Drug-resistant epilepsy affects one-third of patients; the ketogenic diet offers a metabolic alternative, but poor tolerability limits adherence. Ketone esters provide an alternative, though the optimal route is unclear. Prefrontal cortex proteomics was profiled in Kcna1-/- mice, a model of human temporal lobe epilepsy, treated with KE by either oral gavage or subcutaneous injection. Oral gavage predominantly altered proteins associated with synaptic vesicle trafficking, postsynaptic organization, and neurotransmitter transport, a profile consistent with enhanced synaptic plasticity and neuronal signaling. Subcutaneous injection induced widespread downregulation of proteins involved in synaptic structure, mitochondrial translation, and ubiquitination consistent with bioenergetic remodelling and proteostasis.

Despite the availability of nearly 40 approved anti-seizure medications (ASMs), at least one-third of individuals with epilepsy remain refractory to treatment, and many experience life-limiting cognitive or psychiatric side effects. Using a machine learning-guided platform, we identified PDE4 as an underexplored anti-seizure target, which became further validated based on its enriched expression in seizure-relevant brain regions and its potential to modulate excitatory/inhibitory neuronal tone via cAMP signaling. The pan-PDE4 inhibitor crisaborole partially protected against hyperthermia-induced seizures and reduced spontaneous seizures in Scn1a+/- mice, while rolipram and roflumilast showed no efficacy at tolerable doses. SN-2000, a first-in-kind allosteric modulator of PDE4B, was rationally designed for isoform selectivity and brain penetration, and demonstrated versatile reduction of seizure activity across multiple zebrafish and rodent genetic and acquired epilepsy models, with efficacy comparable to standard-of-care ASMs. SN-2000 also demonstrated favorable behavioral outcomes, reducing post-ictal aggression and anxiety-like behaviors, and improving cognitive performance in both wild-type and epileptic mice. These effects were linked to paradoxical regulation of excitatory and neuronal activity in the cortex and thalamus of epileptic mice, respectively, as well as elevated cAMP signaling and downstream pCREB activation. Together, these findings support PDE4B inhibition as a disease-relevant mechanism in epilepsy, and position SN-2000 as a promising therapeutic candidate offering seizure control without the neuropsychiatric burden of existing ASMs and potential pro-cognitive properties. One Sentence Summary SN-2000, a novel allosteric PDE4 inhibitor, reduces seizure activity and shows psychiatric-neutral and pro-cognitive properties in preclinical models ### Competing Interest Statement CDK, JMR, and DMK are employees and shareholders of Stream Neuroscience Inc. MK is an employee of Stream Neuroscience Inc. KI is a shareholder of Stream Neuroscience Inc.

The gut plays a central role in translating dietary signals into systemic health effects, making it a key mediator of the ketogenic diet (KD), a high fat, low carbohydrate regimen. This review synthesizes current knowledge on the interaction between the KD and the gut, emphasizing gut-mediated mechanisms as an interface between dietary interventions and systemic health outcomes, spanning gastrointestinal to neurological health. Topics address gut physiology (gut digestion and absorption, epithelial nutrient sensing, gut motility), intestinal immunity (covering innate, adaptive, and antiviral responses), and extracellular to intracellular processes (i.e. mitochondrial function, stem cell fate, and intestinal circadian rhythm). Special focus is given to the gut microbiome, including both bacterial and fungal communities and how the KD modulates them in conditions such as epilepsy, obesity, traumatic brain injury, and multiple sclerosis. Innovative methods for tailoring the KD, including the use of alternative formulations, ketone esters, and microbiome-focused interventions such as prebiotics and probiotics are examined. Strategies to maximize the diet's benefits while reducing potential side effects are considered. Together, these insights herein offer a comprehensive framework for understanding the interactions between the KD and the gut, a prerequisite for optimizing the overall health benefits of metabolism-based treatments.

