Neocortical disruption and behavioral impairments in rats following in utero RNAi of candidate dyslexia risk gene Kiaa0319

Within the last decade several genes have been identified as candidate risk genes for developmental dyslexia. Recent research using animal models and embryonic RNA interference (RNAi) has shown that a subset of the candidate dyslexia risk genes--DYX1C1, ROBO1, DCDC2, KIAA0319--regulate critical parameters of neocortical development, such as neuronal migration. For example, embryonic disruption of the rodent homolog of DYX1C1 disrupts neuronal migration and produces deficits in rapid auditory processing (RAP) and working memory--phenotypes that have been reported to be associated with developmental dyslexia. In the current study we used a modified prepulse inhibition paradigm to assess acoustic discrimination abilities of male Wistar rats following in utero RNA interference targeting Kiaa0319. We also assessed spatial learning and working memory using a Morris water maze (MWM) and a radial arm water maze. We found that embryonic interference with this gene resulted in disrupted migration of neocortical neurons leading to formation of heterotopia in white matter, and to formation of hippocampal dysplasia in a subset of animals. These animals displayed deficits in processing complex acoustic stimuli, and those with hippocampal malformations exhibited impaired spatial learning abilities. No significant impairment in working memory was detected in the Kiaa0319 RNAi treated animals. Taken together, these results suggest that Kiaa0319 plays a role in neuronal migration during embryonic development, and that early interference with this gene results in an array of behavioral deficits including impairments in rapid auditory processing and simple spatial learning.     

Synaptic and nonsynaptic localization of protocadherin‐γC5 in the rat brain

It has been proposed that γ‐protocadherins (Pcdh‐γs) are involved in the establishment of specific patterns of neuronal connectivity. Contrary to the other Pcdh‐γs, which are expressed in the embryo, Pcdh‐γC5 is expressed postnatally in the brain, coinciding with the peak of synaptogenesis. We have developed an antibody specific for Pcdh‐γC5 to study the expression and localization of Pcdh‐γC5 in brain. Pcdh‐γC5 is highly expressed in the olfactory bulb, corpus striatum, dentate gyrus, CA1 region of the hippocampus, layers I and II of the cerebral cortex, and molecular layer of the cerebellum. Pcdh‐γC5 is expressed in both neurons and astrocytes. In hippocampal neuronal cultures, and in the absence of astrocytes, a significant percentage of synapses, more GABAergic than glutamatergic, have associated Pcdh‐γC5 clusters. Some GABAergic axons show Pcdh‐γC5 in the majority of their synapses. Nevertheless, many Pcdh‐γC5 clusters are not associated with synapses. In the brain, significant numbers of Pcdh‐γC5 clusters are located at contact points between neurons and astrocytes. Electron microscopic immunocytochemistry of the rat brain shows that 1) Pcdh‐γC5 is present in some GABAergic and glutamatergic synapses both pre‐ and postsynaptically; 2) Pcdh‐γC5 is also extrasynaptically localized in membranes and in cytoplasmic organelles of neurons and astrocytes; and 3) Pcdh‐γC5 is also localized in perisynaptic astrocyte processes. The results support the notions that 1) Pcdh‐γC5 plays a role in synaptic specificity and/or synaptic maturation and 2) Pcdh‐γC5 is involved in neuron–neuron synaptic interactions and in neuron–astrocyte interactions, including perisynaptic neuron–astrocyte interactions. J. Comp. Neurol. 518:3439–3463, 2010. © 2010 Wiley‐Liss, Inc.     

Disruption of neuronal migration by RNAi of Dyx1c1 results in neocortical and hippocampal malformations

The brains of individuals with developmental dyslexia have neocortical neuronal migration abnormalities including molecular layer heterotopias, laminar dysplasias, and periventricular nodular heterotopias (PNH). RNA interference (RNAi) of Dyx1c1, a candidate dyslexia susceptibility gene, disrupts neuronal migration in developing embryonic neocortex. Using in utero electroporation, we cotransfected cells in the rat neocortical ventricular zone (VZ) at E14/15 with short hairpin RNA vectors targeting Dyx1c1 along with either plasmids encoding enhanced green fluorescent protein or plasmids encoding monomeric red fluorescent protein only. RNAi of Dyx1c1 resulted in pockets of unmigrated neurons resembling PNH. The pattern of migration of transfected neurons was bimodal, with approximately 20% of the neurons migrating a short distance from the VZ and another 40% that migrated past their expected lamina. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Molecular layer ectopias, which were not related to injection site artifacts, were also seen in 25% of the animals. These results support the hypothesis that targeted disruption of the candidate dyslexia susceptibility gene, Dyx1c1, results in neuronal migration disorders similar to those seen in the brains of dyslexics.     

