Effects of psoralidin on the expression of glutamate decarboxylases and inhibitory synapse development.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter required for excitation/inhibition balance is synthesized by the glutamic acid decarboxylases (GADs) in GABAergic neurons. The levels and activity of GADs are strongly correlated with GABA and neural transmission. Dysregulation of GADs and GABA is associated with various neurological disorders. The study used psoralidin, found in the seeds of Psoralea corylifolia, to investigate its effect on GAD levels and regulatory mechanisms in primary cortical neurons. Psoralidin reduced GAD67 through transcriptional regulation. The reduction was not mediated by the N-methyl-D-aspartate receptor. Additionally, psoralidin attenuated the formation of inhibitory synapses in primary hippocampal neurons.

Survival and functional recovery of primary cortical neurons exposed to actinomycin D.

Actinomycin D (ActD) is an antineoplastic antibiotic that has been commonly used for the treatment of various tumors, including Wilms' tumor, rhabdomyosarcoma, and gestational trophoblastic neoplasia. Recent studies have proposed actinomycin D (ActD) as a novel therapeutic candidate for glioblastoma. ActD significantly reduces tumor growth in recurrent glioblastoma patient-derived mouse models and increases survival by downregulating SOX2 expression. However, ActD treatment of brain tumors can lead to unnecessary exposure of surrounding neurons and normal glial cells to ActD. Cellular and molecular studies are required to estimate and minimize the neurological side effects of ActD. This study investigated the short- and long-term toxicological responses of the primary cortical neurons to ActD. We examined concentration-dependent survival of primary cortical neurons and differential susceptibilities of excitatory, inhibitory neurons, and glial cells to ActD. Distinct alterations in intracellular signaling pathways in cortical neurons were also studied when exposed to ActD. Importantly, we found that primary cortical neurons after ActD discontinuation showed active intracellular signaling pathways responding to extracellular neurotropic factors, but they had extremely poor transcription activity reversibility that was inhibited even by 30-min low-dose ActD exposure. These findings indicate the direct toxicity and extremely poor reversibility of ActD in neurons during chemotherapy for brain tumors.

Reelin Affects Signaling Pathways of a Group of Inhibitory Neurons and the Development of Inhibitory Synapses in Primary Neurons.

Reelin is a secretory protein involved in a variety of processes in forebrain development and function, including neuronal migration, dendrite growth, spine formation, and synaptic plasticity. Most of the function of Reelin is focused on excitatory neurons; however, little is known about its effects on inhibitory neurons and inhibitory synapses. In this study, we investigated the phosphatidylinositol 3-kinase/Akt pathway of Reelin in primary cortical and hippocampal neurons. Individual neurons were visualized using immunofluorescence to distinguish inhibitory neurons from excitatory neurons. Reelin-rich protein supplementation significantly induced the phosphorylation of Akt and ribosomal S6 protein in excitatory neurons, but not in most inhibitory neurons. In somatostatin-expressing inhibitory neurons, one of major subtypes of inhibitory neurons, Reelin-rich protein supplementation induced the phosphorylation of S6. Subsequently, we investigated whether or not Reelin-rich protein supplementation affected dendrite development in cultured inhibitory neurons. Reelin-rich protein supplementation did not change the total length of dendrites in inhibitory neurons in vitro. Finally, we examined the development of inhibitory synapses in primary hippocampal neurons and found that Reelin-rich protein supplementation significantly reduced the density of gephyrin-VGAT-positive clusters in the dendritic regions without changing the expression levels of several inhibitory synapse-related proteins. These findings indicate a new role for Reelin in specific groups of inhibitory neurons and the development of inhibitory synapses, which may contribute to the underlying cellular mechanisms of RELN-associated neurological disorders.

Persistently Elevated mTOR Complex 1-S6 Kinase 1 Disrupts DARPP-32-Dependent D1 Dopamine Receptor Signaling and Behaviors.