BACKGROUND:While initiation and maintenance guidelines for ketogenic diet therapies (KDT) are well established, evidence guiding KDT discontinuation remains limited. We aim to explore practices surrounding KDT weaning and identify key factors influencing clinical decisions. METHODS:An online 24-question survey was distributed via the International Neurological Ketogenic Society (INKS) between March and May 2024. Responses from 53 healthcare professionals (HCPs), including dietitians and physicians, from diverse global regions were analyzed. RESULTS:Most HCPs adopt individualized KDT weaning protocols, often after 2 years of effective KDT, with weaning durations typically lasting 2 to 6 months. Factors influencing discontinuation include seizure control, epilepsy syndrome, diet tolerance, and family preferences. EEG monitoring and ketone tracking during weaning are common practices. For effective KDT, a gradual reduction in dietary ratio is preferred, with various strategies used (e.g., stepwise ratio reduction, liberalizing carbohydrates). Adherence issues and concerns about seizure recurrence are frequent challenges. CONCLUSION:KDT weaning remains a heterogeneous and under-studied aspect of treatment. Despite variations in protocols, common themes include cautious tapering and syndrome-specific considerations. These findings highlight the need for prospective studies to establish evidence-based data for KDT discontinuation.

Infantile epileptic spasms syndrome carries high morbidity and mortality compared with other childhood epilepsy syndromes. High-dose prednisolone is considered the first-line option by many neurologists, with efficacy comparable to adrenocorticotropic hormone (ACTH). The second-line therapy is debated if prednisolone fails to induce remission. The objective of this study was to evaluate the efficacy of adrenocorticotropic hormone versus vigabatrin as second-line antiseizure medication in patients with infantile epileptic spasms syndrome after high-dose prednisolone failure. Thirty-eight patients met the inclusion criteria. Seventeen patients (45%) took vigabatrin and 21 (55%) took adrenocorticotropic hormone. There were no significant differences regarding age of infantile spasms onset ( P = .37) or sex between both groups ( P = .32). Complete control of epileptic spasms was seen in 5 of 17 patients (29.5%) taking vigabatrin and in 4 of 21 (19%) taking adrenocorticotropic hormone ( P = .70). Vigabatrin showed higher efficacy; however, it did not reach statistical significance, possibly because of our sample size. These results may help guide family counseling after the failure of high-dose prednisolone as first-line therapy.

The ketogenic diet (KD) is an established treatment for patients with medically intractable epilepsy and is chiefly characterized by high fat/low-carbohydrate intake and the production of ketone bodies (KB) such as β-hydroxybutyrate (BHB). However, after more than a century of clinical use, the mechanisms underlying its efficacy remain unclear. While prior investigations have examined the effects of the KD and its metabolic substrates on synaptic transmission, few studies have explored a potential connection between astrocytic ion channels and seizure genesis. One essential function of astrocytes is spatial potassium buffering which influences passive potassium conductance (PPC), and when impaired, can result in neuronal hyperexcitability. In the present study, we demonstrate that the KD can mitigate hippocampal astrogliosis in the Kcna1 -null (KO) mouse model of developmental epilepsy. Specifically, we observed a significant increase in GFAP expression in KO mice fed a control diet compared to wild-type (WT) mice, and that the KD prevented this change. Furthermore, we noted a reduction in hippocampal astrocytic PPC in epileptic mice, whereas KD-treated KO animals exhibited nearly normal passive conductance levels. In this regard, we found that while Kir4.1, TREK-1 and TWIK-1 RNA expression levels were not significantly altered by KD treatment in either WT or KO mice, BHB appeared to only minimally affect Kir4.1-mediated currents in transfected HEK cells. Furthermore, bulk RNA-seq analysis of the various treatment groups revealed KD-induced down-regulation of factors linked to hippocampal astrogliosis. Our findings indicate that the KD protects against epilepsy-associated astrogliosis and astrocytic PPC changes, underscoring a novel mechanism of action, and implicate extracellular potassium in its anti-seizure effects. ### Competing Interest Statement The authors have declared no competing interest.

Deficits in social communication and language development are a hallmark of autism spectrum disorder currently with no effective approaches to reduce the negative impact. Interventional studies using animal models have been very limited in demonstrating improved vocal communication. Autism has been proposed to involve metabolic dysregulation. Ketogenic diet (KD) is a metabolism-based therapy for medically intractable epilepsy, and its applications in other neurological conditions have been increasingly tested. However, how KD would affect vocal communication has not been explored. The BTBR mouse strain is widely used to model asocial phenotypes. They display robust and pronounced deficits in vocalization during social interaction, and have metabolic changes implicated in autism. We investigated the effects of KD on ultrasonic vocalizations (USVs) in juvenile and adult BTBR mice during male-female social encounters. After a brief treatment with KD, the number, spectral bandwidth, and much of the temporal structure of USVs were robustly closer to control levels in both juvenile and adult BTBR mice. Composition of call categories and transitioning between individual call subtypes were more effectively altered to more closely align with the control group in juvenile BTBR mice. Together, our data provide further support to the hypothesis that metabolism-based dietary intervention could modify disease expression, including core symptoms, in autism. Future studies should tease apart the molecular mechanisms of KD's effects on vocalization.

