NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination and seizure threshold.

Mutations in nitrogen permease regulator-like 3 (NPRL3), a component of the GATOR1 complex within the mTOR pathway, are associated with epilepsy and malformations of cortical development. Little is known about the effects of NPRL3 loss on neuronal mTOR signalling and morphology, or cerebral cortical development and seizure susceptibility. We report the clinical phenotypic spectrum of a founder NPRL3 pedigree (c.349delG, p.Glu117LysFS; n = 133) among Old Order Mennonites dating to 1727. Next, as a strategy to define the role of NPRL3 in cortical development, CRISPR/Cas9 Nprl3 knockout in Neuro2a cells in vitro and in foetal mouse brain in vivo was used to assess the effects of Nprl3 knockout on mTOR activation, subcellular mTOR localization, nutrient signalling, cell morphology and aggregation, cerebral cortical cytoarchitecture and network integrity. The NPRL3 pedigree exhibited an epilepsy penetrance of 28% and heterogeneous clinical phenotypes with a range of epilepsy semiologies, i.e. focal or generalized onset, brain imaging abnormalities, i.e. polymicrogyria, focal cortical dysplasia or normal imaging, and EEG findings, e.g. focal, multi-focal or generalized spikes, focal or generalized slowing. Whole exome analysis comparing a seizure-free group (n = 37) to those with epilepsy (n = 24) to search for gene modifiers for epilepsy did not identify a unique genetic modifier that explained the variability in seizure penetrance in this cohort. Nprl3 knockout in vitro caused mTOR pathway hyperactivation, cell soma enlargement and the formation of cellular aggregates seen in time-lapse videos that were prevented with the mTOR inhibitors rapamycin or torin1. In Nprl3 knockout cells, mTOR remained localized on the lysosome in a constitutively active conformation, as evidenced by phosphorylation of ribosomal S6 and 4E-BP1 proteins, even under nutrient starvation (amino acid-free) conditions, demonstrating that Nprl3 loss decouples mTOR activation from neuronal metabolic state. To model human malformations of cortical development associated with NPRL3 variants, we created a focal Nprl3 knockout in foetal mouse cortex by in utero electroporation and found altered cortical lamination and white matter heterotopic neurons, effects which were prevented with rapamycin treatment. EEG recordings showed network hyperexcitability and reduced seizure threshold to pentylenetetrazol treatment. NPRL3 variants are linked to a highly variable clinical phenotype which we propose results from mTOR-dependent effects on cell structure, cortical development and network organization.

The role of somatic mutational events in the pathogenesis of epilepsy.

PURPOSE OF REVIEW: There has been rapid progress in defining novel causative gene variants responsible for a large spectrum of human epilepsy syndromes and subtypes. Of particular interest is the discovery that somatic mutations, for example, noninherited mutations occurring in neuroglial progenitor cells during embryonic brain development, are highly linked to malformations of cortical development (MCD) such as focal cortical dysplasia (FCD) type II and hemimegalencephaly. RECENT FINDINGS: Somatic gene variants have been identified in genes encoding regulatory proteins within the mechanistic target of rapamycin (mTOR) signaling cascade and have thus comprised the group classified as mTORopathies. FCD II and hemimegalencephaly often result from mutations in identical genes suggesting that these are spectrum disorders. An exciting recent development has been the identification of somatic mutations causing both FCD Ia and nonlesional neocortical epilepsy. SUMMARY: Defining somatic gene mutations in brain tissue specimens has shed new light on how MCD form and the mechanisms of epileptogenesis associated with MCD. Trials of mTOR inhibitors in tuberous sclerosis complex have demonstrated that inhibition of mTOR activation in mTORopathies can reduce seizure frequency. New somatic mutations found for a variety of epilepsy syndromes may provide new targets for clinical therapeutics.

GATORopathies: The role of amino acid regulatory gene mutations in epilepsy and cortical malformations.

