its homologues, amyloid precursor-like protein 1 (APLP1) or APLP2, fully rescued the shRNA-mediated migration defect. Analysis of an array of mutations and deletions in APP revealed that both the extracellular and cytoplasmic domains of APP are required for efficient rescue. Whereas knock-down of APP inhibited cortical plate entry, overexpression of APP caused accelerated migration of cells past the cortical plate boundary, confirming that normal APP levels are required for correct neuronal migration. In addition, we found that Disabled-1 (Dab1), an adaptor protein with a well established role in cortical cell migration, acts downstream of APP for this function in cortical plate entry. We conclude that full-length APP functions as an important factor for proper migration of neuronal precursors into the cortical plate during the development of the mammalian brain.

DYX1C1 functions in neuronal migration in developing neocortex

Rodent homologues of two candidate dyslexia susceptibility genes, Kiaa0319 and Dcdc2, have been shown to play roles in neuronal migration in developing cerebral neocortex. This functional role is consistent with the hypothesis that dyslexia susceptibility is increased by interference with normal neural development. In this study we report that in utero RNA interference against the rat homolog of another candidate dyslexia susceptibility gene, DYX1C1, disrupts neuronal migration in developing neocortex. The disruption of migration can be rescued by concurrent overexpression of DYX1C1, indicating that the impairment is not due to off-target effects. Transfection of C- and N-terminal truncations of DYX1C1 shows that the C-terminal TPR domains determine DYX1C1 intracellular localization to cytoplasm and nucleus. RNAi rescue experiments using truncated versions of DYX1C1 further indicate that the C-terminus of DYX1C1 is necessary and sufficient to DYX1C1s function in migration. In conclusion, DYX1C1, similar to two other candidate dyslexia susceptibility genes, functions in neuronal migration in rat neocortex.

Developmental Disruptions and Behavioral Impairments in Rats Following In Utero RNAi of Dyx1c1

Developmental malformations of cortex have been shown to co-occur with language, learning, and other cognitive deficits in humans. Rodent models have repeatedly shown that animals with such developmental malformations have deficits related to auditory processing and learning. More specifically, freeze-lesion induced microgyria as well as molecular layer ectopias have been found to impair rapid auditory processing ability in rats and mice. In humans, deficits in rapid auditory processing appear to relate to later impairments of language. Recently, genetic variants of four different genes involved in early brain development have been proposed to associate with an elevated incidence of developmental dyslexia in humans. Three of these, DYX1C1, DCDC2, and KIAA0319, have been shown by in utero RNAi to play a role in neuronal migration in developing neocortex. The present study assessed the effects of in utero RNAi of Dyx1c1 on auditory processing and spatial learning in rats. Results indicate that RNAi of Dyx1c1 is associated with cortical heterotopia and is suggestive of an overall processing deficit of complex auditory stimuli in both juvenile and adult periods (p=.051, one-tail). In contrast, adult data alone reveal a significant processing impairment among RNAi treated subjects compared to shams, indicating an inability for RNAi treated subjects to improve detection of complex auditory stimuli over time (p=.022, one-tail). Further, a subset of RNAi treated rats exhibited hippocampal heterotopia centered in CA1 (in addition to cortical malformations). Malformations of hippocampus were associated with robust spatial learning impairment in this sub-group (p<.01, two-tail). In conclusion, in utero RNAi of Dyx1c1 results in heterogeneous malformations that correspond to distinct behavioral impairments in auditory processing, and spatial learning.

Disruption of postsynaptic GABAA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

We have used RNA interference (RNAi) to knock down the expression of the γ2 subunit of the GABAA receptors (GABAARs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the γ2 subunit, one targeting the coding region and the other one the 3′‐untranslated region (UTR) of the γ2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABAA receptor γ2 subunit and the clustering of other GABAAR subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the γ2 shRNAs had no effect on the clustering of postsynaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD‐95) or presynaptic glutamatergic innervation. A γ2‐enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3′‐UTR targeted by γ2 RNAi, rescued both the postsynaptic clustering of GABAARs and the GABAergic innervation. Decreased GABAAR clustering and GABAergic innervation of pyramidal neurons in the post‐natal rat cerebral cortex was also observed after in utero transfection of these neurons with the γ2 shRNAs. The results indicate that the postsynaptic clustering of GABAARs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.

The Role of DCX and LIS1 in Migration through the Lateral Cortical Stream of Developing Forebrain

During forebrain development the lateral cortical stream (LCS) supplies neurons to structures in the ventral telencephalon including the amygdala and piriform cortex. In the current study, we used spatially directed in utero electroporation and RNAi to investigate mechanisms of migration to the ventral telencephalon. Cells labeled by in utero electroporation of the lateral ventricular zone migrated into the LCS, and entered the lateral neocortex, piriform cortex and amygdala, where they differentiated primarily as pyramidal neurons. RNAi of DCX or LIS1 disrupted migration into amygdala and piriform cortex and caused many neurons to accumulate in the external and amygdalar capsules. RNAi of LIS1 and DCX had similar as well as distinguishable effects on the pattern of altered migration. Combinatorial RNAi of LIS1 and DCX further suggested interaction in the functions of LIS1 and DCX on the morphology and migration of migrating neurons in the LCS. Together, these results confirm that the LCS contributes pyramidal neurons to ventral forebrain structures and reveals that DCX and LIS1 have important functions in this major migratory pathway in the developing forebrain.