Estradiol- and Progesterone-Associated Changes in microRNA-Induced Silencing and Reduced Antiseizure Efficacy of an Antagomir in Female Mice.

About one-third of individuals living with epilepsy have treatment-resistant seizures. Alternative therapeutic strategies are thus urgently needed. One potential novel treatment target is miRNA-induced silencing, which is differentially regulated in epilepsy. Inhibitors (antagomirs) of specific microRNAs (miRNAs) have shown therapeutic promise in preclinical epilepsy studies; however, these studies were mainly conducted in male rodent models, and research into miRNA regulation in females and by female hormones in epilepsy is scarce. This is problematic because female sex and the menstrual cycle can affect the disease course of epilepsy and may, therefore, also alter the efficacy of potential miRNA-targeted treatments. Here, we used the proconvulsant miRNA miR-324-5p and its target, the potassium channel Kv4.2, as an example to test how miRNA-induced silencing and the efficacy of antagomirs in epilepsy are altered in female mice. We showed that Kv4.2 protein is reduced after seizures in female mice similar to male mice; however, in contrast to male mice, miRNA-induced silencing of Kv4.2 is unchanged, and miR-324-5p activity, as measured by the association with the RNA-induced silencing complex, is reduced in females after seizure. Moreover, an miR-324-5p antagomir does not consistently reduce seizure frequency or increase Kv4.2 in female mice. As a possible underlying mechanism, we found that miR-324-5p activity and the silencing of Kv4.2 in the brain were differentially correlated with plasma levels of 17ß-estradiol and progesterone. Our results suggest that hormonal fluctuations in sexually mature female mice influence miRNA-induced silencing and could alter the efficacy of potential future miRNA-based treatments for epilepsy in females.

GABAA Alpha 2,3 Modulation Improves Select Phenotypes in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy-associated phenotypes.

Epilepsy affects all ages, races, genders, and socioeconomic groups. In about one third of patients, epilepsy is uncontrolled with current medications, leaving a vast need for improved therapies. The causes of epilepsy are diverse and not always known but one gene mutated in a small subpopulation of patients is phosphatase and tensin homolog (PTEN). Moreover, focal cortical dysplasia, which constitutes a large fraction of refractory epilepsies, has been associated with signaling defects downstream of PTEN. So far, most preclinical attempts to reverse PTEN deficiency-associated neurological deficits have focused on mTOR, a signaling hub several steps downstream of PTEN. Phosphoinositide 3-kinases (PI3Ks), by contrast, are the direct enzymatic counteractors of PTEN, and thus may be alternative treatment targets. PI3K activity is mediated by four different PI3K catalytic isoforms. Studies in cancer, where PTEN is commonly mutated, have demonstrated that inhibition of only one isoform, p110ß, reduces progression of PTEN-deficient tumors. Importantly, inhibition of a single PI3K isoform leaves critical functions of general PI3K signaling throughout the body intact. Here, we show that this disease mechanism-targeted strategy borrowed from cancer research rescues or ameliorates neuronal phenotypes in male and female mice with neuron-specific PTEN deficiency. These phenotypes include cell signaling defects, protein synthesis aberrations, seizures, and cortical dysplasia. Of note, p110ß is also dysregulated and a promising treatment target in the intellectual disability Fragile X syndrome, pointing towards a shared biological mechanism that is therapeutically targetable in neurodevelopmental disorders of different etiologies. Overall, this work advocates for further assessment of p110ß inhibition not only in PTEN deficiency-associated neurodevelopmental diseases but also other brain disorders characterized by defects in the PI3K/mTOR pathway.

MicroRNA inhibition upregulates hippocampal A-type potassium current and reduces seizure frequency in a mouse model of epilepsy.

Epilepsy is often associated with altered expression or function of ion channels. One example of such a channelopathy is the reduction of A-type potassium currents in the hippocampal CA1 region. The underlying mechanisms of reduced A-type channel function in epilepsy are unclear. Here, we show that inhibiting a single microRNA, miR-324-5p, which targets the pore-forming A-type potassium channel subunit Kv4.2, selectively increased A-type potassium currents in hippocampal CA1 pyramidal neurons in mice. Resting membrane potential, input resistance and other potassium currents were not altered. In a mouse model of acquired chronic epilepsy, inhibition of miR-324-5p reduced the frequency of spontaneous seizures and interictal epileptiform spikes supporting the physiological relevance of miR-324-5p-mediated control of A-type currents in regulating neuronal excitability. Mechanistic analyses demonstrated that microRNA-induced silencing of Kv4.2 mRNA is increased in epileptic mice leading to reduced Kv4.2 protein levels, which is mitigated by miR-324-5p inhibition. By contrast, other targets of miR-324-5p were unchanged. These results suggest a selective miR-324-5p-dependent mechanism in epilepsy regulating potassium channel function, hyperexcitability and seizures.

