Effective knockdown-replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.

Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons-which resulted in growth delay and lethal seizures evident by postnatal week three-and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA.

Hyperactivity of mTORC1- and mTORC2-dependent signaling mediates epilepsy downstream of somatic PTEN loss.

Gene variants that hyperactivate PI3K-mTOR signaling in the brain lead to epilepsy and cortical malformations in humans. Some gene variants associated with these pathologies only hyperactivate mTORC1, but others, such as , , and , hyperactivate both mTORC1- and mTORC2-dependent signaling. Previous work established a key role for mTORC1 hyperactivity in mTORopathies, however, whether mTORC2 hyperactivity contributes is not clear.

Heterozygous expression of a gain-of-function variant has differential effects on SST- and PV-expressing cortical GABAergic neurons.

More than twenty recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (K) channel gene in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a GOF variant (Y777H) on K currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant GOF variants in DEEs.

Hyperactivity of mTORC1 and mTORC2-dependent signaling mediate epilepsy downstream of somatic PTEN loss.

Gene variants that hyperactivate PI3K-mTOR signaling in the brain lead to epilepsy and cortical malformations in humans. Some gene variants associated with these pathologies only hyperactivate mTORC1, but others, such as , , and , hyperactivate both mTORC1- and mTORC2-dependent signaling. Previous work established a key role for mTORC1 hyperactivity in mTORopathies, however, whether mTORC2 hyperactivity contributes is not clear.

Distinct Features of Interictal Activity Predict Seizure Localization and Burden in a Mouse Model of Childhood Epilepsy.

The epileptic brain is distinguished by spontaneous seizures and interictal epileptiform discharges (IEDs). Basic patterns of mesoscale brain activity outside of seizures and IEDs are also frequently disrupted in the epileptic brain and likely influence disease symptoms, but are poorly understood. We aimed to quantify how interictal brain activity differs from that in healthy individuals, and identify what features of interictal activity influence seizure occurrence in a genetic mouse model of childhood epilepsy.

mTORC2 Inhibition Improves Morphological Effects of PTEN Loss, But Does Not Correct Synaptic Dysfunction or Prevent Seizures.

Hyperactivation of PI3K/PTEN-mTOR signaling during neural development is associated with focal cortical dysplasia (FCD), autism, and epilepsy. mTOR can signal through two major hubs, mTORC1 and mTORC2, both of which are hyperactive following PTEN loss of function (LOF). Here, we tested the hypothesis that genetic inactivation of the mTORC2 complex via deletion of is sufficient to rescue morphologic and electrophysiological abnormalities in the dentate gyrus caused by PTEN loss, as well as generalized seizures.

Disruption of mTORC1 rescues neuronal overgrowth and synapse function dysregulated by Pten loss.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of AKT/mTOR signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten loss results in neuronal hypertrophy, hyperexcitability, seizures, and ASD-like behaviors.

Altered Fast Synaptic Transmission in a Mouse Model of DNM1-Associated Developmental Epileptic Encephalopathy.

Developmental epileptic encephalopathies (DEEs) are severe seizure disorders that occur in infants and young children, characterized by developmental delay, cognitive decline, and early mortality. Recent efforts have identified a wide variety of genetic variants that cause DEEs. Among these, variants in the gene have emerged as definitive causes of DEEs, including infantile spasms and Lennox-Gastaut syndrome.

Reduced GABAergic Neuron Excitability, Altered Synaptic Connectivity, and Seizures in a KCNT1 Gain-of-Function Mouse Model of Childhood Epilepsy.

Gain-of-function (GOF) variants in K channels cause severe childhood epilepsies, but there are no mechanisms to explain how increased K currents lead to network hyperexcitability. Here, we introduce a human Na-activated K (K) channel variant (KCNT1-Y796H) into mice and, using a multiplatform approach, find motor cortex hyperexcitability and early-onset seizures, phenotypes strikingly similar to those of human patients. Although the variant increases K currents in cortical excitatory and inhibitory neurons, there is an increase in the K current across subthreshold voltages only in inhibitory neurons, particularly in those with non-fast-spiking properties, resulting in inhibitory-neuron-specific impairments in excitability and action potential (AP) generation.

Genetic inactivation of mTORC1 or mTORC2 in neurons reveals distinct functions in glutamatergic synaptic transmission.

Although mTOR signaling is known as a broad regulator of cell growth and proliferation, in neurons it regulates synaptic transmission, which is thought to be a major mechanism through which altered mTOR signaling leads to neurological disease. Although previous studies have delineated postsynaptic roles for mTOR, whether it regulates presynaptic function is largely unknown. Moreover, the mTOR kinase operates in two complexes, mTORC1 and mTORC2, suggesting that mTOR's role in synaptic transmission may be complex-specific.

Altered excitatory transmission onto hippocampal interneurons in the IQSEC2 mouse model of X-linked neurodevelopmental disease.

Mutations in the X-linked gene IQSEC2 are associated with multiple cases of epilepsy, epileptic encephalopathy, intellectual disability and autism spectrum disorder, the mechanistic understanding and successful treatment of which remain a significant challenge in IQSEC2 and related neurodevelopmental genetic diseases. To investigate disease etiology, we studied behaviors and synaptic function in IQSEC2 deficient mice. Hemizygous Iqsec2 null males exhibit growth deficits, hyperambulation and hyperanxiety phenotypes.

PTEN Loss Increases the Connectivity of Fast Synaptic Motifs and Functional Connectivity in a Developing Hippocampal Network.

Changes in synaptic strength and connectivity are thought to be a major mechanism through which many gene variants cause neurological disease. Hyperactivation of the PI3K-mTOR signaling network, via loss of function of repressors such as PTEN, causes epilepsy in humans and animal models, and altered mTOR signaling may contribute to a broad range of neurological diseases. Changes in synaptic transmission have been reported in animal models of PTEN loss; however, the full extent of these changes, and their effect on network function, is still unknown.

Identification of diverse astrocyte populations and their malignant analogs.

Astrocytes are the most abundant cell type in the brain, where they perform a wide array of functions, yet the nature of their cellular heterogeneity and how it oversees these diverse roles remains shrouded in mystery. Using an intersectional fluorescence-activated cell sorting-based strategy, we identified five distinct astrocyte subpopulations present across three brain regions that show extensive molecular diversity. Application of this molecular insight toward function revealed that these populations differentially support synaptogenesis between neurons.

PTEN is required to maintain luminal epithelial homeostasis and integrity in the adult mammary gland.

In the mammary gland, PTEN loss in luminal and basal epithelial cells results in differentiation defects and enhanced proliferation, leading to the formation of tumors with basal epithelial characteristics. In breast cancer, PTEN loss is associated with a hormone receptor-negative, basal-like subtype that is thought to originate in a luminal epithelial cell. Here, we show that luminal-specific PTEN loss results in distinct effects on epithelial homeostasis and mammary tumor formation.