Reversing frontal disinhibition rescues behavioural deficits in models of CACNA1A-associated neurodevelopment disorders.

CACNA1A deletions cause epilepsy, ataxia, and a range of neurocognitive deficits, including inattention, impulsivity, intellectual deficiency and autism. To investigate the underlying mechanisms, we generated mice carrying a targeted Cacna1a deletion restricted to parvalbumin-expressing (PV) neurons (PVCre;Cacna1ac/+) or to cortical pyramidal cells (PC) (Emx1Cre;Cacna1ac/+). GABA release from PV-expressing GABAergic interneurons (PV-INs) is reduced in PVCre;Cacna1ac/+ mutants, resulting in impulsivity, cognitive rigidity and inattention. By contrast, the deletion of Cacna1a in PCs does not impact cortical excitability or behaviour in Emx1Cre;Cacna1ac/+ mutants. A targeted Cacna1a deletion in the orbitofrontal cortex (OFC) results in reversal learning deficits while a medial prefrontal cortex (mPFC) deletion impairs selective attention. These deficits can be rescued by the selective chemogenetic activation of cortical PV-INs in the OFC or mPFC of PVCre;Cacna1ac/+ mutants. Thus, Cacna1a haploinsufficiency disrupts perisomatic inhibition in frontal cortical circuits, leading to a range of potentially reversible neurocognitive deficits.

Horner Syndrome in Birds of Prey.

Horner syndrome, which results from oculosympathetic denervation, has rarely been reported in birds. A retrospective study was conducted in a raptor rehabilitation program to gain further insight into Horner syndrome in birds. Data from 5128 live raptors admitted over 20 years were reviewed. Horner syndrome was identified in 22 individuals from 13 different species. Raptors from the orders Strigiformes, Accipitriformes, and Falconiformes were affected, with the last order being underrepresented. Ptosis of the upper eyelid was the most commonly reported clinical sign in the birds diagnosed with Horner syndrome, having been noted in 21 cases. Signs of traumatic injury, such as fractures, wounds, and hematomas, were documented in 18 cases. Among the 22 cases, 12 birds were euthanatized, 3 died in treatment, and 7 were successfully released back into the wild.

Both gain-of-function and loss-of-function de novo CACNA1A mutations cause severe developmental epileptic encephalopathies in the spectrum of Lennox-Gastaut syndrome.

OBJECTIVE: Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene. METHODS: We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on CaV 2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling. RESULTS: We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M. SIGNIFICANCE: Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies.

Remodeled cortical inhibition prevents motor seizures in generalized epilepsy.

OBJECTIVE: Deletions of CACNA1A, encoding the α1 subunit of CaV 2.1 channels, cause epilepsy with ataxia in humans. Whereas the deletion of Cacna1a in γ-aminobutyric acidergic (GABAergic) interneurons (INs) derived from the medial ganglionic eminence (MGE) impairs cortical inhibition and causes generalized seizures in Nkx2.1Cre ;Cacna1ac/c mice, the targeted deletion of Cacna1a in somatostatin-expressing INs (SOM-INs), a subset of MGE-derived INs, does not result in seizures, indicating a crucial role of parvalbumin-expressing (PV) INs. Here we identify the cellular and network consequences of Cacna1a deletion specifically in PV-INs. METHODS: We generated PVCre ;Cacna1ac/c mutant mice carrying a conditional Cacna1a deletion in PV neurons and evaluated the cortical cellular and network outcomes of this mutation by combining immunohistochemical assays, in vitro electrophysiology, 2-photon imaging, and in vivo video-electroencephalographic recordings. RESULTS: PVCre ;Cacna1ac/c mice display reduced cortical perisomatic inhibition and frequent absences but only rare motor seizures. Compared to Nkx2.1Cre ;Cacna1ac/c mice, PVCre ;Cacna1ac/c mice have a net increase in cortical inhibition, with a gain of dendritic inhibition through sprouting of SOM-IN axons, largely preventing motor seizures. This beneficial compensatory remodeling of cortical GABAergic innervation is mTORC1-dependent and its inhibition with rapamycin leads to a striking increase in motor seizures. Furthermore, we show that a direct chemogenic activation of cortical SOM-INs prevents motor seizures in a model of kainate-induced seizures. INTERPRETATION: Our findings provide novel evidence suggesting that the remodeling of cortical inhibition, with an mTOR-dependent gain of dendritic inhibition, determines the seizure phenotype in generalized epilepsy and that mTOR inhibition can be detrimental in epilepsies not primarily due to mTOR hyperactivation. Ann Neurol 2018;84:436-451.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Stimulation-induced ectopicity and propagation windows in model damaged axons.