Isoform-selective phosphoinositide 3-kinase inhibition ameliorates a broad range of fragile X syndrome-associated deficits in a mouse model.

Defects in the phosphoinositide 3-kinase (PI3K) pathway are shared characteristics in several brain disorders, including the inherited intellectual disability and autism spectrum disorder, fragile X syndrome (FXS). PI3K signaling therefore could serve as a therapeutic target for FXS and other brain disorders. However, broad inhibition of such a central signal transduction pathway involved in essential cellular functions may produce deleterious side effects. Pharmacological strategies that selectively correct the overactive components of the PI3K pathway while leaving other parts of the pathway intact may overcome these challenges. Here, we provide the first evidence that disease mechanism-based PI3K isoform-specific inhibition may be a viable treatment option for FXS. FXS is caused by loss of the fragile X mental retardation protein (FMRP), which translationally represses specific messenger RNAs, including the PI3K catalytic isoform p110ß. FMRP deficiency increases p110ß protein levels and activity in FXS mouse models and in cells from subjects with FXS. Here, we show that a novel, brain-permeable p110ß-specific inhibitor, GSK2702926A, ameliorates FXS-associated phenotypes on molecular, cellular, behavioral, and cognitive levels in two different FMRP-deficient mouse models. Rescued phenotypes included increased PI3K downstream signaling, protein synthesis rates, and dendritic spine density, as well as impaired social interaction and higher-order cognition. Several p110ß-selective inhibitors, for example, a molecule from the same chemotype as GSK2702926A, are currently being evaluated in clinical trials to treat cancer. Our results suggest that repurposing p110ß inhibitors to treat cognitive and behavioral defects may be a promising disease-modifying strategy for FXS and other brain disorders.

Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma.

Angiosarcoma is an aggressive malignancy of vascular origin that occurs de novo or in the context of previous cancer therapy. Despite multi-modal aggressive treatment including surgical resection, chemotherapy, and radiation, five-year overall survival remains poor at 35%. Due to its rarity, little is known about its molecular pathology and clinical trials have been extremely difficult to conduct. Development of animal models for rare diseases like angiosarcoma is critical to improve our understanding of tumorigenesis and to test novel treatment regimens. A genetically engineered mouse model for angiosarcoma was generated by conditional deletion of Trp53, Pten, and Ptpn12 in endothelial cells. Tumors arising from these mice recapitulate the histology and molecular pathology of the human disease including hyperactivation of the PI3K/mTOR and MAPK signaling pathways. Treatment of tumor-bearing mice with mTOR or MEK inhibitors effectively inactivated signaling and resulted in reduced proliferation and elevated apoptosis leading to tumor regression. The effect of treatment on tumor growth was transient and proliferation was restored after a period of dormancy. However, combined inhibition of mTOR and MEK resulted in profound tumor regression which was sustained for the duration of treatment. These results suggest that angiosarcoma may be effectively treated by this drug combination. .

Induced Telomere Damage to Treat Telomerase Expressing Therapy-Resistant Pediatric Brain Tumors.

Brain tumors remain the leading cause of cancer-related deaths in children and often are associated with long-term sequelae among survivors of current therapies. Hence, there is an urgent need to identify actionable targets and to develop more effective therapies. Telomerase and telomeres play important roles in cancer, representing attractive therapeutic targets to treat children with poor-prognosis brain tumors such as diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG), and high-risk medulloblastoma. We have previously shown that DIPG, HGG, and medulloblastoma frequently express telomerase activity. Here, we show that the telomerase-dependent incorporation of 6-thio-2'deoxyguanosine (6-thio-dG), a telomerase substrate precursor analogue, into telomeres leads to telomere dysfunction-induced foci (TIF) along with extensive genomic DNA damage, cell growth inhibition, and cell death of primary stem-like cells derived from patients with DIPG, HGG, and medulloblastoma. Importantly, the effect of 6-thio-dG is persistent even after drug withdrawal. Treatment with 6-thio-dG elicits a sequential activation of ATR and ATM pathways and induces G2-M arrest. In vivo treatment of mice bearing medulloblastoma xenografts with 6-thio-dG delays tumor growth and increases in-tumor TIFs and apoptosis. Furthermore, 6-thio-dG crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. Together, our findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant telomerase-positive pediatric brain tumors. Mol Cancer Ther; 17(7); 1504-14. ©2018 AACR.