A role of dentate gyrus mechanistic target of rapamycin activation in epileptogenesis in a mouse model of posttraumatic epilepsy.

OBJECTIVE: The mechanistic target of rapamycin (mTOR) pathway has been implicated in promoting epileptogenesis in animal models of acquired epilepsy, such as posttraumatic epilepsy (PTE) following traumatic brain injury (TBI). However, the specific anatomical regions and neuronal populations mediating mTOR's role in epileptogenesis are not well defined. In this study, we tested the hypothesis that mTOR activation in dentate gyrus granule cells promotes neuronal death, mossy fiber sprouting, and PTE in the controlled cortical impact (CCI) model of TBI. METHODS: An adeno-associated virus (AAV)-Cre viral vector was injected into the hippocampus of Rptorflox/flox (regulatory-associated protein of mTOR) mutant mice to inhibit mTOR activation in dentate gyrus granule cells. Four weeks after AAV-Cre or AAV-vehicle injection, mice underwent CCI injury and were subsequently assessed for mTOR pathway activation by Western blotting, neuronal death, and mossy fiber sprouting by immunopathological analysis, and posttraumatic seizures by video-electroencephalographic monitoring. RESULTS: AAV-Cre injection primarily affected the dentate gyrus and inhibited hippocampal mTOR activation following CCI injury. AAV-Cre-injected mice had reduced neuronal death in dentate gyrus detected by Fluoro-Jade B staining and decreased mossy fiber sprouting by ZnT3 immunostaining. Finally, AAV-Cre-injected mice exhibited a decrease in incidence of PTE. SIGNIFICANCE: mTOR pathway activation in dentate gyrus granule cells may at least partly mediate pathological abnormalities and epileptogenesis in models of TBI and PTE. Targeted modulation of mTOR activity in this hippocampal network may represent a focused therapeutic approach for antiepileptogenesis and prevention of PTE.

GABAergic interneurons contribute to the fatal seizure phenotype of CLN2 disease mice.

GABAergic interneuron deficits have been implicated in the epileptogenesis of multiple neurological diseases. While epileptic seizures are a key clinical hallmark of CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), the etiology of these seizures remains elusive. Given that Cln2 R207X/R207X mice display fatal spontaneous seizures and an early loss of several cortical interneuron populations, we hypothesized that those two events might be causally related. To address this hypothesis, we first generated an inducible transgenic mouse expressing lysosomal membrane-tethered TPP1 (TPP1LAMP1) on the Cln2 R207X/R207X genetic background to study the cell-autonomous effects of cell-type-specific TPP1 deficiency. We crossed the TPP1LAMP1 mice with Vgat-Cre mice to introduce interneuron-specific TPP1 deficiency. Vgat-Cre ; TPP1LAMP1 mice displayed storage material accumulation in several interneuron populations both in cortex and striatum, and increased susceptibility to die after PTZ-induced seizures. Secondly, to test the role of GABAergic interneuron activity in seizure progression, we selectively activated these cells in Cln2 R207X/R207X mice using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) in in Vgat-Cre : Cln2 R207X/R207X mice. EEG monitoring revealed that DREADD-mediated activation of interneurons via chronic deschloroclozapine administration accelerated the onset of spontaneous seizures and seizure-associated death in Vgat-Cre : Cln2 R207X/R207X mice, suggesting that modulating interneuron activity can exert influence over epileptiform abnormalities in CLN2 disease. Taken together, these results provide new mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.

A role for δ subunit-containing GABA A receptors on parvalbumin positive neurons in maintaining electrocortical signatures of sleep states.

