NS-Pten knockout mice exhibit sex and hippocampal subregion-specific increases in microglia/macrophage density.

Seizures induce hippocampal subregion dependent enhancements in microglia/macrophage phagocytosis and cytokine release that may contribute to the development of epilepsy. As a model of hyperactive mTOR induced epilepsy, neuronal subset specific phosphatase and tensin homolog (NS-Pten) knockout (KO) mice exhibit hyperactive mTOR signaling in the hippocampus, seizures that progress with age, and enhanced hippocampal microglia/macrophage activation. However, it is unknown where microglia/macrophages are most active within the hippocampus of NS-Pten KO mice. We quantified the density of IBA1 positive microglia/macrophages in the CA1, CA2/3, and dentate gyrus of NS-Pten KO and wildtype (WT) male and female mice at 4, 10, and 15 weeks of age. NS-Pten KO mice exhibited an overall increase in the number of IBA1 positive microglia/macrophages in each subregion and in the entire hippocampus. After accounting for differences in size, the whole hippocampus of NS-Pten KO mice still exhibited an increased density of IBA1 positive microglia/macrophages. Subregion analyses showed that this increase was restricted to the dentate gyrus of both male and female NS-Pten KO mice and to the CA1 of male NS-Pten KO mice. These data suggest enhanced microglia/macrophage activity may occur in the NS-Pten KO mice in a hippocampal subregion and sex-dependent manner. Future work should seek to determine whether these region-specific increases in microgliosis play a role in the progression of epilepsy in this model.

Narvaiz DA ，Blandin KJ ，Sullens DG ，Womble PD ，Pilcher JB ，O'Neill G ，Wiley TA ，Kwok EM ，Chilukuri SV ，Lugo JN ... -  《-》

Neuronal subset-specific phosphatase and tensin homolog knockout mice exhibit age and brain region-associated alterations in microglia/macrophage activation.

Seizures induce brain region-dependent enhancements in microglia/macrophage activation. Neuronal subset-specific phosphatase and tensin homolog (PTEN) knockout (KO) mice display hyperactive mammalian target of rapamycin (mTOR) signaling in the hippocampus, cerebellum, and cortex followed by seizures that increase in severity with age. To determine if KO mice also exhibit alterations in the spatiotemporal activation pattern of microglia, we used flow cytometry to compare the percentage of major histocompatibility complex-II activated microglia/macrophages between KO and wildtype (WT) mice at 5, 10, and 15 weeks of age. At 5 weeks, microglia/macrophage activation was greater in the cortex, P < 0.001, cerebellum, P < 0.001, and hippocampus, P < 0.001, of KO compared to WT mice. At 10 weeks, activation was greatest in the cortex of KO mice, P < 0.001, in the cerebellum of WT mice, P < 0.001, but similar in the hippocampus, P > 0.05. By 15 weeks, activation in the hippocampus was more than 25 times greater in KO mice compared to WT mice, P < 0.001. We show that hyperactive mTOR signaling is associated with an altered spatiotemporal pattern of microglia/macrophage activation in the brain and induces an enhanced neuroimmune response in the hippocampus.

Narvaiz DA ，Sullens DG ，Santana-Coelho D ，Lugo JN ... -  《-》

mTOR inhibition suppresses established epilepsy in a mouse model of cortical dysplasia.

