Rasmussen encephalitis (RE) is a rare pediatric neuroinflammatory disease of unknown etiology characterized by intractable seizures, and progressive atrophy usually confined to one cerebral hemisphere. Surgical removal or disconnection of the affected cerebral hemisphere is currently the only intervention that effectively stops the seizures. Histopathological evaluation of resected brain tissue has shown that activated brain resident macrophages (microglia) and infiltrating T cells are involved in the inflammatory reaction. Here, we report that T cells isolated from seven RE brain surgery specimens express the resident memory T cell (TRM) marker CD103. CD103 was expressed by >50% of CD8(+) αβ T cells and γδ T cells irrespective of the length of time from seizure onset to surgery, which ranged from 0.3 to 8.4 years. Only ~10% of CD4(+) αβ were CD103(+), which was consistent with the observation that few CD4(+) T cells are found in RE brain parenchyma. Clusters of T cells in brain parenchyma, which are a characteristic of RE histopathology, stained for CD103. Less than 10% of T cells isolated from brain specimens from eight surgical cases of focal cortical dysplasia (FCD), a condition that is also characterized by intractable seizures, were CD103(+). In contrast to the RE cases, the percent of CD103(+) T cells increased with the length of time from seizure onset to surgery. In sections of brain tissue from the FCD cases, T cells were predominantly found around blood vessels, and did not stain for CD103. The presence of significant numbers of TRM cells in RE brain irrespective of the length of time between clinical presentation and surgical intervention supports the conclusion that a cellular immune response to an as yet unidentified antigen(s) occurs at an early stage of the disease. Reactivated TRM cells may contribute to disease progression.

Rasmussen encephalitis (RE) is a rare pediatric neuroinflammatory disease characterized by intractable seizures and unilateral brain atrophy. T cell infiltrates in affected brain tissue and the presence of circulating autoantibodies in some RE patients have indicated that RE may be an autoimmune disease. The strongest genetic links to autoimmunity reside in the MHC locus, therefore, we determined the human leukocyte antigen (HLA) class I and class II alleles carried by a cohort of 24 RE surgery cases by targeted in-depth genomic sequencing. Compared with a reference population the allelic frequency of three alleles, DQA1*04:01:01, DQB1*04:02:01, and HLA-C*07:02:01:01 indicated that they might confer susceptibility to the disease. It has been reported that HLA-C*07:02 is a risk factor for Graves disease. Further, eight patients in the study cohort carried HLA-A*03:01:01:01, which has been linked to susceptibility to multiple sclerosis. Four patients carried a combination of three HLA class II alleles that has been linked to type 1 diabetes (DQA1*05:01:01:01~DQB1*02:01:01~DRB1*03:01:01:01), and five patients carried a combination of HLA class II alleles that has been linked to the risk of contracting multiple sclerosis (DQA1*01:02:01:01, DQB1*06:02:01, DRB1*15:01:01:01). We also analyzed the diversity of αβ T cells in brain and blood specimens from 14 of these RE surgery cases by sequencing the third complementarity regions (CDR3s) of rearranged T cell receptor β genes. A total of 31 unique CDR3 sequences accounted for the top 5% of all CDR3 sequences in the 14 brain specimens. Thirteen of these sequences were found in sequencing data from healthy blood donors; the remaining 18 sequences were patient specific. These observations provide evidence for the clonal expansion of public and private T cells in the brain, which might be influenced by the RE patient's HLA haplotype.

Rasmussen encephalitis (RE) is a rare neuroinflammatory disease characterized by intractable seizures and progressive atrophy on one side of the cerebrum. Perivascular cuffing and clusters of T cells in the affected cortical hemisphere are indicative of an active cellular immune response.

It remains controversial whether neuronal damage and synaptic reorganization found in some forms of epilepsy are the result of an initial injury and potentially contributory to the epileptic condition or are the cumulative affect of repeated seizures. A number of reports of human and animal pathology suggest that at least some neuronal loss precedes the onset of seizures, but there is debate over whether there is further damage over time from intermittent seizures. In support of this latter hypothesis are MRI studies in people that show reduced hippocampal volumes and cortical thickness with longer durations of the disease. In this study we addressed the question of neuronal loss from intermittent seizures using kindled rats (no initial injury) and rats with limbic epilepsy (initial injury).

