Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

APC sets the Wnt tone necessary for cerebral cortical progenitor development.

Adenomatous polyposis coli (APC) regulates the activity of ß-catenin, an integral component of Wnt signaling. However, the selective role of the APC-ß-catenin pathway in cerebral cortical development is unknown. Here we genetically dissected the relative contributions of APC-regulated ß-catenin signaling in cortical progenitor development, a necessary early step in cerebral cortical formation. Radial progenitor-specific inactivation of the APC-ß-catenin pathway indicates that the maintenance of appropriate ß-catenin-mediated Wnt tone is necessary for the orderly differentiation of cortical progenitors and the resultant formation of the cerebral cortex. APC deletion deregulates ß-catenin, leads to high Wnt tone, and disrupts Notch1 signaling and primary cilium maintenance necessary for radial progenitor functions. ß-Catenin deregulation directly disrupts cilium maintenance and signaling via Tulp3, essential for intraflagellar transport of ciliary signaling receptors. Surprisingly, deletion of ß-catenin or inhibition of ß-catenin activity in APC-null progenitors rescues the APC-null phenotype. These results reveal that APC-regulated ß-catenin activity in cortical progenitors sets the appropriate Wnt tone necessary for normal cerebral cortical development.

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex.

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.

Disrupted-in-Schizophrenia-1 expression is regulated by beta-site amyloid precursor protein cleaving enzyme-1-neuregulin cascade.

Neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia-1 (DISC1) are promising susceptibility factors for schizophrenia. Both are multifunctional proteins with roles in a variety of neurodevelopmental processes, including progenitor cell proliferation, migration, and differentiation. Here, we provide evidence linking these factors together in a single pathway, which is mediated by ErbB receptors and PI3K/Akt. We show that signaling by NRG1 and NRG2, but not NRG3, increase expression of an isoform of DISC1 in vitro. Receptors ErbB2 and ErbB3, but not ErbB4, are responsible for transducing this effect, and PI3K/Akt signaling is also required. In NRG1 knockout mice, this DISC1 isoform is selectively reduced during neurodevelopment. Furthermore, a similar decrease in DISC1 expression is seen in beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) knockout mice, in which NRG1/Akt signaling is reportedly impaired. In contrast to neuronal DISC1 that was reported and characterized, expression of DISC1 in other types of cells in the brain has not been addressed. Here we demonstrate that DISC1, like NRG and ErbB proteins, is expressed in neurons, astrocytes, oligodendrocytes, microglia, and radial progenitors. These findings may connect NRG1, ErbBs, Akt, and DISC1 in a common pathway, which may regulate neurodevelopment and contribute to susceptibility to schizophrenia.

A Comprehensive Analysis of Cerebellar Volumes in the 22q11.2 Deletion Syndrome.

BACKGROUND: The presence of a 22q11.2 microdeletion (22q11.2 deletion syndrome [22q11DS]) ranks among the greatest known genetic risk factors for the development of psychotic disorders. There is emerging evidence that the cerebellum is important in the pathophysiology of psychosis. However, there is currently limited information on cerebellar neuroanatomy in 22q11DS specifically. METHODS: High-resolution 3T magnetic resonance imaging was acquired in 79 individuals with 22q11DS and 70 typically developing control subjects (N = 149). Lobar and lobule-level cerebellar volumes were estimated using validated automated segmentation algorithms, and subsequently group differences were compared. Hierarchical clustering, principal component analysis, and graph theoretical models were used to explore intercerebellar relationships. Cerebrocerebellar structural connectivity with cortical thickness was examined via linear regression models. RESULTS: Individuals with 22q11DS had, on average, 17.3% smaller total cerebellar volumes relative to typically developing subjects (p < .0001). The lobules of the superior posterior cerebellum (e.g., VII and VIII) were particularly affected in 22q11DS. However, all cerebellar lobules were significantly smaller, even after adjusting for total brain volumes (all cerebellar lobules p < .0002). The superior posterior lobule was disproportionately associated with cortical thickness in the frontal lobes and cingulate cortex, brain regions known be affected in 22q11DS. Exploratory analyses suggested that the superior posterior lobule, particularly Crus I, may be associated with psychotic symptoms in 22q11DS. CONCLUSIONS: The cerebellum is a critical but understudied component of the 22q11DS neuroendophenotype.

Glycogen synthase kinase-3 regulates endoplasmic reticulum (ER) stress-induced CHOP expression in neuronal cells.