OBJECTIVE:Despite growing interest in the potential use of exogenous ketones for the treatment of epilepsy, their impact on seizures and the gut microbiome and mycobiome remain unclear. METHODS:Here, we examined the effects of both oral gavage and subcutaneous (SC) injection of a ketone ester (KE) in spontaneously epileptic Kcna1-null (KO) mice that model seminal aspects of human temporal lobe epilepsy. Electroencephalographic recordings and biochemical analyses were performed in KE-treated KO mice. Fecal microbial and fungal communities were profiled to determine whether the antiseizure activity of KE involves changes in the gut microbiome. RESULTS:We found that exogenous KE administration by SC injection was more effective than oral gavage in terms of rendering antiseizure effects while generating similar degrees of ketonemia. However, reductions in mean daily seizure counts were accompanied by overall alterations in the fecal bacterial microbiome. Either oral or SC injection imposed a greater impact on the microbiome in male than female mice. In males, oral KE decreased Bacteroidota phylum and genera of Ligilactobacillus and Muribaculaceae, whereas SC injection decreased Bacteroides, Lactobacillus, and Lachnospiraceae. The fecal mycobiome was affected by KE injection to a greater degree than by oral gavage, and more in females than in males, as reflected by an increase in Ascomycota and Saccharomyces. Correlation analysis between microbiome and seizure counts revealed that in mice receiving KE injection, the seizure count was positively correlated with an amplicon sequencing variant of Lactobacillus (Spearman rho = .64, p = .03) and tended toward a negative correlation with Saccharomyces (Spearman rho = -.57, p = .057). SIGNIFICANCE:Our findings demonstrate that exogenous ketone administration alone can induce antiseizure effects equally via different routes of administration, and that they induce differential shifts in both the bacterial microbiome and mycobiome.

A 6-year-old boy presented with 4 months progressive verbal comprehension difficulties, declining school performance, strabismus, and visual impairment. General examination noted moderate hypotension and hyperpigmented skin. Pertinent neurologic examination abnormalities included difficulty with comprehension, decreased visual acuity (20/50 bilaterally), disconjugate gaze, bilateral dysmetria, and unsteady gait. CSF protein was elevated (1.82 g/L). Brain MRI showed posterior cerebrum and brainstem abnormalities consistent with adrenoleukodystrophy(1) (figure). Morning cortisol was decreased (21 nM) and adrenocorticotrophic hormone stimulation test confirmed adrenal insufficiency. Diagnosis was confirmed by very long chain fatty acid analysis (elevated C26:0/C22:0 and C24:0/C22:0 ratios) and identification of pathogenic ABCD1 mutation.

Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack of preventative treatments. Based on the observation that discrete seizures lead to local and postictal vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain we reasoned those similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of seizure-induced premature mortality, ibuprofen and nicardipine extended life while systemic drug levels remained high enough to be effective. We also examined the potential role of spreading depolarization in the acute model of seizure-induced premature mortality. These data provide a proof-of-principle for the neurovascular hypothesis of SUDEP rather than spreading depolarization and the use of currently available drugs to prevent it.

Background Deficits in social communication and language development is a hallmark of autism spectrum disorder currently with no cure. Interventional studies using animal models have been very limited in demonstrating improved vocal communication. Autism has been proposed to involve metabolic dysregulation. Ketogenic diet (KD) is a metabolism-based therapy for medically intractable epilepsy, and its applications in other neurological conditions have been increasingly tested. However, how it would affect vocal communication has not been explored. The BTBR mouse strain is considered a model of idiopathic autism. They display robust deficits in vocalization during social interaction, and have metabolic changes implicated in autism. Methods We investigated the effects of KD on ultrasonic vocalizations (USVs) in juvenile and adult BTBR mice during male-female social encounters. Results After a brief treatment with KD, the amount, spectral bandwidth, and much of the temporal structure of USVs were robustly improved in both juvenile and adult BTBR mice. Composition of call categories and transitioning between individual call subtypes was more effectively improved in juvenile BTBR mice. Limitations Although sharing certain attributes, mouse vocalization is unlikely to model all aspects in the development and deficits of human language. KD is highly restrictive and can be difficult to administer, especially for many people with autism who have narrow food selections. Side effects and potential influence on development should also be considered. Future studies are required to tease apart the molecular mechanisms of KD’s effects on vocalization. Conclusions Together, our data provide further support to the hypothesis that metabolism-based dietary intervention could modify disease expression, including core symptoms, in autism.