Pathological high-frequency oscillations (HFOs), specifically fast ripples (FRs, >250 Hz), are pathognomonic of an active epileptogenic zone. However, the origin of FRs remains unknown. Here we explored the correlation between FRs recorded with intraoperative pre-resection electrocorticography (ECoG) and spontaneous synaptic activity recorded ex vivo from cortical tissue samples resected for the treatment of pharmacoresistant epilepsy. The cohort included 47 children (ages 0.22-9.99 yr) with focal cortical dysplasias (CD types I and II), tuberous sclerosis complex (TSC) and non-CD pathologies. Whole-cell patch clamp recordings were obtained from pyramidal neurons and interneurons in cortical regions that were positive or negative for pathological HFOs, defined as FR band oscillations (250-500 Hz) at ECoG. The frequency of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and IPSCs, respectively) was compared between HFO+ and HFO- regions. Regardless of pathological substrate, regions positive for FRs displayed significantly increased frequencies of sIPSCs compared with regions negative for FRs. In contrast, the frequency of sEPSCs was similar in both regions. In about one third of cases (n = 17), pacemaker GABA synaptic activity (PGA) was observed. In the vast majority (n = 15), PGA occurred in HFO+ areas. Further, fast-spiking interneurons displayed signs of hyperexcitability exclusively in HFO+ areas. These results indicate that, in pediatric epilepsy patients, increased GABA synaptic activity is associated with interictal FRs in the epileptogenic zone and suggest an active role of GABAergic interneurons in the generation of pathological HFOs. Increased GABA synaptic activity could serve to dampen excessive excitability of cortical pyramidal neurons in the epileptogenic zone, but it could also promote neuronal network synchrony.

In the present study, we characterized the effects of bath application of the proconvulsant drug 4-aminopyridine (4-AP) alone or in combination with GABAA and/or GABAB receptor antagonists, in cortical dysplasia (CD type I and CD type IIa/b), tuberous sclerosis complex (TSC), and non-CD cortical tissue samples from pediatric epilepsy surgery patients. Whole-cell patch clamp recordings in current and voltage clamp modes were obtained from cortical pyramidal neurons (CPNs), interneurons, and balloon/giant cells. In pyramidal neurons, bath application of 4-AP produced an increase in spontaneous synaptic activity as well as rhythmic membrane oscillations. In current clamp mode, these oscillations were generally depolarizing or biphasic and were accompanied by increased membrane conductance. In interneurons, membrane oscillations were consistently depolarizing and accompanied by bursts of action potentials. In a subset of balloon/giant cells from CD type IIb and TSC cases, respectively, 4-AP induced very low-amplitude, slow membrane oscillations that echoed the rhythmic oscillations from pyramidal neurons and interneurons. Bicuculline reduced the amplitude of membrane oscillations induced by 4-AP, indicating that they were mediated principally by GABAA receptors. 4-AP alone or in combination with bicuculline increased cortical excitability but did not induce seizure-like discharges. Ictal activity was observed in pyramidal neurons and interneurons from CD and TSC cases only when phaclofen, a GABAB receptor antagonist, was added to the 4-AP and bicuculline solution. These results emphasize the critical and permissive role of GABAB receptors in the transition to an ictal state in pediatric CD tissue and highlight the importance of these receptors as a potential therapeutic target in pediatric epilepsy.

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases, including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain, we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin, which led to a non-cell autonomous migration defect in neighboring cells, due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy.