Endoplasmic reticulum (ER) stress, often resulting from cellular accumulation of misfolded proteins, occurs in many neurodegenerative disorders, in part because of the relatively long lifetime of neurons. Excessive accumulation of misfolded proteins activates the unfolded protein response (UPR) that dampens protein synthesis and promotes removal of misfolded proteins to support survival of ER-stressed cells. However, the UPR also initiates apoptotic signaling to kill cells if recovery is not achieved. Thus, there is much interest in identifying determinants of the life-death switch and interventions that promote recovery and survival. One intervention that has consistently been shown to protect cells from ER stress-induced apoptosis is application of inhibitors of glycogen synthase kinase-3 (GSK3). Therefore, we examined where in the UPR pathway GSK3 inhibitors intercede to impede signaling towards apoptosis. Apoptosis following UPR activation can be mediated by activation of two transcription factors, ATF4 and ATF6, that activate expression of the death-inducing transcription factor C/EBP homologous protein (CHOP/GADD153) following ER stress. We found that ER stress activated ATF6 and ATF4, but these responses were not inhibited by pretreatment with GSK3 inhibitors. However, inhibition of GSK3 effectively reduced the expression of CHOP, and this was apparent in several types of neural-related cells and was evident after application of several structurally diverse GSK3 inhibitors. Therefore, reduction of CHOP activation provides one mechanism by which inhibitors of GSK3 are capable of shifting cell fate towards survival instead of apoptosis following ER stress.

An End-To-End Pipeline for Fully Automatic Morphological Quantification of Mouse Brain Structures From MRI Imagery.

Segmentation of mouse brain magnetic resonance images (MRI) based on anatomical and/or functional features is an important step towards morphogenetic brain structure characterization of murine models in neurobiological studies. State-of-the-art image segmentation methods register image volumes to standard presegmented templates or well-characterized highly detailed image atlases. Performance of these methods depends critically on the quality of skull-stripping, which is the digital removal of tissue signal exterior to the brain. This is, however, tedious to do manually and challenging to automate. Registration-based segmentation, in addition, performs poorly on small structures, low resolution images, weak signals, or faint boundaries, intrinsic to in vivo MRI scans. To address these issues, we developed an automated end-to-end pipeline called DeepBrainIPP (deep learning-based brain image processing pipeline) for 1) isolating brain volumes by stripping skull and tissue from T2w MRI images using an improved deep learning-based skull-stripping and data augmentation strategy, which enables segmentation of large brain regions by atlas or template registration, and 2) address segmentation of small brain structures, such as the paraflocculus, a small lobule of the cerebellum, for which DeepBrainIPP performs direct segmentation with a dedicated model, producing results superior to the skull-stripping/atlas-registration paradigm. We demonstrate our approach on data from both in vivo and ex vivo samples, using an in-house dataset of 172 images, expanded to 4,040 samples through data augmentation. Our skull stripping model produced an average Dice score of 0.96 and residual volume of 2.18%. This facilitated automatic registration of the skull-stripped brain to an atlas yielding an average cross-correlation of 0.98. For small brain structures, direct segmentation yielded an average Dice score of 0.89 and 5.32% residual volume error, well below the tolerance threshold for phenotype detection. Full pipeline execution is provided to non-expert users via a Web-based interface, which exposes analysis parameters, and is powered by a service that manages job submission, monitors job status and provides job history. Usability, reliability, and user experience of DeepBrainIPP was measured using the Customer Satisfaction Score (CSAT) and a modified PYTHEIA Scale, with a rating of excellent. DeepBrainIPP code, documentation and network weights are freely available to the research community.

Schizophrenia-related microdeletion causes defective ciliary motility and brain ventricle enlargement via microRNA-dependent mechanisms in mice.

Progressive ventricular enlargement, a key feature of several neurologic and psychiatric diseases, is mediated by unknown mechanisms. Here, using murine models of 22q11-deletion syndrome (22q11DS), which is associated with schizophrenia in humans, we found progressive enlargement of lateral and third ventricles and deceleration of ciliary beating on ependymal cells lining the ventricular walls. The cilia-beating deficit observed in brain slices and in vivo is caused by elevated levels of dopamine receptors (Drd1), which are expressed in motile cilia. Haploinsufficiency of the microRNA-processing gene Dgcr8 results in Drd1 elevation, which is brought about by a reduction in Drd1-targeting microRNAs miR-382-3p and miR-674-3p. Replenishing either microRNA in 22q11DS mice normalizes ciliary beating and ventricular size. Knocking down the microRNAs or deleting their seed sites on Drd1 mimicked the cilia-beating and ventricular deficits. These results suggest that the Dgcr8-miR-382-3p/miR-674-3p-Drd1 mechanism contributes to deceleration of ciliary motility and age-dependent ventricular enlargement in 22q11DS.

GSK3 beta N-terminus binding to p53 promotes its acetylation.