Background Extracellular signal-regulated kinase (ERK/MAPK) pathway in the brain is hypothesized to be a critical convergent node in the development of autism spectrum disorder. We reasoned that selectively targeting this pathway could reverse core autism-like phenotype in animal models.Methods Here we tested a clinically relevant, selective inhibitor of ERK pathway, PD325901 (Mirdametinib), in a mouse model of idiopathic autism, the BTBR mice.Findings We report that treating juvenile mice with PD325901 reduced ERK pathway activation, dose and duration -dependently reduced core disease-modeling deficits in sociability, vocalization and repetitive behavior, and reversed abnormal EEG signals. Further analysis revealed that subchronic treatment did not affect weight gain, locomotion, or neuronal density in the brain. Parallel treatment in the C57BL/6J mice did not alter their phenotype.Interpretation Our data indicate that selectively inhibiting ERK pathway using PD325901 is beneficial in the BTBR model, thus further support the notion that ERK pathway is critically involved in the pathophysiology of autism. These results suggest that a similar approach could be applied to animal models of syndromic autism with dysre-gulated ERK signaling, to further test selectively targeting ERK pathway as a new approach for treating autism.Funding : This has beenwork was supported by Alberta Children's Hospital Research Foundation (JMR & NC), University of Calgary Faculty of Veterinary Medicine (NC), Kids Brain Health Network (NC), and Natural Sciences and Engineering Research Council of Canada (NC).Copyright (c) 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2023;91:

Prolonged severe hypoxia follows brief seizures and represents a mechanism underlying several negative postictal manifestations without interventions. Approximately 50% of the postictal hypoxia phenomenon can be accounted for by arteriole vasoconstriction. What accounts for the rest of the drop in unbound oxygen is unclear. Here, we determined the effect of pharmacological modulation of mitochondrial function on tissue oxygenation in the hippocampus of rats after repeatedly evoked seizures. Rats were treated with mitochondrial uncoupler 2,4 dinitrophenol (DNP) or antioxidants. Oxygen profiles were recorded using a chronically implanted oxygen-sensing probe, before, during, and after seizure induction. Mitochondrial function and redox tone were measured using in vitro mitochondrial assays and immunohistochemistry. Postictal cognitive impairment was assessed using the novel object recognition task. Mild mitochondrial uncoupling by DNP raised hippocampal oxygen tension and ameliorated postictal hypoxia. Chronic DNP also lowered mitochondrial oxygen-derived reactive species and oxidative stress in the hippocampus during postictal hypoxia. Uncoupling the mitochondria exerts therapeutic benefits on postictal cognitive dysfunction. Finally, antioxidants do not affect postictal hypoxia, but protect the brain from associated cognitive deficits. We provided evidence for a metabolic component of the prolonged oxygen deprivation that follow seizures and its pathological sequelae. Furthermore, we identified a molecular underpinning of this metabolic component, which involves excessive oxygen conversion into reactive species. Mild mitochondrial uncoupling may be a potential therapeutic strategy to treat the postictal state where seizure control is absent or poor.

Lennox-Gastaut syndrome (LGS) is a severe, chronic, complex form of early childhood-onset epilepsy characterized by multiple seizure types, generalized slow (<2.5 Hz) spike-and-wave activity and other electroencephalography abnormalities, and cognitive impairment. A key treatment goal is early seizure control, and several anti-seizure medications (ASMs) are available. Due to the low success rate in achieving seizure control with monotherapy and an absence of efficacy data supporting any particular combination of ASMs for treating LGS, a rational approach to selection of appropriate polytherapy should be applied to maximize benefit to patients. Such "rational polytherapy" involves consideration of factors including safety (including boxed warnings), potential drug-drug interactions, and complementary mechanisms of action. Based on the authors' clinical experience, rufinamide offers a well-considered first adjunctive therapy for LGS, particularly in combination with clobazam and other newer agents for LGS, and may be particularly useful for reducing the frequency of tonicatonic seizures associated with LGS.