JNK signaling is required for proper tangential migration and laminar allocation of cortical interneurons.

The precise migration of cortical interneurons is essential for the formation and function of cortical circuits, and disruptions to this key developmental process are implicated in the etiology of complex neurodevelopmental disorders, including schizophrenia, autism and epilepsy. We have recently identified the Jun N-terminal kinase (JNK) pathway as an important mediator of cortical interneuron migration in mice, regulating the proper timing of interneuron arrival into the cortical rudiment. In the current study, we demonstrate a vital role for JNK signaling at later stages of corticogenesis, when interneurons transition from tangential to radial modes of migration. Pharmacological inhibition of JNK signaling in ex vivo slice cultures caused cortical interneurons to rapidly depart from migratory streams and prematurely enter the cortical plate. Similarly, genetic loss of JNK function led to precocious stream departure ex vivo, and stream disruption, morphological changes and abnormal allocation of cortical interneurons in vivo These data suggest that JNK signaling facilitates the tangential migration and laminar deposition of cortical interneurons, and further implicates the JNK pathway as an important regulator of cortical development.

PIK3R2/Pik3r2 Activating Mutations Result in Brain Overgrowth and EEG Changes.

OBJECTIVE: Mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) complex have been associated with a broad spectrum of brain and organ overgrowth syndromes. For example, mutations in phosphatidylinositol-3-kinase regulatory subunit 2 (PIK3R2) have been identified in human patients with megalencephaly polymicrogyria polydactyly hydrocephalus (MPPH) syndrome, which includes brain overgrowth. To better understand the pathogenesis of PIK3R2-related mutations, we have developed and characterized a murine model. METHODS: We generated a knock-in mouse model for the most common human PIK3R2 mutation, p.G373R (p.G367R in mice) using CRISPR/Cas9. The mouse phenotypes, including brain size, seizure activity, cortical lamination, cell proliferation/size/density, interneuron migration, and PI3K pathway activation, were analyzed using standard methodologies. For human patients with PIK3R2 mutations, clinical data (occipitofrontal circumference [OFC] and epilepsy) were retrospectively obtained from our clinical records (published / unpublished). RESULTS: The PI3K-AKT pathway was hyperactivated in these mice, confirming the p.G367R mutation is an activating mutation in vivo. Similar to human patients with PIK3R2 mutations, these mice have enlarged brains. We found cell size to be increased but not cell numbers. The embryonic brain showed mild defects in cortical lamination, although not observed in the mature brain. Furthermore, electroencephalogram (EEG) recordings from mutant mice showed background slowing and rare seizures, again similar to our observations in human patients. INTERPRETATION: We have generated a PIK3R2 mouse model that exhibits megalencephaly and EEG changes, both of which overlap with human patients. Our data provide novel insight into the pathogenesis of the human disease caused by PIK3R2 p.G373R mutation. We anticipate this model will be valuable in testing therapeutic options for human patients with MPPH. ANN NEUROL 2020;88:1077-1094.

Cortical interneurons require Jnk1 to enter and navigate the developing cerebral cortex.

Proper assembly of cortical circuitry relies on the correct migration of cortical interneurons from their place of birth in the ganglionic eminences to their place of terminal differentiation in the cerebral cortex. Although molecular mechanisms mediating cortical interneuron migration have been well studied, intracellular signals directing their migration are largely unknown. Here we illustrate a novel and essential role for c-Jun N-terminal kinase (JNK) signaling in guiding the pioneering population of cortical interneurons into the mouse cerebral cortex. Migrating cortical interneurons express Jnk proteins at the entrance to the cortical rudiment and have enriched expression of Jnk1 relative to noninterneuronal cortical cells. Pharmacological blockade of JNK signaling in ex vivo slice cultures resulted in dose-dependent and highly specific disruption of interneuron migration into the nascent cortex. Time-lapse imaging revealed that JNK-inhibited cortical interneurons advanced slowly and assumed aberrant migratory trajectories while traversing the cortical entry zone. In vivo analyses of JNK-deficient embryos supported our ex vivo pharmacological data. Deficits in interneuron migration were observed in Jnk1 but not Jnk2 single nulls, and those migratory deficits were further exacerbated when homozygous loss of Jnk1 was combined with heterozygous reduction of Jnk2. Finally, genetic ablation of Jnk1 and Jnk2 from cortical interneurons significantly perturbed migration in vivo, but not in vitro, suggesting JNK activity functions to direct their guidance rather than enhance their motility. These data suggest JNK signaling, predominantly mediated by interneuron expressed Jnk1, is required for guiding migration of cortical interneurons into and within the developing cerebral cortex.