BACKGROUND: The serine-threonine kinase mTORC1 (mechanistic target of rapamycin complex 1) is essential for normal cell function but is aberrantly activated in the brain in both genetic-developmental and sporadic diseases and is associated with a spectrum of neuropsychiatric symptoms. The underlying molecular mechanisms of cognitive and neuropsychiatric symptoms remain controversial. METHODS: The present study examines behaviors in transgenic models that express Rheb, the most proximal known activator of mTORC1, and profiles striatal phosphoproteomics in a model with persistently elevated mTORC1 signaling. Biochemistry, immunohistochemistry, electrophysiology, and behavior approaches are used to examine the impact of persistently elevated mTORC1 on D1 dopamine receptor (D1R) signaling. The effect of persistently elevated mTORC1 was confirmed using D1-Cre to elevate mTORC1 activity in D1R neurons. RESULTS: We report that persistently elevated mTORC1 signaling blocks canonical D1R signaling that is dependent on DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein). The immediate downstream effector of mTORC1, ribosomal S6 kinase 1 (S6K1), phosphorylates and activates DARPP-32. Persistent elevation of mTORC1-S6K1 occludes dynamic D1R signaling downstream of DARPP-32 and blocks multiple D1R responses, including dynamic gene expression, D1R-dependent corticostriatal plasticity, and D1R behavioral responses including sociability. Candidate biomarkers of mTORC1-DARPP-32 occlusion are increased in the brain of human disease subjects in association with elevated mTORC1-S6K1, supporting a role for this mechanism in cognitive disease. CONCLUSIONS: The mTORC1-S6K1 intersection with D1R signaling provides a molecular framework to understand the effects of pathological mTORC1 activation on behavioral symptoms in neuropsychiatric disease.

Amyloid-like oligomerization of AIMP2 contributes to α-synuclein interaction and Lewy-like inclusion.

Lewy bodies are pathological protein inclusions present in the brain of patients with Parkinson's disease (PD). These inclusions consist mainly of α-synuclein with associated proteins, such as parkin and its substrate aminoacyl transfer RNA synthetase complex-interacting multifunctional protein-2 (AIMP2). Although AIMP2 has been suggested to be toxic to dopamine neurons, its roles in α-synuclein aggregation and PD pathogenesis are largely unknown. Here, we found that AIMP2 exhibits a self-aggregating property. The AIMP2 aggregate serves as a seed to increase α-synuclein aggregation via specific and direct binding to the α-synuclein monomer. The coexpression of AIMP2 and α-synuclein in cell cultures and in vivo resulted in the rapid formation of α-synuclein aggregates with a corresponding increase in toxicity. Moreover, accumulated AIMP2 in mouse brain was largely redistributed to insoluble fractions, correlating with the α-synuclein pathology. Last, we found that α-synuclein preformed fibril (PFF) seeding, adult Parkin deletion, or oxidative stress triggered a redistribution of both AIMP2 and α-synuclein into insoluble fraction in cells and in vivo. Supporting the pathogenic role of AIMP2, AIMP2 knockdown ameliorated the α-synuclein aggregation and dopaminergic cell death in response to PFF or 6-hydroxydopamine treatment. Together, our results suggest that AIMP2 plays a pathological role in the aggregation of α-synuclein in mice. Because AIMP2 insolubility and coaggregation with α-synuclein have been seen in the PD Lewy body, targeting pathologic AIMP2 aggregation might be useful as a therapeutic strategy for neurodegenerative α-synucleinopathies.

Luteolin prevents liver from tunicamycin-induced endoplasmic reticulum stress via nuclear factor erythroid 2-related factor 2-dependent sestrin 2 induction.

Endoplasmic reticulum (ER) stress designates a cellular response to the accumulation of misfolded proteins, which is related to disease progression in the liver. Luteolin (3',4',5,7-tetrahydroxyflavone) is a phytochemical found frequently in medicinal herbs. Although luteolin has been reported to possess the therapeutic potential to prevent diverse stage of liver diseases, its role in hepatic ER stress has not been established. Thus, the present study aimed to determine the role of luteolin in tunicamycin (Tm)-induced ER stress, and to identify the relevant mechanisms involved in its hepatoprotective effects. In hepatocyte-derived cells and primary hepatocytes, luteolin significantly decreased Tm- or thapsigargin-mediated C/EBP homologous protein (CHOP) expression. In addition, luteolin reduced the activation of three canonical signaling pathways related to the unfolded protein response, and decreased mRNA levels of glucose-regulated protein 78, ER DNA J domain-containing protein 4, and asparagine synthetase. Luteolin also significantly upregulated sestrin 2 (SESN2), and luteolin-mediated CHOP inhibition was blocked in SESN2 (+/-) cells. Moreover, luteolin resulted in phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), as well as increased nuclear Nrf2 expression. Deletion of the antioxidant response element in the human SESN2 promoter inhibited increased luciferase activation by luteolin, suggesting that Nrf2 is a critical transcription factor for luteolin-dependent SESN2 expression. In a Tm-mediated liver injury model, luteolin decreased serum alanine aminotransferase and aspartate aminotransferase activities, prevented degenerative changes and apoptosis of hepatocytes, and inhibited CHOP and glucose-regulated protein 78 expression in hepatic tissues. Therefore, luteolin may be an effective phytochemical to manage ER stress-related liver injury.

Enhanced phosphorylation of S6 protein in mouse cortical layer V and subplate neurons.