GABA A receptors containing δ subunits have been shown to mediate tonic/slow inhibition in the CNS. These receptors are typically found extrasynaptically and are activated by relatively low levels of ambient GABA in the extracellular space. In the mouse neocortex, δ subunits are expressed on the surface of some pyramidal cells as well as on parvalbumin positive (PV+) interneurons. An important function of PV+ interneurons is the organization of coordinated network activity that can be measured by EEG; however, it remains unclear what role tonic/slow inhibitory control of PV+ neurons may play in shaping oscillatory activity. After confirming a loss of functional δ mediated tonic currents in PV cells in cortical slices from mice lacking Gabrd in PV+ neurons (PV δcKO), we performed EEG recordings to survey network activity across wake and sleep states. PV δcKO mice showed altered spectral content of EEG during NREM and REM sleep that was a result of increased oscillatory activity in NREM and the emergence of transient high amplitude bursts of theta frequency activity during REM. Viral reintroduction of Gabrd to PV+ interneurons in PV δcKO mice rescued REM EEG phenotypes, supporting an important role for δ subunit mediated inhibition of PV+ interneurons for maintaining normal REM cortical oscillations. Significance statement: The impact on cortical EEG of inhibition on PV+ neurons was studied by deleting a GABA A receptor subunit selectively from these neurons. We discovered unexpected changes at low frequencies during sleep that were rescued by viral reintroduction.

Periodic and aperiodic changes to cortical EEG in response to pharmacological manipulation.

Cortical electroencephalograms (EEGs) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/f) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than excitation/inhibition ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of excitation/inhibition ratio, we analyzed video-EEG during active exploration in mice of both sexes during various pharmacological manipulations with the fitting oscillations and one over f (FOOOF) algorithm. We found that GABAA receptor (GABAAR)-positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABAAR antagonists at subconvulsive doses) did not follow the prediction. To tilt excitation/inhibition ratio more selectively toward excitation, we suppressed the activity of parvalbumin-positive interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of cortical excitation/inhibition ratio.NEW & NOTEWORTHY Neuropsychiatric illness may be associated with altered excitation/inhibition balance. A single electroencephalogram (EEG) parameter, the aperiodic exponent of power spectra, may predict the ratio between excitation and inhibition. Here, we use cortical EEGs in mice to evaluate this hypothesis, using pharmacological manipulations of known mechanism. We show that the aperiodic exponent of EEG power spectra is not a reliable marker of excitation/inhibition ratio. Thus, alternative markers of this ratio must be sought.

Cerebral vascular and blood brain-barrier abnormalities in a mouse model of epilepsy and tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. METHODS: Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. In vivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. RESULTS: VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. SIGNIFICANCE: Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC.

Spatial and amplitude dynamics of neurostimulation: Insights from the acute intrahippocampal kainate seizure mouse model.

OBJECTIVE: Neurostimulation is an emerging treatment for patients with drug-resistant epilepsy, which is used to suppress, prevent, and terminate seizure activity. Unfortunately, after implantation and despite best clinical practice, most patients continue to have persistent seizures even after years of empirical optimization. The objective of this study is to determine optimal spatial and amplitude properties of neurostimulation in inhibiting epileptiform activity in an acute hippocampal seizure model. METHODS: We performed high-throughput testing of high-frequency focal brain stimulation in the acute intrahippocampal kainic acid mouse model of status epilepticus. We evaluated combinations of six anatomic targets and three stimulus amplitudes. RESULTS: We found that the spike-suppressive effects of high-frequency neurostimulation are highly dependent on the stimulation amplitude and location, with higher amplitude stimulation being significantly more effective. Epileptiform spiking activity was significantly reduced with ipsilateral 250 µA stimulation of the CA1 and CA3 hippocampal regions with 21.5% and 22.2% reductions, respectively. In contrast, we found that spiking frequency and amplitude significantly increased with stimulation of the ventral hippocampal commissure. We further found spatial differences with broader effects from CA1 versus CA3 stimulation. SIGNIFICANCE: These findings demonstrate that the effects of therapeutic neurostimulation in an acute hippocampal seizure model are highly dependent on the location of stimulation and stimulus amplitude. We provide a platform to optimize the anti-seizure effects of neurostimulation, and demonstrate that an exploration of the large electrical parameter and location space can improve current modalities for treating epilepsy. PLAIN LANGUAGE SUMMARY: In this study, we tested how electrical pulses in the brain can help control seizures in mice. We found that the electrode's placement and the stimulation amplitude had a large effect on outcomes. Some brain regions, notably nearby CA1 and CA3, responded positively with reduced seizure-like activities, while others showed increased activity. These findings emphasize that choosing the right spot for the electrode and adjusting the strength of electrical pulses are both crucial when considering neurostimulation treatments for epilepsy.