Objective: To develop a novel software method (AR2) for reducing muscle contamination of ictal scalp electroencephalogram (EEG), and validate this method on the basis of its performance in comparison to a commercially available software method (AR1) to accurately depict seizure-onset location. Methods: A blinded investigation used 23 EEG recordings of seizures from 8 patients. Each recording was uninterpretable with digital filtering because of muscle artifact and processed using AR1 and AR2 and reviewed by 26 EEG specialists. EEG readers assessed seizure-onset time, lateralization, and region, and specified confidence for each determination. The two methods were validated on the basis of the number of readers able to render assignments, confidence, the intra-class correlation (ICC), and agreement with other clinical findings. Results: Among the 23 seizures, two-thirds of the readers were able to delineate seizure-onset time in 10 of 23 using AR1, and 15 of 23 using AR2 (p<0.01). Fewer readers could lateralize seizure-onset (p<0.05). The confidence measures of the assignments were low (probable-unlikely), but increased using AR2 (p<0.05). The ICC for identifying the time of seizure-onset was 0.15 (95% confidence interval (CI), 0.11-0.18) using AR1 and 0.26 (95% CI 0.21-0.30) using AR2.  The EEG interpretations were often consistent with behavioral, neurophysiological, and neuro-radiological findings, with left sided assignments correct in 95.9% (CI 85.7-98.9%, n=4) of cases using AR2, and 91.9% (77.0-97.5%) (n=4) of cases using AR1. Conclusions: EEG artifact reduction methods for localizing seizure-onset does not result in high rates of interpretability, reader confidence, and inter-reader agreement. However, the assignments by groups of readers are often congruent with other clinical data. Utilization of the AR2 software method may improve the validity of ictal EEG artifact reduction.

While most forms of Parkinson's Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.

Rasmussen encephalitis (RE) is a rare complex inflammatory disease, primarily seen in young children, that is characterized by severe partial seizures and brain atrophy. Surgery is currently the only effective treatment option. To identify genes specifically associated with the immunopathology in RE, RNA transcripts of genes involved in inflammation and autoimmunity were measured in brain tissue from RE surgeries and compared with those in surgical specimens of cortical dysplasia (CD), a major cause of intractable pediatric epilepsy.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Microglia are well known to play a critical role in maintaining brain homeostasis. However, their role in epileptogenesis has yet to be determined. Here, we demonstrate that elevated mTOR signaling in mouse microglia leads to phenotypic changes, including an amoeboid-like morphology, increased proliferation, and robust phagocytosis activity, but without a significant induction of pro-inflammatory cytokines. We further provide evidence that these noninflammatory changes in microglia disrupt homeostasis of the CNS, leading to reduced synapse density, marked microglial infiltration into hippocampal pyramidal layers, moderate neuronal degeneration, and massive proliferation of astrocytes. Moreover, the mice thus affected develop severe early-onset spontaneous recurrent seizures (SRSs). Therefore, we have revealed an epileptogenic mechanism that is independent of the microglial inflammatory response. Our data suggest that microglia could be an opportune target for epilepsy prevention.

CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.

Glioblastoma (GBM) is characterized by extensive microvascular hyperproliferation. In addition to supplying blood to the tumor, GBM vessels also provide trophic support to glioma cells and serve as conduits for migration into the surrounding brain, promoting recurrence. Here, we enrich CD31-expressing glioma vascular cells (GVCs) and A2B5-expressing glioma tumor cells (GTCs) from primary GBM and use RNA sequencing to create a comprehensive molecular interaction map of the secreted and extracellular factors elaborated by GVCs that can interact with receptors and membrane molecules on GTCs. To validate our findings, we utilize functional assays, including a hydrogel-based migration assay and in vivo mouse models to demonstrate that one identified factor, the little-studied integrin binding sialoprotein (IBSP), enhances tumor growth and promotes the migration of GTCs along the vasculature. This perivascular niche interactome will serve as a resource to the research community in defining the potential functions of the GBM vasculature.

Comparative analyses have identified genomic regions potentially involved in human evolution but do not directly assess function. Human accelerated regions (HARs) represent conserved genomic loci with elevated divergence in humans. If some HARs regulate human-specific social and behavioral traits, then mutations would likely impact cognitive and social disorders. Strikingly, rare biallelic point mutations-identified by whole-genome and targeted "HAR-ome" sequencing-showed a significant excess in individuals with ASD whose parents share common ancestry compared to familial controls, suggesting a contribution in 5% of consanguineous ASD cases. Using chromatin interaction sequencing, massively parallel reporter assays (MPRA), and transgenic mice, we identified disease-linked, biallelic HAR mutations in active enhancers for CUX1, PTBP2, GPC4, CDKL5, and other genes implicated in neural function, ASD, or both. Our data provide genetic evidence that specific HARs are essential for normal development, consistent with suggestions that their evolutionary changes may have altered social and/or cognitive behavior. PAPERCLIP.