The prevalence in human cancers of mutations in p53 exemplifies its crucial role as a tumor suppressor transcription factor. Previous studies have shown that the constitutively active serine/threonine kinase glycogen synthase kinase-3beta (GSK3 beta) associates with the C-terminal basic domain of p53 and regulates its actions. In this study we identified the GSK3 beta N-terminal amino acids 78-92 as necessary for its association with p53. Inhibitors of GSK3 impaired the acetylation of p53 at Lys373 and Lys382 near the GSK3 beta binding region in p53, indicating that GSK3 beta facilitates p53 acetylation. We also found that acetylation of p53 reduced its association with GSK3 beta, as well as with GSK3alpha. These results indicate that the N-terminal region of GSK3 beta binds p53, this association promotes the acetylation of p53, and subsequently acetylated p53 dissociates from GSK3.

Schizophrenia-Related Microdeletion Impairs Emotional Memory through MicroRNA-Dependent Disruption of Thalamic Inputs to the Amygdala.

Individuals with 22q11.2 deletion syndrome (22q11DS) are at high risk of developing psychiatric diseases such as schizophrenia. Individuals with 22q11DS and schizophrenia are impaired in emotional memory, anticipating, recalling, and assigning a correct context to emotions. The neuronal circuits responsible for these emotional memory deficits are unknown. Here, we show that 22q11DS mouse models have disrupted synaptic transmission at thalamic inputs to the lateral amygdala (thalamo-LA projections). This synaptic deficit is caused by haploinsufficiency of the 22q11DS gene Dgcr8, which is involved in microRNA processing, and is mediated by the increased dopamine receptor Drd2 levels in the thalamus and by reduced probability of glutamate release from thalamic inputs. This deficit in thalamo-LA synaptic transmission is sufficient to cause fear memory deficits. Our results suggest that dysregulation of the Dgcr8-Drd2 mechanism at thalamic inputs to the amygdala underlies emotional memory deficits in 22q11DS.

Restoring auditory cortex plasticity in adult mice by restricting thalamic adenosine signaling.

Circuits in the auditory cortex are highly susceptible to acoustic influences during an early postnatal critical period. The auditory cortex selectively expands neural representations of enriched acoustic stimuli, a process important for human language acquisition. Adults lack this plasticity. Here we show in the murine auditory cortex that juvenile plasticity can be reestablished in adulthood if acoustic stimuli are paired with disruption of ecto-5'-nucleotidase-dependent adenosine production or A1-adenosine receptor signaling in the auditory thalamus. This plasticity occurs at the level of cortical maps and individual neurons in the auditory cortex of awake adult mice and is associated with long-term improvement of tone-discrimination abilities. We conclude that, in adult mice, disrupting adenosine signaling in the thalamus rejuvenates plasticity in the auditory cortex and improves auditory perception.

Hypoxia activates glycogen synthase kinase-3 in mouse brain in vivo: protection by mood stabilizers and imipramine.

BACKGROUND: Glycogen synthase kinase-3 (GSK3), which is primarily regulated by an inhibitory phosphorylation of an N-terminal serine, has been implicated as contributing to mood disorders by the finding that it is inhibited by the mood stabilizer lithium. METHODS: This study tested if the antidepressant imipramine or the mood stabilizers lithium and sodium valproate regulated pathophysiological serine-dephosphorylation of GSK3 caused by hypoxia in mouse brain in vivo. RESULTS: Hypoxia caused rapid serine-dephosphorylation of both isoforms of GSK3, GSK3beta and GSK3alpha, in mouse cerebral cortex, hippocampus, and striatum. Pretreatment of mice with imipramine, sodium valproate, or lithium attenuated hypoxia-induced serine-dephosphorylation of GSK3beta and GSK3alpha in all three brain regions. CONCLUSIONS: These results demonstrate that imipramine and mood stabilizers are capable of blocking pathophysiologically induced serine-dephosphorylation of GSK3, supporting the hypothesis that stabilization of serine-phosphorylation of GSK3 contributes to their therapeutic effects.

Differential regulation of microtubule severing by APC underlies distinct patterns of projection neuron and interneuron migration.

Coordinated migration of distinct classes of neurons to appropriate positions leads to the formation of functional neuronal circuitry in the cerebral cortex. The two major classes of cortical neurons, interneurons and projection neurons, utilize distinctly different modes (radial versus tangential) and routes of migration to arrive at their final positions in the cerebral cortex. Here, we show that adenomatous polyposis coli (APC) modulates microtubule (MT) severing in interneurons to facilitate tangential mode of interneuron migration, but not the glial-guided, radial migration of projection neurons. APC regulates the stability and activity of the MT-severing protein p60-katanin in interneurons to promote the rapid remodeling of neuronal processes necessary for interneuron migration. These findings reveal how severing and restructuring of MTs facilitate distinct modes of neuronal migration necessary for laminar organization of neurons in the developing cerebral cortex.

Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex.

Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia, we show that migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron's migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may in part underlie the neurological defects in Joubert syndrome patients.