Hyperactivation of the mechanistic target of rapamycin (mTOR; also known as mammalian target of rapamycin) pathway has been demonstrated in human cortical dysplasia (CD) as well as in animal models of epilepsy. Although inhibition of mTOR signaling early in epileptogenesis suppressed epileptiform activity in the neuron subset-specific Pten knockout (NS-Pten KO) mouse model of CD, the effects of mTOR inhibition after epilepsy is fully established were not previously examined in this model. Here, we investigated whether mTOR inhibition suppresses epileptiform activity and other neuropathological correlates in adult NS-Pten KO mice with severe and well-established epilepsy. The progression of epileptiform activity, mTOR pathway dysregulation, and associated neuropathology with age in NS-Pten KO mice were evaluated using video-electroencephalography (EEG) recordings, Western blotting, and immunohistochemistry. A cohort of NS-Pten KO mice was treated with the mTOR inhibitor rapamycin (10 mg/kg i.p., 5 days/week) starting at postnatal week 9 and video-EEG monitored for epileptiform activity. Western blotting and immunohistochemistry were performed to evaluate the effects of rapamycin on the associated pathology. Epileptiform activity worsened with age in NS-Pten KO mice, with parallel increases in the extent of hippocampal mTOR complex 1 and 2 (mTORC1 and mTORC2, respectively) dysregulation and progressive astrogliosis and microgliosis. Rapamycin treatment suppressed epileptiform activity, improved baseline EEG activity, and increased survival in severely epileptic NS-Pten KO mice. At the molecular level, rapamycin treatment was associated with a reduction in both mTORC1 and mTORC2 signaling and decreased astrogliosis and microgliosis. These findings reveal a wide temporal window for successful therapeutic intervention with rapamycin in the NS-Pten KO mouse model, and they support mTOR inhibition as a candidate therapy for established, late-stage epilepsy associated with CD and genetic dysregulation of the mTOR pathway.

Nguyen LH ，Brewster AL ，Clark ME ，Regnier-Golanov A ，Sunnen CN ，Patil VV ，D'Arcangelo G ，Anderson AE ... -  《-》

mTOR-dependent alterations of Kv1.1 subunit expression in the neuronal subset-specific Pten knockout mouse model of cortical dysplasia with epilepsy.

Nguyen LH ，Anderson AE 《Scientific Reports》

PTEN deletion increases hippocampal granule cell excitability in male and female mice.

Deletion of the mTOR pathway inhibitor PTEN from postnatally-generated hippocampal dentate granule cells causes epilepsy. Here, we conducted field potential, whole cell recording and single cell morphology studies to begin to elucidate the mechanisms by which granule cell-specific PTEN-loss produces disease. Cells from both male and female mice were recorded to identify sex-specific effects. PTEN knockout granule cells showed altered intrinsic excitability, evident as a tendency to fire in bursts. PTEN knockout granule cells also exhibited increased frequency of spontaneous excitatory synaptic currents (sEPSCs) and decreased frequency of inhibitory currents (sIPSCs), further indicative of a shift towards hyperexcitability. Morphological studies of PTEN knockout granule cells revealed larger dendritic trees, more dendritic branches and an impairment of dendrite self-avoidance. Finally, cells from both female control and female knockout mice received more sEPSCs and more sIPSCs than corresponding male cells. Despite the difference, the net effect produced statistically equivalent EPSC/IPSC ratios. Consistent with this latter observation, extracellularly evoked responses in hippocampal slices were similar between male and female knockouts. Both groups of knockouts were abnormal relative to controls. Together, these studies reveal a host of physiological and morphological changes among PTEN knockout cells likely to underlie epileptogenic activity. Hyperactivation of the mTOR pathway is associated with numerous neurological diseases, including autism and epilepsy. Here, we demonstrate that deletion of the mTOR negative regulator, PTEN, from a subset of hippocampal dentate granule impairs dendritic patterning, increases excitatory input and decreases inhibitory input. We further demonstrate that while granule cells from female mice receive more excitatory and inhibitory input than males, PTEN deletion produces mostly similar changes in both sexes. Together, these studies provide new insights into how the relatively small number (≈200,000) of PTEN knockout granule cells instigates the development of the profound epilepsy syndrome evident in both male and female animals in this model.

Santos VR ，Pun RYK ，Arafa SR ，LaSarge CL ，Rowley S ，Khademi S ，Bouley T ，Holland KD ，Garcia-Cairasco N ，Danzer SC ... -  《-》