Periodic and aperiodic changes to cortical EEG in response to pharmacological manipulation.

Cortical electroencephalograms (EEG) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/ f ) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition (E/I) balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than E/I ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of E/I ratio, we analyzed active exploration in mice using video EEG following various pharmacological manipulations with the Fitting Oscillations & One Over F (FOOOF) algorithm. We found that GABA A receptor (GABA A R) positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABA A R antagonists at sub-convulsive doses) did not follow the prediction. To tilt E/I ratio more selectively toward excitation, we suppressed the activity of parvalbumin (PV) interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations and studies demonstrating increased cortical activity following PV suppression, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of E/I ratio. Alternatively, the concept of E/I state may be sufficiently oversimplified that it cannot be mapped readily onto an EEG parameter. Significance StateBment: Neuropsychiatric illness is widely prevalent and debilitating. Causes are not well understood, but some hypotheses point toward altered excitation/inhibition (E/I) balance. Here, we use cortical electroencephalograms (EEG) in mice, given applicability of cortical EEG across species, and evaluate the impact of validated drugs, including anxiolytics (pentobarbital and diazepam), along with novel rapid-acting antidepressants (ketamine and allopregnanolone). We focus on analyzing the aperiodic component of EEG power spectra, which may be associated with changes in E/I ratio. We show that aperiodic exponent of EEG power spectra is not a reliable marker of E/I ratio. Moreover, the concept of E/I ratio may be too broad and complex to be defined by an EEG parameter.

Sleep-wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice.

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here, we demonstrated that PR-domain containing protein 13 (Prdm13)+ neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not in old mice. Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control. Furthermore, DMH-specific Prdm13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during SD. These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep fragmentation and increased sleep attempts during SD, whereas these effects of DR were abrogated in DMH-Prdm13-KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated increased sleep attempts during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging.

Gene therapy ameliorates spontaneous seizures associated with cortical neuron loss in a Cln2R207X mouse model.

Although a disease-modifying therapy for classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) exists, poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2R207X mice, which carry one of the most common pathogenic mutations in human patients but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities, including spontaneous seizures, providing a robust, quantifiable, and clinically relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord and differed markedly from the staging seen in mouse models of other forms of neuronal ceroid lipofuscinosis. Neonatal administration of adeno-associated virus serotype 9-mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the life span of Cln2R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging preclinical efficacy of therapeutic interventions for CLN2 disease.

Sleep deprivation exacerbates microglial reactivity and Aß deposition in a TREM2-dependent manner in mice.

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer's disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-ß (Aß) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression. Sleep deprivation not only enhanced TREM2-dependent Aß plaque deposition compared with 5xFAD mice with normal sleeping patterns but also induced microglial reactivity that was independent of the presence of parenchymal Aß plaques. We investigated lysosomal morphology using transmission electron microscopy and found abnormalities particularly in mice without Aß plaques and also observed lysosomal maturation impairments in a TREM2-dependent manner in both microglia and neurons, suggesting that changes in sleep modified neuro-immune cross-talk. Unbiased transcriptome and proteome profiling provided mechanistic insights into functional pathways triggered by sleep deprivation that were unique to TREM2 and Aß pathology and that converged on metabolic dyshomeostasis. Our findings highlight that sleep deprivation directly affects microglial reactivity, for which TREM2 is required, by altering the metabolic ability to cope with the energy demands of prolonged wakefulness, leading to further Aß deposition, and underlines the importance of sleep modulation as a promising future therapeutic approach.