Prenatal diagnosis of congenital femoral deficiency and fibular hemimelia.

OBJECTIVES: Routine ultrasonography can detect congenital femoral deficiency (CFD) and fibular hemimelia (FH), but prenatal detection rate and its relation to deformity severity have never been reported. Whether mothers prefer prenatal diagnosis is also unknown. We aimed to determine whether mothers prefer prenatal diagnosis, to report detection rates for CFD and/or FH, and to correlate detection rates to severity of limb shortening. METHODS: Surveys were mailed to 171 mothers who gave birth to children with CFD/FH between 2000 and 2008. Bilateral femoral and tibial lengths were measured on postnatal radiographs. We calculated corresponding femoral/tibial lengths at gestational weeks 20 and 30. RESULTS: Sixty-five surveys were returned, and 56 radiographs were reviewed. Most mothers (63%) preferred prenatal diagnosis as it enables prenatal counseling. Congenital limb shortening was detected in 24 cases (37%) and was not detected in 41 cases (63%). Detection rate was 52% (12 of 23) in CFD cases, 23% (three of 13) in FH cases, and 30% (six of 20) in combined cases. CFD cases with severe shortening had a higher detection rate. CONCLUSIONS: Ultrasonographers should measure both femoral and tibial lengths. Unilateral shortening should result in pediatric orthopedic consultation to estimate limb-length discrepancy at maturity and discuss treatment.

ARX regulates cortical interneuron differentiation and migration.

Mutations in aristaless-related homeobox ( ARX ) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARX's role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3 , Cspr3 ), fate specification (e.g., Nkx2.1 , Maf , Mef2c ), and migration (e.g., Nkx2.1 , Lmo1 , Cxcr4 , Nrg1 , ErbB4 ). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

Report of Sugar-Sweetened Beverages Offered in Pennsylvania Childcare Centers.

The study objective was to quantify sugar-sweetened beverage (SSB) offerings to children in Pennsylvania (PA) childcare centers and determine whether this information is communicated to parents. In October 2014, a SurveyMonkey link was sent to 4461 PA childcare centers. The 518 respondents represented 88% of PA counties. 279 centers (54%) serve SSBs. 330 (65%) of childcare centers provide parents a report of their child's daily intake. Of 185 centers serving SSBs and providing a daily intake report, 91% include SSB consumption. In total, 38% of centers (103/272) offer but do not report SSB consumption. In 96% of centers, parents may request their child not receive SSBs. In conclusion, though more than half of PA childcare centers surveyed offer SSBs, those providing daily intake reports usually include SSB consumption. Requiring daily intake reports may be a strategy to increase parental awareness of items consumed outside the home.

Neonatal conductive hearing loss disrupts the development of the Cat-315 epitope on perineuronal nets in the rat superior olivary complex.

The critical period is a postnatal window characterized by a high level of experience-dependent neuronal plasticity in the central nervous system and sensory deprivation during this period significantly impacts neurological function. Perineuronal nets (PNNs) are specialized aggregates of the extracellular matrix which ensheath neuronal cell bodies, primary dendrites and axon hillocks and function in neuronal protection and stabilize synapses. PNNs are generally not present at birth, but reach adult-like patterns by the end of the third or fourth postnatal week. Their appearance is believed to mark the close of the critical period and sensory deprivation during this epoch disrupts development of PNNs. Here we investigate the postnatal development of two PNN markers (Wisteria floribunda agglutinin [WFA] and Cat-315) and the effect of neonatal conductive hearing loss (CHL) on their development. Our data indicates that these PNN markers are not present in the superior olivary complex (SOC) at birth, but develop over the first four postnatal weeks in different temporal patterns and also that neonatal CHL results in a significant decrease in the number of SOC neurons associated with Cat-315 reactive PNNs.