Infantile spasms are seizures manifesting within a spectrum of epileptic encephalopathies of infancy that often lead to cognitive impairment. Their current therapies, including adrenocorticotropic hormone (ACTH), high dose steroids, or vigabatrin, are not always effective and may be associated with serious side effects. Overactivation of the TORC1 complex of the mTOR pathway is implicated in the pathogenesis of certain genetic and acquired disorders that are linked with infantile spasms, like tuberous sclerosis. Here, we tested the therapeutic potential of rapamycin, a TORC1 inhibitor, as a potential treatment for infantile spasms in the multiple-hit rat model of ACTH-refractory symptomatic infantile spasms, which is not linked to tuberous sclerosis. Rapamycin or vehicle were given after spasms appeared. Their effects on spasms, other seizures, performance in Barnes maze, and expression of the phosphorylated S6 ribosomal protein (pS6: a TORC1 target) in the cortex, using immunofluorescence, were compared. Rapamycin suppressed spasms dose-dependently and improved visuospatial learning, although it did not reduce the frequency of other emerging seizures. High-dose pulse rapamycin effected acute and sustained suppression of spasms and improved cognitive outcome, without significant side effects. Therapeutically effective rapamycin doses normalized the pS6 expression, which was increased in perilesional cortical regions of pups with spasms. These findings support that pathological overactivation of TORC1 may be implicated in the pathogenesis of infantile spasms, including those that are not linked to tuberous sclerosis. Furthermore, a high-dose, pulse rapamycin treatment is a promising, well tolerated and disease-modifying new therapy for infantile spasms, including those refractory to ACTH.

The incidence of seizures is particularly high in the early ages of life. The immaturity of inhibitory systems, such as GABA, during normal brain development and its further dysregulation under pathological conditions that predispose to seizures have been speculated to play a major role in facilitating seizures. Seizures can further impair or disrupt GABAA signaling by reshuffling the subunit composition of its receptors or causing aberrant reappearance of depolarizing or hyperpolarizing GABAA receptor currents. Such effects may not result in epileptogenesis as frequently as they do in adults. Given the central role of GABAA signaling in brain function and development, perturbation of its physiological role may interfere with neuronal morphology, differentiation, and connectivity, manifesting as cognitive or neurodevelopmental deficits. The current GABAergic antiepileptic drugs, while often effective for adults, are not always capable of stopping seizures and preventing their sequelae in neonates. Recent studies have explored the therapeutic potential of chloride cotransporter inhibitors, such as bumetanide, as adjunctive therapies of neonatal seizures. However, more needs to be known so as to develop therapies capable of stopping seizures while preserving the age- and sex-appropriate development of the brain.

Intracellular pH (pH i ) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell-cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pH i affects junctional conductance (g j ) and g j dependence on transjunctional voltage (V j ). To characterize V j gating, we fit the g j -V j relation using a stochastic four-state model containing one V j -sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V j across it. Using the model, we estimated gating parameters characterizing sensitivity to V j and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH i changes from 7.2 to ∼8.0 shifted g j -V j dependence of Cx45 aHCs along the V j axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g j dependence on V j . In contrast, acidification shifted g j -V j dependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal g j achieved under alkaline conditions showed that only ∼4% of channels were functional. The acid dissociation constant (pK a ) of g j -pH i dependence of Cx45/Cx45 GJs was ∼7. The pK a of heterotypic Cx45/Cx43-EGFP GJs was lower, ∼6.7, between the pK a s of Cx45 and Cx43-EGFP (∼6.5) homotypic GJs. In summary, pH i significantly modulates junctional conductance of Cx45 by affecting both V j gating and number of functional channels.cell-cell coupling | pH-dependent gating | EGFP | hemichannel | connexon C hanges in intracellular pH (pH i ) take place under different physiological and pathological conditions, and H + ions have a broad effect on cell function including cell-cell electrical and metabolic communication mediated by gap junctions (GJs) and paracrine signaling through nonjunctional/unapposed hemichannels (1-4). Modest pH i changes have been observed under normal physiological conditions [e.g., changes of neuronal activity or the resting potential (5, 6)], and greater changes occur under pathological conditions such as hypoxia, ischemia, or epilepsy (4,7,8).During the last decade, significant progress has been made toward understanding the molecular mechanisms of pH-dependent modulation of GJs and hemichannels (2, 9-12). Several domains in the cytoplasmic loop and C terminus of connexin43 (Cx43) appear to be involved in pH-dependent gating (2, 9, 11, 12). Furthermore, pH-dependent interaction of connexins with other cytoplasmic proteins may be important in the remodeling of connexins and in protection from lesion spread after local ischemic injury (13,14).GJs provide channels with an inner diameter of ∼1.4 nm between the interiors of the coupled cells. This link allows the spread of electrical potential and small metabolites. Each GJ cha...

SUMMARY Objective Infantile spasms (IS) have poor outcomes and limited treatment options, including vigabatrin, a GABA aminotransferase inactivator. Vigabatrin has been associated with retinal toxicity. A high affinity vigabatrin analogue (CPP-115, Catalyst Pharmaceutical Partners) has shown lower risk of retinal toxicity. Here, we test the efficacy of CPP-115 in reducing spasms and its tolerability in the multiple-hit rat model of IS, in which daily vigabatrin reduced spasms only for one day, but was not well tolerated. Methods Male rats were treated with the protocol of the multiple-hit model of IS at postnatal day 3 (PN3). Using a randomized, blinded, vehicle-controlled, dose-response study design, CPP-115 [0.1, 1, or 5 mg/kg intraperitoneally (i.p.)] or vehicle were given daily (PN4-12) or as single injection (PN7) after spasms onset. Intermittent video- or video-EEG monitoring was done. Secondary endpoints included: daily weights, survival, performance on open field activity, surface righting time, and negative geotaxis (PN3-20), horizontal bar (PN13-20), Barnes maze (PN16-19). Statistics used a linear mixed model of raw or normalized log-transformed data, taking into account the repeated observations on each animal. Results The lower CPP-115 doses (0.1–1 mg/kg/day, PN4-12) reduced spasms between PN6-7 without increasing mortality. CPP-115 at 5 mg/kg/day (PN4-12) reduced spasms earlier (PN5), but was eventually lethal. A single CPP-115 injection (1mg/kg i.p.) decreased electroclinical spasms acutely but transiently. CPP-115 transiently improved the probability to >50% reduction of spasms, but did not accelerate spasms cessation. CPP-115 did not alter neurodevelopmental outcomes or visuospatial learning. Significance We provide proof-of-concept evidence that CPP-115, a vigabatrin analogue, decreases spasms in the multiple-hit rat model of IS at considerably lower and better tolerated doses than vigabatrin did in our previous studies. Further optimization of the treatment protocol is needed. CPP-115 may be a promising new candidate treatment for IS with better tolerability than vigabatrin.