Spatial and Amplitude Dynamics of Neurostimulation: Insights from the Acute Intrahippocampal Kainate Seizure Mouse Model.

Objective: Neurostimulation is an emerging treatment for patients with medically refractory epilepsy, which is used to suppress, prevent, and terminate seizure activity. Unfortunately, after implantation and despite best clinical practice, most patients continue to have persistent seizures even after years of empirical optimization. The objective of this study is to determine optimal spatial and amplitude properties of neurostimulation in inhibiting epileptiform activity in an acute hippocampal seizure model. Methods: We performed high-throughput testing of high-frequency focal brain stimulation in the acute intrahippocampal kainic acid mouse model of temporal lobe epilepsy. We evaluated combinations of six anatomic targets and three stimulus amplitudes. Results: We found that the spike-suppressive effects of high-frequency neurostimulation are highly dependent on the stimulation amplitude and location, with higher amplitude stimulation being significantly more effective. Epileptiform spiking activity was significantly reduced with ipsilateral 250 µA stimulation of the CA1 and CA3 hippocampal regions with 21.5% and 22.2% reductions, respectively. In contrast, we found that spiking frequency and amplitude significantly increased with stimulation of the ventral hippocampal commissure. We further found spatial differences with broader effects from CA1 versus CA3 stimulation. Significance: These findings demonstrate that the effects of therapeutic neurostimulation in an acute hippocampal seizure model are highly dependent on the location of stimulation and stimulus amplitude. We provide a platform to optimize the anti-seizure effects of neurostimulation, and demonstrate that an exploration of the large electrical parameter and location space can improve current modalities for treating epilepsy. Key Points: Evaluated spatial and temporal parameters of neurostimulation in a mouse model of acute seizuresBrief bursts of high-frequency (100 Hz) stimulation effectively interrupted epileptiform activity.The suppressive effect was highly dependent on stimulation amplitude and was maximal at the ipsilateral CA1 and CA3 regions.Pro-excitatory effects were identified with high-amplitude high-frequency stimulation at the ventral hippocampal commissure and contralateral CA1.

Non-sedative cortical EEG signatures of allopregnanolone and functional comparators.

Neurosteroids that positively modulate GABAA receptors are among a growing list of rapidly acting antidepressants, including ketamine and psychedelics. To develop increasingly specific treatments with fewer side effects, we explored the possibility of EEG signatures in mice, which could serve as a cross-species screening tool. There are few studies of the impact of non-sedative doses of rapid antidepressants on EEG in either rodents or humans. Here we hypothesize that EEG features may separate a rapid antidepressant neurosteroid, allopregnanolone, from other GABAA positive modulators, pentobarbital and diazepam. Further, we compared the actions GABA modulators with those of ketamine, an NMDA antagonist and prototype rapid antidepressant. We examined EEG spectra during active exploration at two cortical locations and examined cross-regional and cross-frequency interactions. We found that at comparable doses, the effects of allopregnanolone, despite purported selectivity for certain GABAAR subtypes, was indistinguishable from pentobarbital during active waking exploration. The actions of diazepam had recognizable common features with allopregnanolone and pentobarbital but was also distinct, consistent with subunit selectivity of benzodiazepines. Finally, ketamine exhibited no distinguishing overlap with allopregnanolone in the parameters examined. Our results suggest that rapid antidepressants with different molecular substrates may remain separated at the level of large-scale ensemble activity, but the studies leave open the possibility of commonalities in more discrete circuits and/or in the context of a dysfunctional brain.

Functional neuropathology of neonatal hypoxia-ischemia by single-mouse longitudinal electroencephalography.