Neural tissue injuries render voltage-gated Na+ channels (Nav) leaky, thereby altering excitability, disrupting propagation and causing neuropathic pain related ectopic activity. In both recombinant systems and native excitable membranes, membrane damage causes the kinetically-coupled activation and inactivation processes of Nav channels to undergo hyperpolarizing shifts. This damage-intensity dependent change, called coupled left-shift (CLS), yields a persistent or "subthreshold" Nav window conductance. Nodes of Ranvier simulations involving various degrees of mild CLS showed that, as the system's channel/pump fluxes attempt to re-establish ion homeostasis, the CLS elicits hyperexcitability, subthreshold oscillations and neuropathic type action potential (AP) bursts. CLS-induced intermittent propagation failure was studied in simulations of stimulated axons, but pump contributions were ignored, leaving open an important question: does mild-injury (small CLS values, pumps functioning well) render propagation-competent but still quiescent axons vulnerable to further impairments as the system attempts to cope with its normal excitatory inputs? We probe this incipient diffuse axonal injury scenario using a 10-node myelinated axon model. Fully restabilized nodes with mild damage can, we show, become ectopic signal generators ("ectopic nodes") because incoming APs stress Na+ / K+ gradients, thereby altering spike thresholds. Comparable changes could contribute to acquired sodium channelopathies as diverse as epileptic phenomena and to the neuropathic amplification of normally benign sensory inputs. Input spike patterns, we found, propagate with good fidelity through an ectopically firing site only when their frequencies exceed the ectopic frequency. This "propagation window" is a robust phenomenon, occurring despite Gaussian noise, large jitter and the presence of several consecutive ectopic nodes.

The genetic landscape of infantile spasms.

Infantile spasms (IS) is an early-onset epileptic encephalopathy of unknown etiology in ∼40% of patients. We hypothesized that unexplained IS cases represent a large collection of rare single-gene disorders. We investigated 44 children with unexplained IS using comparative genomic hybridisation arrays (aCGH) (n = 44) followed by targeted sequencing of 35 known epilepsy genes (n = 8) or whole-exome sequencing (WES) of familial trios (n = 18) to search for rare inherited or de novo mutations. aCGH analysis revealed de novo variants in 7% of patients (n = 3/44), including a distal 16p11.2 duplication, a 15q11.1q13.1 tetrasomy and a 2q21.3-q22.2 deletion. Furthermore, it identified a pathogenic maternally inherited Xp11.2 duplication. Targeted sequencing was informative for ARX (n = 1/14) and STXBP1 (n = 1/8). In contrast, sequencing of a panel of 35 known epileptic encephalopathy genes (n = 8) did not identify further mutations. Finally, WES (n = 18) was very informative, with an excess of de novo mutations identified in genes predicted to be involved in neurodevelopmental processes and/or known to be intolerant to functional variations. Several pathogenic mutations were identified, including de novo mutations in STXBP1, CASK and ALG13, as well as recessive mutations in PNPO and ADSL, together explaining 28% of cases (5/18). In addition, WES identified 1-3 de novo variants in 64% of remaining probands, pointing to several interesting candidate genes. Our results indicate that IS are genetically heterogeneous with a major contribution of de novo mutations and that WES is significantly superior to targeted re-sequencing in identifying detrimental genetic variants involved in IS.

Touch responsiveness in zebrafish requires voltage-gated calcium channel 2.1b.

The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish.

A mutation in the RNF170 gene causes autosomal dominant sensory ataxia.

Autosomal dominant sensory ataxia is a rare genetic condition that results in a progressive ataxia that is caused by degeneration of the posterior columns of the spinal cord. To date only two families have been clinically ascertained with this condition, both from Maritime Canada. We previously mapped both families to chromosome 8p12-8q12 and have now screened the majority of annotated protein-coding genes in the shared haplotype region by direct DNA sequencing. We have identified a putative pathogenic mutation in the gene encoding ring-finger protein RNF170, a potential ubiquitin ligase. This mutation is a rare non-synonymous change in a well-conserved residue and is predicted to be pathogenic by SIFT, PolyPhen, PANTHER and Align-GVD. Microinjection of wild-type or mutant orthologous messenger RNAs into zebrafish (Danio rerio) embryos confirmed that the mutation dominantly disrupts normal embryonic development. Together these results suggest that the mutation in RNF170 is causal for the sensory ataxia in these families.

Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia.

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts--bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.