Direct visualization of microtubules using a genetic tool to analyse radial progenitor-astrocyte continuum in brain.

Microtubule cytoskeletal dynamics of cortical progenitors and astroglial cells have critical roles in the emergence of normal functional organization of cerebral cortex and in disease processes such as tumorigenesis. However, tools to efficiently visualize these events are lacking. Here we describe a mouse genetic model to efficiently visualize and analyse radial progenitors, their astroglial progeny, and the microtubule cytoskeleton of these cells in the developing and adult brain. Using this tool, we demonstrate altered microtubule organization and capture dynamics in adenomatous polyposis coli-deficient radial progenitors. Further, using multiphoton microscopy, we show the utility of this tool in real-time imaging of astrocytes in living mouse brain and the short-term stable nature of astrocytes in cerebral cortex. Thus, this model will help explore the dynamics of radial progenitor/astrocyte development or dysfunction and the influence of microtubule functions during these events.

Going tubular in the rostral migratory stream: neurons remodel astrocyte tubes to promote directional migration in the adult brain.

Newly generated neuroblasts from the subventricular zone of the adult brain migrate as neuronal chains within a network of astroglial tubes in the rostral migratory stream. This highly directed, rapid migration channels new neurons to the olfactory bulb. In this issue of Neuron, Kaneko et al. demonstrate that migrating neurons dynamically remodel the morphology and organization of astroglial tubes to promote long distance, directional migration of neurons in the adult brain.

Blocked inhibitory serine-phosphorylation of glycogen synthase kinase-3alpha/beta impairs in vivo neural precursor cell proliferation.

BACKGROUND: Adult neurogenesis augments neuronal plasticity, and deficient neurogenesis might contribute to mood disorders and schizophrenia and impede treatment responses. Because these diseases might be associated with inadequately controlled glycogen synthase kinase-3 (GSK3), we tested whether blocked inhibitory serine-phosphorylation of GSK3 impairs neurogenesis. METHODS: Neural precursor cell (NPC) proliferation was measured by dentate gyrus bromodeoxyuridine (BrdU) labeling in GSK3alpha/beta(21A/21A/9A/9A) knockin mice with serine-to-alanine mutations to block inhibitory serine-phosphorylation of GSK3 while it remains within the physiological range, because GSK3 is not overexpressed. RESULTS: There was a drastic 40% impairment in neurogenesis in vivo in GSK3 knockin mice compared with wild-type mice. Impaired neurogenesis could be due to effects of GSK3 in NPCs or in surrounding cells that modulate NPCs. In vitro proliferation was equivalent for NPCs from GSK3 knockin and wild-type mice, suggesting an in vivo deficiency in GSK3 knockin mice of external support for NPC proliferation. Measurements of two neurotrophins that promote neurogenesis demonstrated less hippocampal vascular endothelial growth factor but not brain-derived growth factor in GSK3 knockin mice than wild-type mice, reinforcing the possibility that insufficient environmental support in GSK3 knockin mice might contribute to impaired neurogenesis. In vivo chronic co-administration of lithium and fluoxetine, which each increase inhibitory serine-phosphorylation of wild-type GSK3, increased NPC proliferation in wild-type but not GSK3 knockin mice. CONCLUSIONS: Blocked inhibitory control of GSK3 impaired neurogenesis and the capacity of therapeutic drugs to stimulate neurogenesis, likely through deficient environmental factors that support neurogenesis, which might contribute to psychiatric diseases and responses to therapeutic drugs.

Neural precursor cells are protected from apoptosis induced by trophic factor withdrawal or genotoxic stress by inhibitors of glycogen synthase kinase 3.

Mechanisms controlling the survival of neural precursor cells (NPCs) are critical during brain development, in adults for neuron replenishment, and after transplantation for neuron replacement. This investigation found that glycogen synthase kinase 3 (GSK3) promotes apoptotic signaling in cultured NPCs derived from embryonic mouse brain subjected to two common apoptotic conditions, trophic factor withdrawal and genotoxic stress. Trophic factor withdrawal activated GSK3 and the key apoptosis mediators Bax and caspase-3. Pharmacological inhibition of GSK3 activity produced dramatic reductions in the activation of Bax and caspase-3 and NPC death after trophic factor withdrawal. Trophic factor withdrawal-induced apoptosis was delayed in Bax knock-out NPCs, but GSK3 inhibitors provided additional protection. Genotoxic stress induced by camptothecin treatment of NPCs stabilized p53, which formed a complex with GSK3beta and activated Bax and caspase-3. Camptothecin-induced activation of caspase-3 was reduced by GSK3 inhibitors in both bax(+)(/)(+) and bax(-/-) NPCs. Thus, NPCs are sensitive to loss of trophic factors and genotoxic stress, and inhibitors of GSK3 are capable of enhancing NPC survival.