The mammalian neocortex is composed of six major layers of neurons. Each group of neurons in the cortical layers has distinct characteristics based on the expression of specific genes and connectivity patterns of neural circuits. Neuronal subtype transition and regional identity acquisition are established by temporal cues and interaction between several transcription factors during neurogenesis. The impairment of cortical lamination or neural circuits results in a wide range of neurodevelopmental disorders such as autism, schizophrenia, and certain forms of childhood epilepsy. Despite continuous efforts to classify neurons with the aid of genetic and epigenetic analyses, the neuron-specific properties associated with post-transcriptional modification remain unclear. In the present study, the distribution of phosphorylated S6-positive layers across the neocortex was examined using several layer markers. The development of pS6 S235/236 layers in layer V and the subplate was spatiotemporally regulated in the mouse brain. In addition, enhanced phosphorylation of ribosomal protein S6 in Ctip2-positive layer V neurons in vivo was sustained under in-vitro conditions using a culture of primary cortical neurons.

Induction of E6AP by microRNA-302c dysregulation inhibits TGF-ß-dependent fibrogenesis in hepatic stellate cells.

Hepatic stellate cells (HSCs) are essential for liver fibrosis. E6 associated protein (E6AP) is one of the E3-ubiquitin-protein ligase and has been studied in proliferation and cellular stress. Currently, no information is available on the role of E6AP on transforming growth factor-ß (TGF-ß) signaling and hepatic fibrogenesis. This study examined whether E6AP is overexpressed in activated HSCs, and if so, its effect on hepatic fibrogenesis and the molecular mechanism. E6AP was expressed higher in HSCs than hepatocytes, and was up-regulated in activated HSCs, HSCs from the livers of carbon tetrachloride-injected mice, or TGF-ß-treated LX-2 cells. The TGF-ß-mediated E6AP up-regulation was not due to altered mRNA level nor protein stability. Thus, we performed microRNA (miRNA, miR) analysis and found that miR-302c was dysregulated in TGF-ß-treated LX-2 cells or activated primary HSCs. We revealed that miR-302c was a modulator of E6AP. E6AP overexpression inhibited TGF-ß-induced expression of plasminogen activator inhibitor-1 in LX-2 cells, albeit it was independent of Smad pathway. Additionally, E6AP inhibited TGF-ß-mediated phosphorylation of mitogen-activated protein kinases. To conclude, E6AP overexpression due to decreased miR-302c in HSCs attenuated hepatic fibrogenesis through inhibition of the TGF-ß-induced mitogen-activated protein kinase signaling pathway, implying that E6AP and other molecules may contribute to protection against liver fibrosis.

The regulation of glutamic acid decarboxylases in GABA neurotransmission in the brain.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter that is required for the control of synaptic excitation/inhibition and neural oscillation. GABA is synthesized by glutamic acid decarboxylases (GADs) that are widely distributed and localized to axon terminals of inhibitory neurons as well as to the soma and, to a lesser extent, dendrites. The expression and activity of GADs is highly correlated with GABA levels and subsequent GABAergic neurotransmission at the inhibitory synapse. Dysregulation of GADs has been implicated in various neurological disorders including epilepsy and schizophrenia. Two isoforms of GADs, GAD67 and GAD65, are expressed from separate genes and have different regulatory processes and molecular properties. This review focuses on the recent advances in understanding the structure of GAD, its transcriptional regulation and post-transcriptional modifications in the central nervous system. This may provide insights into the pathological mechanisms underlying neurological diseases that are associated with GAD dysfunction.

Cell-Based Screen Using Amyloid Mimic ß23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of α-Synuclein and Huntingtin Aggregates.

Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (ß23) expression model to screen potential lead compounds inhibiting ß23-induced toxicity. Highthroughput screening identified several natural compounds as nuclear ß23 inhibitors, including peucedanocoumarin III (PCIII). Interestingly, PCIII accelerates disaggregation and proteasomal clearance of both nuclear and cytosolic ß23 aggregates and protects SH-SY5Y cells from toxicity induced by ß23 expression. Of translational relevance, PCIII disassembled fibrils and enhanced clearance of cytosolic and nuclear protein aggregates in cellular models of huntingtin and α-synuclein aggregation. Moreover, cellular toxicity was diminished with PCIII treatment for polyglutamine (PolyQ)-huntingtin expression and α-synuclein expression in conjunction with 6-hydroxydopamine (6-OHDA) treatment. Importantly, PCIII not only inhibited α-synuclein aggregation but also disaggregated preformed α-synuclein fibrils in vitro . Taken together, our results suggest that a Tet-Off ß23 cell model could serve as a robust platform for screening effective lead compounds inhibiting nuclear or cytosolic protein aggregates. Brain-permeable PCIII or its derivatives could be beneficial for eliminating established protein aggregates.

Correction to: Psoralidin Stimulates Expression of Immediate-Early Genes and Synapse Development in Primary Cortical Neurons.