The mechanistic target of rapamycin (mTOR) pathway has been implicated in a growing number of malformations of cortical development (MCD) associated with intractable epilepsy. Mutations in single genes encoding mTOR pathway regulatory proteins have been linked to MCD such as focal cortical dysplasia (FCD) types IIa and IIb, hemimegalencephaly (HME), and megalencephaly. Recent studies have demonstrated that the GATOR1 protein complex, comprised of DEPDC5, NPRL3, and NPRL2, plays a pivotal role in regulating mTOR signaling in response to cellular amino acid levels and that mutations in DEPDC5, NPRL3, or NPRL2 are linked to FCD, HME, and seizures. Histopathological analysis of FCD and HME tissue specimens resected from individuals harboring DEPDC5, NPRL3, or NPRL2 gene mutations reveals hyperactivation of mTOR pathway signaling. Family pedigrees carrying mutations in either DEPDC5 or NPRL3 share clinical phenotypes of epilepsy and MCD, as well as intellectual and neuropsychiatric disabilities. Interestingly, some individuals with seizures associated with DEPDC5, NPRL3, or NPRL2 variants exhibit normal brain imaging suggesting either occult MCD or a role for these genes in non-lesional neocortical epilepsy. Mouse models resulting from knockdown or knockout of either Depdc5 or Nprl3 exhibit altered cortical lamination, neuronal dysmorphogenesis, and enhanced neuronal excitability as reported in models resulting from direct mTOR activation through expression of its canonical activator RHEB. The role of the GATOR1 proteins in regulating mTOR signaling suggest plausible options for mTOR inhibition in the treatment of epilepsy associated with mutations in DEPDC5, NPRL3, or NPRL2.

DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

Mutations in DEPDC5 and NPRL3 subunits of GATOR1, a modulator of mechanistic target of rapamycin (mTOR), are linked to malformations of cortical development (MCD). Brain specimens from these individuals reveal abnormal cortical lamination, altered cell morphology, and hyperphosphorylation of ribosomal S6 protein (PS6), a marker for mTOR activation. While numerous studies have examined GATOR1 subunit function in non-neuronal cell lines, few have directly assessed loss of GATOR1 subunit function in neuronal cell types. We hypothesized that DEPDC5 or NPRL3 shRNA-mediated knockdown (DEPDC5/NPRL3 KD) leads to inappropriate functional activation of mTOR and mTOR-dependent alterations in neuronal morphology. Neuronal size was determined in human specimens harboring DEPDC5 or NPRL3 mutations resected for epilepsy treatment. DEPDC5/NPRL3 KD effects on cell size, filopodial extension, subcellular mTOR complex 1 (mTORC1) localization, and mTORC1 activation during nutrient deprivation were assayed in mouse neuroblastoma cells (N2aC) and mouse subventricular zone derived neural progenitor cells (mNPCs). mTORC1-dependent effects of DEPDC5/NPRL3 KD were determined using the mTOR inhibitor rapamycin. Changes in mTOR subcellular localization and mTORC1 pathway activation following DEPDC5/NPRL3 KD were determined by examining the proximity of mTOR to the lysosomal surface during amino acid starvation. Neurons exhibiting PS6 immunoreactivity (Ser 235/236) in human specimens were 1.5× larger than neurons in post-mortem control samples. DEPDC5/NPRL3 KD caused mTORC1, but not mTORC2, hyperactivation, soma enlargement, and increased filopodia in N2aC and mNPCs compared with wildtype cells. DEPDC5/NPRL3 KD led to inappropriate mTOR localization at the lysosome along with constitutive mTOR activation following amino acid deprivation. DEPDC5/NPRL3 KD effects on morphology and functional mTOR activation were reversed by rapamycin. mTOR-dependent effects of DEPDC5/NPRL3 KD on morphology and subcellular localization of mTOR in neurons suggests that loss-of-function in GATOR1 subunits may play a role in MCD formation during fetal brain development.

Intracellular and circulating neuronal antinuclear antibodies in human epilepsy.

There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, the human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from the brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in the serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n=4/4). In the brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated albumin. All subcellular fractions from brain resections of patients with epilepsy contained extravasated IgGs (n=10/10), but epileptogenic cortex, where seizures originated from, displayed the highest levels of chromatin-bound IgGs. In the nuclear IgG pool, anti-histone autoantibodies were identified by two independent immunodetection methods. HEp-2 assay and ELISA confirmed the presence of anti-histone (n=5/8) and anti-chromatin antibodies in the serum from patients with epilepsy. We developed a multi-step approach to unmask autoantigens in the brain and sera of patients with epilepsy. This approach revealed antigen-bound antinuclear antibodies in neurons and free antinuclear IgGs in the serum of patients with epilepsy. Conditions with blood-brain barrier disruption but not seizures, were characterized by extravasated but not chromatin-bound IgGs. Our results show that the pool of intracellular IgG in the brain of patients with epilepsy consists of nucleus-specific autoantibodies targeting chromatin and histones. Seizures may be the trigger of neuronal uptake of antinuclear antibodies.

A role for inflammation in status epilepticus is revealed by a review of current therapeutic approaches.