Focal cortical dysplasia (FCD) and hemimegalencephaly (HME) are epileptogenic neurodevelopmental malformations caused by mutations in mTOR pathway genes. Deep sequencing of these genes in FCD/HME brain tissue identified an etiology in 27 of 66 cases (41%). Radiographically indistinguishable lesions are caused by somatic activating mutations in AKT3, MTOR, and PIK3CA and germline loss-of-function mutations in DEPDC5, NPRL2, and TSC1/2, including TSC2 mutations in isolated HME demonstrating a "two-hit" model. Mutations in the same gene cause a disease continuum from FCD to HME to bilateral brain overgrowth, reflecting the progenitor cell and developmental time when the mutation occurred. Single-cell sequencing demonstrated mTOR activation in neurons in all lesions. Conditional Pik3ca activation in the mouse cortex showed that mTOR activation in excitatory neurons and glia, but not interneurons, is sufficient for abnormal cortical overgrowth. These data suggest that mTOR activation in dorsal telencephalic progenitors, in some cases specifically the excitatory neuron lineage, causes cortical dysplasia.

Using a targeted transcriptomics approach, we have analyzed resected brain tissue from a cohort of 53 pediatric epilepsy surgery cases, and have found that there is a spectrum of involvement of both the innate and adaptive immune systems as evidenced by the differential expression of immune-specific genes in the affected brain tissue. The specimens with the highest expression of immune-specific genes were from two Rasmussen encephalitis cases, which is known to be a neuro-immunological disease, but also from tuberous sclerosis complex (TSC), focal cortical dysplasia, and hemimegalencephaly surgery cases. We obtained T cell receptor (TCR) Vβ chain sequence data from brain tissue and blood from patients with the highest levels of T cell transcripts. The clonality indices and the frequency of the top 50 Vβ clonotypes indicated that T cells in the brain were clonally restricted. The top 50 Vβ clonotypes comprised both public and private (patient specific) clonotypes, and the TCR Vβ chain third complementarity region (CDR3) of the most abundant public Vβ clonotype in each brain sample was strikingly similar to a CDR3 that recognizes an immunodominant epitope in either human cytomegalovirus or Epstein Barr virus, or influenza virus A. We found that the frequency of 14 of the top 50 brain Vβ clonotypes from a TSC surgery case had significantly increased in brain tissue removed to control recurrent seizures 11 months after the first surgery. Conversely, we found that the frequency in the blood of 18 of the top 50 brain clonotypes from a second TSC patient, who was seizure free, had significantly decreased 5 months after surgery indicating that T cell clones found in the brain had contracted in the periphery after removal of the brain area associated with seizure activity and inflammation. However, the frequency of a public and a private clonotype significantly increased in the brain after seizures recurred and the patient underwent a second surgery. Combined single cell gene expression and TCR sequencing of brain-infiltrating leukocytes from the second surgery showed that the two clones were CD8 effector T cells, indicating that they are likely to be pathologically relevant.

