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.

Proper axonal and dendritic bundling is essential for the establishment of neuronal connections and the synchronization of synaptic inputs, respectively. Cell adhesion molecules of the L1-CAM (L1-cell adhesion molecule) family regulate axon guidance and fasciculation, neuron migration, dendrite morphology, and synaptic plasticity. It remains unclear how these molecules play so many different roles. Here we show that polarized axon-dendrite targeting of an avian L1-CAM protein, NgCAM (neuron-glia cell adhesion molecule), can regulate the switch of bundling of the two major compartments of rat hippocampal neurons. Using a new in-vitro model for studying neurite-neurite interactions, we found that expressed axonal NgCAM induced robust axonal bundling via the trans-homophilic interaction of immunoglobulin domains. Interestingly, dendritic bundling was induced by the dendritic targeting of NgCAM, caused by either deleting its fibronectin repeats or blocking activities of protein kinases. Consistent with the NgCAM results, expression of mouse L1-CAM also induced axonal bundling and blocking kinase activities disrupted its axonal targeting. Furthermore, the trans-homophilic interaction stabilized the bundle formation, probably through recruiting NgCAM proteins to contact sites and promoting guided axon outgrowth. Taken together, our results suggest that precise localization of L1-CAM is important for establishing proper cell-cell contacts in neural circuits.

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.

Action potentials (APs) propagating along axons require the activation of voltage-gated Na(+) (Nav) channels. How Nav channels are transported into axons is unknown. We show that KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with small interfering RNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing AP firing. Live-cell imaging showed that fluorescently tagged AnkG or Nav1.2 cotransported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav, but not Kv1.2, channels in mouse cerebellum. These results indicate that AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport before anchoring them to the AIS and nodes of Ranvier.

Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here, we report that KIF5/kinesin I transports Kv3.1 voltage-gated K(+) (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull-down assays with exogenously expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Overexpression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by RNA interference significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn(2+)-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, coexpression of the YFP (yellow fluorescent protein)-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels colocalize and comove with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons.

The present study examined synaptic communication between direct and indirect output pathway striatal medium-sized spiny neurons (MSNs) and their target structures, the substantia nigra pars reticulata (SNr) and the external globus pallidus (GPe) in two mouse models of Huntington's disease (HD). Cre recombination, optogenetics, and whole-cell patch-clamp recordings were used to determine alterations in intrinsic and synaptic properties of SNr and GPe neurons from both male and female symptomatic R6/2 (>60 d) and presymptomatic (2 months) or symptomatic (10-12 months) YAC128 mice. Cell membrane capacitance was decreased, whereas input resistance was increased in SNr neurons from R6/2, but not YAC128 mice. The amplitude of GABAergic responses evoked by optogenetic stimulation of direct pathway terminals was reduced in SNr neurons of symptomatic mice of both models. A decrease in spontaneous GABA synaptic activity, in particular large-amplitude events, in SNr neurons also was observed. Passive membrane properties of GPe neurons were not different between R6/2 or YAC128 mice and their control littermates. Similarly, the amplitude of GABA responses evoked by activation of indirect pathway MSN terminals and the frequency of spontaneous GABA synaptic activity were similar in HD and control animals. In contrast, the decay time of the evoked GABA response was significantly longer in cells from HD mice. Interestingly, activation of indirect pathway MSNs within the striatum evoked larger-amplitude responses in direct pathway MSNs. Together, these results demonstrate differential alterations in responses evoked by direct and indirect pathway terminals in SNr and GPe leading to striatal output imbalance and motor dysfunction.SIGNIFICANCE STATEMENT Previous work on Huntington's disease (HD) focused on striatal medium-sized spiny neurons (MSNs) almost exclusively. Little is known about the effects that alterations in the striatum have on output structures of the direct and indirect pathways, the substantia nigra pars reticulata (SNr) and the external segment of the globus pallidus (GPe), respectively. We combined electrophysiological and optogenetic methods to examine responses evoked by selective activation of terminals of direct and indirect pathway MSNs in SNr and GPe neurons in two mouse models of HD. We show a differential disruption of synaptic communication between the direct and indirect output pathways of the striatum with their target regions leading to an imbalance of striatal output, which will contribute to motor dysfunction.

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.

The cortico-basal ganglia-thalamo-cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1-4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico-basal ganglia-thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

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.

The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

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.