Stephen J. Moss

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe developmental and epileptic encephalopathy characterized by early onset drug-resistant seizures and later cognitive and social impairments. Existing therapies primarily involve antiseizure medications, which have sedative side effects and lack effective treatments for behavioral impairments. Potassium chloride cotransporter (KCC2) activity is regulated by phosphorylation and is a crucial component of the GABAergic inhibitory system. However, KCC2 dysfunction in CDD remains poorly understood. Here, to investigate potential KCC2 dysfunction, we used a constitutive Cdkl5 knockout mouse model of CDD. We used liquid chromatography coupled with tandem mass spectrometry and quantitative analysis to examine the unbiased phosphorylation of KCC2. We observed aberrant KCC2 phosphorylation and reduced expression, suggesting reduced KCC2 activity. Examining developmental KCC2 changes revealed significant alterations in key phosphorylation residues and decreased expression between postnatal days 14 and 21. Treatment with the KCC2 activator (OV350) between p10 and p21 saw a significant reduction in infantile spasms compared to vehicle-treated Cdkl5 knockout mice. Remarkably, when these mice were adults, the mice that received OV350 as pups had reduced seizure susceptibility and their cognitive and behavioral deficits were alleviated. These findings indicate that enhancing KCC2 function during a critical developmental window may be a promising therapeutic strategy for CDD and other developmental and epileptic encephalopathies.

Rett syndrome is an X-linked neurodevelopmental disorder resulting from mutations in the MeCP2 gene, leading to intellectual disability, impaired motor coordination, decreased sociability, and seizures. Central to the underlying pathophysiology are deficits in synaptic inhibition, which are mediated by hyperpolarizing GABA _A R currents. These events develop postnatally and are dependent upon increased neuronal Cl ^- extrusion mediated by SLC12A5 (KCC2). Therefore, we tested whether its activation modifies the disease phenotypes evident in female MeCP2 ^+/- mice, using OV350, a direct activator of KCC2. OV350 rapidly induced a sustained reduction in EEG power, accompanied by a decrease in the severity of epileptic discharges. Increased motor coordination, sociability, and spatial memory were also observed. Deficits in KCC2 phosphorylation were also seen in MeCP2 ^+/- mice, consistent with reductions in its activity that were also ameliorated by OV350. Thus, KCC2 activation may be efficacious in limiting the impact of Rett syndrome and other neurodevelopmental disorders.

Introduction: The potassium chloride co-transporter 2 (KCC2) is the principal Cl⁻ extrusion mechanism employed by mature neurons in the central nervous system (CNS) and plays a critical role in determining the efficacy of fast synaptic inhibition mediated by type A γ-aminobutyric acid receptors (GABA_ARs) to protect against epileptogenesis. It has previously been demonstrated that epileptic seizures down-regulate KCC2 and induce neuronal apoptosis through the extrinsic apoptotic pathway. However, the mechanism by which neuronal death is induced by KCC2 loss remains unknown. We have previously demonstrated that C1q copurifies with KCC2 in comparable amounts. C1q is responsible for synaptic elimination in the brain during development, aging and neurodegeneration.Methods: Here, we studied apoptotic induction in models of KCC2 loss of function and demonstrated the importance of C1q in this process using a constitutive C1qKO mouse model. We characterized the activation of different apoptotic pathways by measuring caspase 8 and caspase 9 cleavage as markers of extrinsic and intrinsic apoptosis, respectively.Results: This study demonstrates in vitro, ex vivo and following seizures in vivo, that reduced KCC2 function coincides with neuronal death by activating the extrinsic apoptotic pathway, which is contingent upon complement C1q. Moreover, kainic acid (KA)- and glutamate-induced excitotoxicity also selectively activates the extrinsic apoptotic pathway which is contingent upon C1q.Discussion: These results strongly support the hypothesis that the KCC2/C1q protein complex plays a critical role in the apoptotic process that occurs following loss of KCC2 function.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a developmental and epileptic encephalopathy (DEE) characterized by severe drug-resistant epileptic disorders beginning in early childhood, along with cognitive and social impairments in later childhood and adulthood. Existing pharmacological therapies for CDD primarily focus on anti-seizure medications, which often have associated sedative side effects. In addition, there are currently no effective treatments for cognitive or behavioral impairments associated with this disorder. Postnatal development expression of CDKL5 has a similar timeline as the developmental activity of the potassium chloride co-transporter (KCC2), the maturation of which is a prerequisite for the developmental switch to fast synaptic hyperpolarizing inhibition mediated by g-aminobutyric acid type A receptors (GABA _A R). This developmental GABA switch is determined by changes in the phosphorylation of multiple residues in KCC2. During this initial postnatal period, dramatic changes occur as major neuronal circuits are formed, laying down the initial pathways important for memory consolidation and behavioral processing. Currently, a knowledge gap exists in understanding KCC2 dysfunction in CDD. In adult Cdkl5 KO mice we found aberrant KCC2 phosphorylation and expression, such that KCC2 phosphorylation profile appeared immature. We examined the developmental changes in KCC2 and observed significant alterations in the phosphorylation of key residues and decreased KCC2 expression from p14 to p21. Because KCC2 loss-of-function has been strongly correlated with excessive neuronal excitation, cognitive and behavioral impairments, we examined seizure susceptibility, spatial memory, and social interaction in adult Cdkl5 KO mice following once daily administration of the KCC2 activator (OV350), or vehicle, to infant Cdkl5 KO mice. We found that adult Cdkl5 KO mice are more susceptible to kainate-induced seizures, show poor sociability and deficits in spatial learning and memory compared to WT mice. Twelve days of OV350 treatment as infants (p10 to p21) prevented the development of benzodiazepine-resistant seizures and alleviated cognitive and behavioral deficits in adult Cdkl5 KO mice. In contrast, 12 days of OV350 treatment in adult Cdkl5 KO mice had limited ability to alleviate cognitive and behavioral deficits. In summary, this study demonstrates that enhancing KCC2 function may be a potential therapeutic target for CDD and other DEEs. However, early intervention during critical developmental windows is crucial for optimal outcomes.