Tuberous Sclerosis Complex (TSC) and cortical dysplasia Type IIB (CDIIB) share histopathologic features that suggest similar epileptogenic mechanisms. This study compared the morphological and electrophysiological properties of cortical cells in tissue from pediatric TSC (n=20) and CDIIB (n=20) patients using whole-cell patch clamp recordings and biocytin staining. Cell types were normal-appearing and dysmorphic-cytomegalic pyramidal neurons, interneurons, and giant/balloon cells, including intermediate neuronal-glial cells. In the cortical mantle, giant/balloon cells occurred more frequently in TSC than in CDIIB cases, whereas cytomegalic pyramidal neurons were found more frequently in CDIIB. Cell morphology and membrane properties were similar in TSC and CDIIB cases. Except for giant/balloon and intermediate cells, all neuronal cell types fired action potentials and displayed spontaneous postsynaptic currents. However, the frequency of spontaneous glutamatergic postsynaptic currents in normal pyramidal neurons and interneurons was significantly lower in CDIIB compared with TSC cases and the GABAergic activity was higher in all neuronal cell types in CDIIB. Further, acutely dissociated pyramidal neurons displayed higher sensitivity to exogenous application of GABA in CDIIB compared with TSC cases. These results indicate that, in spite of similar histopathologic features and basic cell membrane properties, TSC and CDIIB display differences in the topography of abnormal cells, excitatory and inhibitory synaptic network properties, and GABA(A) receptor sensitivity. These differences support the notion that the mechanisms of epileptogenesis could differ in patients with TSC and CDIIB. Consequently, pharmacologic therapies should take these findings into consideration.

The present study was designed to examine the potential cellular antiseizure mechanisms of everolimus, a mechanistic target of rapamycin (mTOR) pathway blocker, in pediatric epilepsy cases. Cortical tissue samples obtained from pediatric patients (n = 11, ages 0.67-6.75 years) undergoing surgical resections for the treatment of their pharmacoresistant epilepsy were examined electrophysiologically in ex vivo slices. The cohort included mTOR-mediated pathologies (tuberous sclerosis complex [TSC] and severe cortical dysplasia [CD]) as well as non-mTOR-mediated pathologies (tumor and perinatal infarct). Bath application of everolimus (2 μm) had practically no effect on spontaneous inhibitory postsynaptic activity. In contrast, long-term application of everolimus reduced spontaneous excitatory postsynaptic activity, burst discharges induced by blockade of γ-aminobutyric acid A (GABAA) receptors, and epileptiform activity generated by 4-aminopyridine, a K+ channel blocker. The antiseizure effects were more pronounced in TSC and CD cases, whereas in non-mTOR-mediated pathologies, the effects were subtle at best. These results support further clinical trials of everolimus in mTOR pathway-mediated pathologies and emphasize that the effects require sustained exposure over time.

Brain-infiltrating lymphocytes (BILs) were isolated from resected brain tissue from 10 pediatric epilepsy patients who had undergone surgery for Hemimegalencephaly (HME) (n = 1), Tuberous sclerosis complex (TSC) (n = 2), Focal cortical dysplasia (FCD) (n = 4), and Rasmussen encephalitis (RE) (n = 3). Peripheral blood mononuclear cells (PBMCs) were also isolated from blood collected at the time of the surgery. Cells were immunostained with a panel of 20 antibody markers, and analyzed by mass cytometry. To identify and quantify the immune cell types in the samples, an unbiased clustering method was applied to the entire data set. More than 85 percent of the CD45+ cells isolated from resected RE brain tissue comprised T cells; by contrast NK cells and myeloid cells constituted 80-95 percent of the CD45+ cells isolated from the TSC and the FCD brain specimens. Three populations of myeloid cells made up >50 percent of all of the myeloid cells in all of the samples of which a population of HLA-DR+ CD11b+ CD4- cells comprised the vast majority of myeloid cells in the BIL fractions from the FCD and TSC cases. CD45RA+ HLA-DR- CD11b+ CD16+ NK cells constituted the major population of NK cells in the blood from all of the cases. This subset also comprised the majority of NK cells in BILs from the resected RE and HME brain tissue, whereas NK cells defined as CD45RA- HLA-DR+ CD11b- CD16- cells comprised 86-96 percent of the NK cells isolated from the FCD and TSC brain tissue. Thirteen different subsets of CD4 and CD8 αβ T cells and γδ T cells accounted for over 80% of the CD3+ T cells in all of the BIL and PBMC samples. At least 90 percent of the T cells in the RE BILs, 80 percent of the T cells in the HME BILs and 40-66 percent in the TSC and FCD BILs comprised activated antigen-experienced (CD45RO+ HLA-DR+ CD69+) T cells. We conclude that even in cases where there is no evidence for an infection or an immune disorder, activated peripheral immune cells may be present in epileptogenic areas of the brain, possibly in response to seizure-driven brain inflammation.