OBJECTIVE: Neonatal cerebral hypoxia-ischemia (HI) results in symptomatic seizures and long-term neurodevelopmental disability. The Rice-Vannucci model of rodent neonatal HI has been used extensively to examine and translate the functional consequences of acute and chronic HI-induced encephalopathy. Yet, longitudinal electrophysiological characterization of this brain injury model has been limited by the size of the neonatal mouse's head and postnatal maternal dependency. We overcome this challenge by employing a novel method of longitudinal single-mouse electroencephalography (EEG) using chronically implanted subcranial electrodes in the term-equivalent mouse pup. We characterize the neurophysiological disturbances occurring during awake and sleep states in the acute and chronic phases following newborn brain injury. METHODS: C57BL/6 mice underwent long-term bilateral subcranial EEG and electromyographic electrode placement at postnatal day 9 followed by unilateral carotid cauterization and exposure to 40 minutes of hypoxia the following day. EEG recordings were obtained prior, during, and intermittently after the HI procedure from postnatal day 10 to weaning age. Quantitative EEG and fast Fourier transform analysis were used to evaluate seizures, cortical cerebral dysfunction, and disturbances in vigilance states. RESULTS: We observed neonatal HI-provoked electrographic focal and bilateral seizures during or immediately following global hypoxia and most commonly contralateral to the ischemic injury. Spontaneous chronic seizures were not seen. Injured mice developed long-term asymmetric EEG background attenuation in all frequencies and most prominently during non-rapid eye movement (NREM) sleep. HI mice also showed transient impairments in vigilance state duration and transitions during the first 2 days following injury. SIGNIFICANCE: The functional burden of mouse neonatal HI recorded by EEG resembles closely that of the injured human newborn. The use of single-mouse longitudinal EEG in this immature model can advance our understanding of the developmental and pathophysiological mechanisms of neonatal cerebral injury and help translate novel therapeutic strategies against this devastating condition.

Effects of chronic cannabidiol in a mouse model of naturally occurring neuroinflammation, neurodegeneration, and spontaneous seizures.

Cannabidiol (CBD) has gained attention as a therapeutic agent and is purported to have immunomodulatory, neuroprotective, and anti-seizure effects. Here, we determined the effects of chronic CBD administration in a mouse model of CLN1 disease (Cln1-/-) that simultaneously exhibits neuroinflammation, neurodegeneration, and spontaneous seizures. Proteomic analysis showed that putative CBD receptors are expressed at similar levels in the brains of Cln1-/- mice compared to normal animals. Cln1-/- mice received an oral dose (100 mg/kg/day) of CBD for six months and were evaluated for changes in pathological markers of disease and seizures. Chronic cannabidiol administration was well-tolerated, high levels of CBD were detected in the brain, and markers of astrocytosis and microgliosis were reduced. However, CBD had no apparent effect on seizure frequency or neuron survival. These data are consistent with CBD having immunomodulatory effects. It is possible that a higher dose of CBD could also reduce neurodegeneration and seizure frequency.

Upregulation of the pathogenic transcription factor SPI1/PU.1 in tuberous sclerosis complex and focal cortical dysplasia by oxidative stress.