The effects of Kiaa0319 knockdown on cortical and subcortical anatomy in male rats

Developmental dyslexia is a disorder characterized by a specific deficit in reading despite adequate overall intelligence and educational resources. The neurological substrate underlying these significant behavioral impairments is not known. Studies of post mortem brain tissue from male and female dyslexic individuals revealed focal disruptions of neuronal migration concentrated in the left hemisphere, along with aberrant symmetry of the right and left the planum temporale, and changes in cell size distribution within the medial geniculate nucleus of the thalamus ( 0100 and 0130). More recent neuroimaging studies have identified several changes in the brains of dyslexic individuals, including regional changes in gray matter, changes in white matter, and changes in patterns of functional activation. In a further effort to elucidate the etiology of dyslexia, epidemiological and genetic studies have identified several candidate dyslexia susceptibility genes. Some recent work has investigated associations between some of these genetic variants and structural changes in the brain. Variants of one candidate dyslexia susceptibility gene, KIAA0319, have been linked to morphological changes in the cerebellum and functional activational changes in the superior temporal sulcus ( 0140 and 0250). Animal models have been used to create a knockdown of Kiaa0319 (the rodent homolog of the human gene) via in utero RNA interference in order to study the gene's effects on brain development and behavior. Studies using this animal model have demonstrated that knocking down the gene leads to focal disruptions of neuronal migration in the form of ectopias and heterotopias, similar to those observed in the brains of human dyslexics. However, further changes to the structure of the brain have not been studied following this genetic disruption. The current study sought to determine the effects of embryonic Kiaa0319 knockdown on volume of the cortex and hippocampus, as well as midsagittal area of the corpus callosum in male rats. Results demonstrate that Kiaa0319 knockdown did not change the volume of the cortex or hippocampus, but did result in a significant reduction in the midsagittal area of the corpus callosum. Taken in the context of previous reports of behavioral deficits following Kiaa0319 knockdown ( Szalkowski et al., 2012), and reports that reductions of corpus callosum size are related to processing deficits in humans ( Paul, 2011), these results suggest that Kiaa0319 has a specific involvement in neural systems important for temporal processing.     

In vivo clonal overexpression of neuroligin 3 and neuroligin 2 in neurons of the rat cerebral cortex: Differential effects on GABAergic synapses and neuronal migration

We studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vesicular gamma‐aminobutyric acid (GABA) transporter (vGAT) and glutamic acid decarboxylase (GAD)65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent. In contrast, overexpression of NL3 or NL2 after IUEP does not affect vesicular glutamate transporter 1 (vGlut1) in the glutamatergic contacts that the NL3 or NL2‐overexpressing neurons receive. The NL3 or NL2‐overexpressing neurons do not show increased innervation by parvalbumin‐containing GABAergic terminals or increased parvalbumin in the same terminals that show increased vGAT. These results indicate that the observed increase in vGAT and GAD65 is not due to increased GABAergic innervation but to increased expression of vGAT and GAD65 in the GABAergic contacts that NL3 or NL2‐overexpressing neurons receive. The majority of bright vGAT puncta contacting the NL3‐overexpressing neurons have no gephyrin juxtaposed to them, indicating that many of these contacts are nonsynaptic. This contrasts with the majority of the NL2‐overexpressing neurons, which show plenty of synaptic gephyrin clusters juxtaposed to vGAT. Besides having an effect on GABAergic contacts, overexpression of NL3 interferes with the neuronal radial migration, in the cerebral cortex, of the neurons overexpressing NL3. J. Comp. Neurol. 523:1359–1378, 2015. © 2015 Wiley Periodicals, Inc.     

In vivo transgenic expression of collybistin in neurons of the rat cerebral cortex

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ‐aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABA_A receptors (GABA_ARs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CB_SH3? or CB_SH3+ in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2_SH3? or CB2_SH3+ in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CB_SH3? or CB_SH3+ DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291–1311, 2017. © 2016 Wiley Periodicals, Inc.     

Increasing doublecortin expression promotes migration of human embryonic stem cell-derived neurons

Human embryonic stem cell-derived neuronal progenitors (hNPs) provide a potential source for cellular replacement following neurodegenerative diseases. One of the greatest challenges for future neuron replacement therapies will be to control extensive cell proliferation and stimulate cell migration of transplanted cells. The doublecortin (DCX) gene encodes the protein DCX, a microtubule-associated protein essential for the migration of neurons in the human brain. In this study, we tested whether increasing the expression of DCX in hNPs would favorably alter their proliferation and migration. Migration and proliferation of hNPs was compared between hNPs expressing a bicistronic DCX/IRES-GFP transgene and those expressing a green fluorescent protein (GFP) transgene introduced by piggyBac-mediated transposition. The DCX-transfected hNPs showed a significant decrease in their proliferation and migrated significantly further on two different substrates, Matrigel and brain slices. Additionally, a dense network of nestin-positive (+) and vimentin+ fibers were found to extend from neurospheres transplanted onto brain slices, and this fiber growth was increased from neurospheres containing DCX-transfected hNPs. In summary, our results show that increased DCX expression inhibits proliferation and promotes migration of hNPs. Stem Cells2012;30:1852-1862.     

How long does it take to diagnose young-onset dementia? A comparison with late-onset dementia

Neurological Sciences - Dementia occurring in young people may be difficult to recognize. We compared the time to diagnosis between young- (YOD, age < 65) and late-onset dementia...     

Pure word deafness: a case report of an atypical manifestation of Alzheimer’s disease

Neurological Sciences - Auditory agnosia refers to the impairments in sound recognition despite intact hearing and written language abilities. When auditory agnosia is specific to spoken language,...