A significant number of patients with epilepsy fail to respond to currently available antiepileptic drugs. This suggests a need for alternative approaches to reduce the occurrence of seizures in these patients. Recent data have shown that in addition to well-known neuronal mechanism, seizures may be a consequence of misguided inflammatory response and blood-brain barrier disruption. Both peripheral and brain proinflammatory events have been demonstrated to govern the onset of status epilepticus. Evidence deriving from the experimental and clinical realms supports the notion that a role for proinflammatory and cerebrovascular events in seizure disorders is broader than previously suspected. As a result, methods to pharmacologically reduce blood-brain barrier permeability and reduce inflammation have emerged as means to reduce seizure burden. For instance, corticosteroids have been shown to be beneficial and the same agents may be able to further reduce seizure burden in conjunction with currently prescribed antiepileptic drugs.

Rapid modeling of an ultra-rare epilepsy variant in wild-type mice by in utero prime editing.

Generating animal models for individual patients within clinically-useful timeframes holds great potential toward enabling personalized medicine approaches for genetic epilepsies. The ability to rapidly incorporate patient-specific genomic variants into model animals recapitulating elements of the patient's clinical manifestations would enable applications ranging from validation and characterization of pathogenic variants to personalized models for tailoring pharmacotherapy to individual patients. Here, we demonstrate generation of an animal model of an individual epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A, without the need for germline transmission and breeding. Using in utero prime editing in the brain of wild-type mice, our approach yielded high in vivo editing precision and induced frequent, spontaneous seizures which mirrored specific elements of the patient's clinical presentation. Leveraging the speed and versatility of this approach, we introduce PegAssist, a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. The capability to produce individualized animal models rapidly and cost-effectively will reduce barriers to access for precision medicine, and will accelerate drug development by offering versatile in vivo platforms to identify compounds with efficacy against rare neurological conditions.

Viral expression of constitutively active AKT3 induces CST axonal sprouting and regeneration, but also promotes seizures.

Increasing the intrinsic growth potential of neurons after injury has repeatedly been shown to promote some level of axonal regeneration in rodent models. One of the most studied pathways involves the activation of the PI3K/AKT/mTOR pathways, primarily by reducing the levels of PTEN, a negative regulator of PI3K. Likewise, activation of signal transducer and activator of transcription 3 (STAT3) has previously been shown to boost axonal regeneration and sprouting within the injured nervous system. Here, we examined the regeneration of the corticospinal tract (CST) after cortical expression of constitutively active (ca) Akt3 and STAT3, both separately and in combination. Overexpression of caAkt3 induced regeneration of CST axons past the injury site independent of caSTAT3 overexpression. STAT3 demonstrated improved axon sprouting compared to controls and contributed to a synergistic improvement in effects when combined with Akt3 but failed to promote axonal regeneration as an individual therapy. Despite showing impressive axonal regeneration, animals expressing Akt3 failed to show any functional improvement and deteriorated with time. During this period, we observed progressive Akt3 dose-dependent increase in behavioral seizures. Histology revealed increased phosphorylation of ribosomal S6 protein within the unilateral cortex, increased neuronal size, microglia activation and hemispheric enlargement (hemimegalencephaly).

BK channel properties correlate with neurobehavioral severity in three KCNMA1-linked channelopathy mouse models.

KCNMA1 forms the pore of BK K+ channels, which regulate neuronal and muscle excitability. Recently, genetic screening identified heterozygous KCNMA1 variants in a subset of patients with debilitating paroxysmal non-kinesigenic dyskinesia, presenting with or without epilepsy (PNKD3). However, the relevance of KCNMA1 mutations and the basis for clinical heterogeneity in PNKD3 has not been established. Here, we evaluate the relative severity of three KCNMA1 patient variants in BK channels, neurons, and mice. In heterologous cells, BKN999S and BKD434G channels displayed gain-of-function (GOF) properties, whereas BKH444Q channels showed loss-of-function (LOF) properties. The relative degree of channel activity was BKN999S > BKD434G>WT > BKH444Q. BK currents and action potential firing were increased, and seizure thresholds decreased, in Kcnma1N999S/WT and Kcnma1D434G/WT transgenic mice but not Kcnma1H444Q/WT mice. In a novel behavioral test for paroxysmal dyskinesia, the more severely affected Kcnma1N999S/WT mice became immobile after stress. This was abrogated by acute dextroamphetamine treatment, consistent with PNKD3-affected individuals. Homozygous Kcnma1D434G/D434G mice showed similar immobility, but in contrast, homozygous Kcnma1H444Q/H444Q mice displayed hyperkinetic behavior. These data establish the relative pathogenic potential of patient alleles as N999S>D434G>H444Q and validate Kcnma1N999S/WT mice as a model for PNKD3 with increased seizure propensity.