Tuberous sclerosis complex (TSC) is a congenital disorder characterized by cortical malformations and concomitant epilepsy caused by loss-of-function mutations in the mTOR suppressors TSC1 or TSC2. While the underlying molecular changes caused by mTOR activation in TSC have previously been investigated, the drivers of these transcriptional change have not been fully elucidated. A better understanding of the perturbed transcriptional regulation could lead to the identification of novel pathways for therapeutic intervention not only in TSC, but other genetic epilepsies in which mTOR activation plays a key role, such as focal cortical dysplasia 2b (FCD). Here, we analyzed RNA sequencing data from cortical tubers and a tsc2-/- zebrafish. We identified differential expression of the transcription factors (TFs) SPI1/PU.1, IRF8, GBX2, and IKZF1 of which SPI1/PU.1 and IRF8 targets were enriched among the differentially expressed genes. Furthermore, for SPI1/PU.1 these findings were conserved in TSC zebrafish model. Next, we confirmed overexpression of SPI1/PU.1 on the RNA and protein level in a separate cohort of surgically resected TSC tubers and FCD tissue, in fetal TSC tissue, and a Tsc1GFAP-/- mouse model of TSC. Subsequently, we validated the expression of SPI1/PU.1 in dysmorphic cells with mTOR activation in TSC tubers. In fetal TSC, we detected SPI1/PU.1 expression prenatally and elevated RNA Spi1 expression in Tsc1GFAP-/- mice before the development of seizures. Finally, in vitro, we identified that in astrocytes and neurons SPI1 transcription was driven by H2 O2 -induced oxidative stress, independent of mTOR. We identified SPI1/PU.1 as a novel TF involved in the pro-inflammatory gene expression of malformed cells in TSC and FCD 2b. This transcriptional program is activated in response to oxidative stress and already present prenatally. Importantly, SPI1/PU.1 protein appears to be strictly limited to malformed cells, as we did not find SPI1/PU.1 protein expression in mice nor in our in vitro models.

Astrocyte deletion of α2-Na/K ATPase triggers episodic motor paralysis in mice via a metabolic pathway.

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.

Early developmental electroencephalography abnormalities, neonatal seizures, and induced spasms in a mouse model of tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is one of the most common genetic causes of epilepsy. Seizures in TSC typically first present in infancy or early childhood, including focal seizures and infantile spasms. Infantile spasms in TSC are particularly characteristic in its strong responsiveness to vigabatrin. Although a number of mouse models of epilepsy in TSC have been described, there are very limited electroencephalographic (EEG) or seizure data during the preweanling neonatal and infantile-equivalent mouse periods. Tsc1GFAP CKO mice are a well-characterized mouse model of epilepsy in TSC, but whether these mice have seizures during early development has not been documented. The objective of this study was to determine whether preweanling Tsc1GFAP CKO mice have developmental EEG abnormalities or seizures, including spasms. METHODS: Longitudinal video-EEG and electromyographic recordings were performed serially on Tsc1GFAP CKO and control mice from postnatal days 9-21 and analyzed for EEG background abnormalities, sleep-wake vigilance states, and spontaneous seizures. Spasms were also induced with varying doses of N-methyl-D-aspartate (NMDA). RESULTS: The interictal EEG of Tsc1GFAP CKO mice had excessive discontinuity and slowing, suggesting a delayed developmental progression compared with control mice. Tsc1GFAP CKO mice also had increased vigilance state transitions and fragmentation. Tsc1GFAP CKO mice had spontaneous focal seizures in the early neonatal period and a reduced threshold for NMDA-induced spasms, but no spontaneous spasms were observed. SIGNIFICANCE: Neonatal Tsc1GFAP CKO mice recapitulate early developmental aspects of EEG abnormalities, focal seizures, and an increased propensity for spasms. This mouse model may be useful for early mechanistic and therapeutic studies of epileptogenesis in TSC.

Hypothalamic orexin and mechanistic target of rapamycin activation mediate sleep dysfunction in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disease related to hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and manifested by neurological symptoms, such as epilepsy and sleep disorders. The pathophysiology of sleep dysfunction is poorly understood and is likely multifactorial, but may involve intrinsic biological regulators in the brain. Here, we characterized a mouse model of sleep disorders in TSC and investigated mechanisms of sleep dysfunction in this conditional knockout model involving inactivation of the Tsc1 gene in neurons and astrocytes (Tsc1GFAPCKO mice). Sleep studies utilizing EEG, EMG, and behavioral analysis found that Tsc1GFAPCKO mice have decreased REM sleep and impaired sleep-wake differentiation between light and dark phases. mTOR activity and orexin expression were increased in hypothalamic sections and cultured hypothalamic neurons from Tsc1GFAPCKO mice. Both the sleep abnormalities and increased orexin expression in Tsc1GFAPCKO mice were reversed by rapamycin treatment, indicating their dependence on mTOR activation. An orexin antagonist, suvorexant, also restored normal REM levels in Tsc1GFAPCKO mice. These results identify a novel mechanistic link between mTOR and orexin in the hypothalamus related to sleep dysfunction and suggest a targeted therapeutic approach to sleep disorders in TSC.