Summary: Neuroactive steroids (NAS), which are synthesized in the brain from progesterone, exert potent effects on behavior and are used to treat postpartum depression, yet how these compounds induce sustained modifications in neuronal activity are ill-defined. Here, we examined the efficacy of NAS for membrane progesterone receptors (mPRs) δ and ε, members of a family of GPCRs for progestins that are expressed in the CNS. NAS increase PKC activity via G _q activation of mPRδ with EC50s between 3-11nM. In contrast, they activate G _s via mPRε to potentiate PKA activity with similar potencies. NAS also induced rapid internalization of only mPRδ. In the forebrain of female mice, mPRδ expression levels were 8-fold higher than males. Consistent with this, activation of PKC by NAS was evident in acute brain slices from female mice. Collectively, our results suggests that NAS may exert sex-specific effects on intracellular signaling in the brain via activation of mPRs.

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain which mediates its rapid effects on neuronal excitability via ionotropic GABA_Areceptors. GABA levels in the brain are critically dependent upon GABA-aminotransferase (GABA-AT) which promotes its degradation. Vigabatrin, a low-affinity GABA-AT inhibitor, exhibits anticonvulsant efficacy, but its use is limited due to cumulative ocular toxicity. OV329 is a rationally designed, next-generation GABA-AT inhibitor with enhanced potency. We demonstrate that sustained exposure to OV329 in mice reduces GABA-AT activity and subsequently elevates GABA levels in the brain. Parallel increases in the efficacy of GABAergic inhibition were evident, together with elevations in electroencephalographic delta power. Consistent with this, OV329 exposure reduced the severity of status epilepticus and the development of benzodiazepine refractory seizures. Thus, OV329 may be of utility in treating seizure disorders and associated pathologies that result from neuronal hyperexcitability.

Estrogens, specifically 17β-estradiol (estradiol), can modulate synaptic function by regulating the expression and localisation of synaptic proteins. However, the mechanisms underlying estradiol’s regulation of synaptic protein expression, and whether if they occur in a sex specific manner, is not well understood. In this study, using sex-specific hippocampal slice cultures and mixed-sex primary hippocampal neurons, we investigated whether local protein synthesis is required for estradiol- induced synaptic protein expression. Estradiol rapidly increased the rate of protein synthesis and the number of actively translating ribosomes along dendrites and near synapses in both male and female hippocampal neurons. Importantly, these effects occurred independently of gene transcription. Moreover, estradiol also increased the abundance of nascent proteins localised to synapses, independently of gene transcription. Specifically, estradiol increased the synaptic expression of GluN2B- containing N -methyl-D-aspartate receptors and PSD-95 in male and female hippocampus. Mechanistically, mTOR signalling was required for estradiol-induced increases in overall local protein synthesis only in male but not female hippocampus. Consistent with this, mTOR signalling mediated estradiol increases in GluN2B in male, but not female, hippocampus. Conversely, mTOR inhibition, blocked estradiol-induced increased PSD-95 expression in both male and female hippocampus. Collectively, these data suggest that the rapid modulation of local protein synthesis by estradiol is required for changes in the synaptic proteome in male and female hippocampus, and that the requirement of the mTOR signalling pathway in these effects occur in both a sex-specific and protein-dependent manner, with this signalling pathway have a greater role in male compared to female hippocampus.