So far, only 70 patients around the world have been diagnosed with a newly identified rare syndrome known as KCNMA1-linked channelopathy. The condition is characterised by seizures and abnormal movements which include frequent 'drop attacks', a sudden and debilitating loss of muscle control that causes patients to fall without warning. The disease is associated with mutations in the gene for KCNMA1, a member of a class of proteins important for controlling nerve cell activity and brain function. However, due to the limited number of people affected by the condition, it is difficult to link a particular mutation to the observed symptoms; the basis for the drop attacks therefore remains unknown. Park et al. set out to 'model' KCNMA1-linked channelopathy in the laboratory, in order to determine which mutations in the KCNMA1 gene caused these symptoms. Three groups of mice were each genetically engineered to carry either one of the two most common mutations in the gene for KCNMA1, or a very rare mutation associated with the movement symptoms. Behavioural experiments and studies of nerve cell activity revealed that the mice carrying mutations that made the KCNMA1 protein more active developed seizures more easily and became immobilized, showing the mouse version of drop attacks. Giving these mice the drug dextroamphetamine, which works in some human patients, stopped the immobilizing attacks altogether. These results show for the first time which specific genetic changes cause the main symptoms of KCNMA1-linked channelopathy. Park et al. hope that this knowledge will deepen our understanding of this disease and help develop better treatments.

Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 knockout.

TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been comprehensively investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.

Focal Cortical Dysplasia: Gene Mutations, Cell Signaling, and Therapeutic Implications.

Focal cortical dysplasias (FCDs) are malformations of cortical development (MCDs) that are highly associated with medication-resistant epilepsy and are the most common cause of neocortical epilepsy in children. FCDs are a heterogeneous group of developmental disorders caused by germline or somatic mutations that occur in genes regulating the PI3K/Akt/mTOR pathway-a key pathway in neuronal growth and migration. Accordingly, FCDs are characterized by abnormal cortical lamination, cell morphology (e.g., cytomegaly), and cellular polarity. In some FCD subtypes, balloon cells express proteins typically seen in neuroglial progenitor cells. Because recurrent intractable seizures are a common feature of FCDs, epileptogenic electrophysiological properties are also observed in addition to local inflammation. Here, we will summarize the current literature regarding FCDs, addressing the current classification system, histopathology, molecular genetics, electrophysiology, and transcriptome and cell signaling changes.

Sending Mixed Signals: The Expanding Role of Molecular Cascade Mutations in Malformations of Cortical Development and Epilepsy.

Advances in gene sequencing techniques have led to a dramatic increase in the number of signaling cascade and cytoskeletal assembly mutations associated with malformations of cortical development and epilepsy. At the forefront of this research are novel mutations found in regulators of the PI3K/AKT/mTOR cascade and tubulin-associated malformations of cortical development. However, there is limited understanding of the consequences of these newly discovered germline and somatic mutations on cellular function or how these changes in cell biology may lead to areas-large or small-of malformed cortex and recurrent spontaneous seizures. We summarize and discuss what is currently known in this field in an effort to shine light on vast gaps in our knowledge of relatively common causes of cortical malformations.

Anti-NR2 antibodies, blood-brain barrier, and cognitive dysfunction.

Cognitive dysfunction (CD) is one of the most common neuropsychiatric manifestations of systemic lupus erythematosus (SLE). In animal models, antibodies to NR2 subunit of N-methyl D-aspartate receptor (anti-NR2) cause memory impairment, but only with blood-brain barrier (BBB) disruption or intrathecal administration. Several studies have failed to find association of aNR2 with CD, but none have assessed BBB integrity. S100B, an astrocyte-specific protein, has been used as biomarker of BBB disruption in traumatic brain injury and some neurodegenerative disorders. Antibodies to this immunologically privileged protein (anti-S100B) might indicate preceding BBB disruption. We hypothesized that aNR2 antibody is pathogenic in SLE patients only with BBB disruption. Demographic, clinical, and laboratory data was collected from patients with SLE. Total throughput score (TTS) of the Automated Neuropsychological Assessment Metrics (ANAM) was used as primary outcome measure. CD was defined as TTS < 1.5 SD below an age-, sex-, and race-matched RA population mean. Serum was analyzed by established ELISA techniques. Fifty-seven patients were evaluated and 12 had CD. Age, ethnicity, and family income were significantly different between the two groups (p < 0.05). In a multiple regression model adjusting for other variables, no significant effects of anti-NR2, S100B, or anti-S100B on TTS were found. Even at high levels of S100B and anti-S100B, no significant influence of anti-NR2 on TTS was found. The anti-NR2 was not associated with CD in SLE even in context of potential BBB disruption. This suggests that, if pathogenic, these antibodies may be produced intrathecally.