Adipose tissue NAD+ biosynthesis is required for regulating adaptive thermogenesis and whole-body energy homeostasis in mice.

Nicotinamide adenine dinucleotide (NAD+) is a critical coenzyme for cellular energy metabolism. The aim of the present study was to determine the importance of brown and white adipose tissue (BAT and WAT) NAD+ metabolism in regulating whole-body thermogenesis and energy metabolism. Accordingly, we generated and analyzed adipocyte-specific nicotinamide phosphoribosyltransferase (Nampt) knockout (ANKO) and brown adipocyte-specific Nampt knockout (BANKO) mice because NAMPT is the rate-limiting NAD+ biosynthetic enzyme. We found ANKO mice, which lack NAMPT in both BAT and WAT, had impaired gene programs involved in thermogenesis and mitochondrial function in BAT and a blunted thermogenic (rectal temperature, BAT temperature, and whole-body oxygen consumption) response to acute cold exposure, prolonged fasting, and administration of ß-adrenergic agonists (norepinephrine and CL-316243). In addition, the absence of NAMPT in WAT markedly reduced adrenergic-mediated lipolytic activity, likely through inactivation of the NAD+-SIRT1-caveolin-1 axis, which limits an important fuel source fatty acid for BAT thermogenesis. These metabolic abnormalities were rescued by treatment with nicotinamide mononucleotide (NMN), which bypasses the block in NAD+ synthesis induced by NAMPT deficiency. Although BANKO mice, which lack NAMPT in BAT only, had BAT cellular alterations similar to the ANKO mice, BANKO mice had normal thermogenic and lipolytic responses. We also found NAMPT expression in supraclavicular adipose tissue (where human BAT is localized) obtained from human subjects increased during cold exposure, suggesting our finding in rodents could apply to people. These results demonstrate that adipose NAMPT-mediated NAD+ biosynthesis is essential for regulating adaptive thermogenesis, lipolysis, and whole-body energy metabolism.

Mild chronic perturbation of inhibition severely alters hippocampal function.

Pentameric GABAA receptors mediate a large share of CNS inhibition. The γ2 subunit is a typical constituent. At least 11 mutations in the γ2 subunit cause human epilepsies, making the role of γ2-containing receptors in brain function of keen basic and translational interest. How small changes to inhibition may cause brain abnormalities, including seizure disorders, is unclear. In mice, we perturbed fast inhibition with a point mutation T272Y (T6'Y in the second membrane-spanning domain) to the γ2 subunit. The mutation imparts resistance to the GABAA receptor antagonist picrotoxin, allowing verification of mutant subunit incorporation. We confirmed picrotoxin resistance and biophysical properties in recombinant receptors. T6'Y γ2-containing receptors also exhibited faster deactivation but unaltered steady-state properties. Adult T6'Y knockin mice exhibited myoclonic seizures and abnormal cortical EEG, including abnormal hippocampal-associated theta oscillations. In hippocampal slices, picrotoxin-insensitive inhibitory synaptic currents exhibited fast decay. Excitatory/inhibitory balance was elevated by an amount expected from the IPSC alteration. Partial pharmacological correction of γ2-mediated IPSCs with diazepam restored total EEG power toward baseline, but had little effect on the abnormal low-frequency peak in the EEG. The results suggest that at least part of the abnormality in brain function arises from the acute effects of truncated inhibition.