The original version of this article unfortunately contained a mistake. The funding information was incorrect in the Acknowledgement section of this article. The corrected text is given below.

Psoralidin Stimulates Expression of Immediate-Early Genes and Synapse Development in Primary Cortical Neurons.

Upon synaptic stimulation and glutamate release, glutamate receptors are activated to regulate several downstream effectors and signaling pathways resulting in synaptic modification. One downstream intracellular effect, in particular, is the expression of immediate-early genes (IEGs), which have been proposed to be important in synaptic plasticity because of their rapid expression following synaptic activation and key role in memory formation. In this study, we screened a natural compound library in order to find a compound that could induce the expression of IEGs in primary cortical neurons and discovered that psoralidin, a natural compound isolated from the seeds of Psoralea corylifolia, stimulated synaptic modulation. Psoralidin activated mitogen-activated protein kinase (MAPK) signaling, which in turn induced the expression of neuronal IEGs, particularly Arc, Egr-1, and c-fos. N-methyl-D-aspartate (NMDA) receptors activation and extracellular calcium influx were implicated in the psoralidin-induced intracellular changes. In glutamate dose-response curve, psoralidin shifted glutamate EC50 to lower values without enhancing maximum activity. Interestingly, psoralidin increased the density, area, and intensity of excitatory synapses in primary hippocampal neurons, which were mediated by NMDA receptor activation and MAPK signaling. These results suggest that psoralidin triggers synaptic remodeling through activating NMDA receptor and subsequent MAPK signaling cascades and therefore could possibly serve as an NMDA receptor modulator.

Hypoxia regulates the level of glutamic acid decarboxylase enzymes and interrupts inhibitory synapse stability in primary cultured neurons.

Gamma-aminobutyric acid (GABA) is the main neurotransmitter of inhibitory synaptic transmission, which is critical for oscillatory activity and synchronization of neurons in neural networks. GABA is synthesized by glutamic acid decarboxylase (GAD) enzymes in the inhibitory neuron and, thus, the deregulation of GAD enzymes and subsequent change of GABAergic activity are involved in various neurological and neuropsychiatric diseases. Under hypoxic conditions, neurons undergo neuropathological alterations which can be subtle or severe. Many studies have focused on the alteration of excitatory neurons by hypoxic injury, while inhibitory neuronal changes have not been well determined. Here, we demonstrated that hypoxic conditions decrease the expression of inhibitory neuron-related proteins, including GAD enzymes, through transcript downregulation and proteasomal degradation. Hif-1α induction and glutamate release under hypoxic conditions were implicated in the mechanism of GAD enzyme level reduction. Surprisingly, these conditions altered the density and size of inhibitory synapses, which was irreversible by reoxygenation, but was mediated by glutamate activity. Our findings suggest that potential implication of the compositional and structural alterations of inhibitory neuron in the pathogenesis of various hypoxic injuries.

CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation.

Aging is considered the major risk factor for neurodegenerative diseases including Parkinson's disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.

Hydrocortisone-induced parkin prevents dopaminergic cell death via CREB pathway in Parkinson's disease model.

Dysfunctional parkin due to mutations or post-translational modifications contributes to dopaminergic neurodegeneration in Parkinson's disease (PD). Overexpression of parkin provides protection against cellular stresses and prevents dopamine cell loss in several PD animal models. Here we performed an unbiased high-throughput luciferase screening to identify chemicals that can increase parkin expression. Among promising parkin inducers, hydrocortisone possessed the most favorable profiles including parkin induction ability, cell protection ability, and physicochemical property of absorption, distribution, metabolism, and excretion (ADME) without inducing endoplasmic reticulum stress. We found that hydrocortisone-induced parkin expression was accountable for cell protection against oxidative stress. Hydrocortisone-activated parkin expression was mediated by CREB pathway since gRNA to CREB abolished hydrocortisone's ability to induce parkin. Finally, hydrocortisone treatment in mice increased brain parkin levels and prevented 6-hydroxy dopamine induced dopamine cell loss when assessed at 4 days after the toxin's injection. Our results showed that hydrocortisone could stimulate parkin expression via CREB pathway and the induced parkin expression was accountable for its neuroprotective effect. Since glucocorticoid is a physiological hormone, maintaining optimal levels of glucocorticoid might be a potential therapeutic or preventive strategy for Parkinson's disease.

VPS35 regulates parkin substrate AIMP2 toxicity by facilitating lysosomal clearance of AIMP2.

Vacuolar protein sorting-associated protein 35 (VPS35) is involved in retrograde transport of proteins from endosomes to trans-Golgi network. Gene mutations in VPS35 are linked to autosomal dominant late-onset Parkinson's disease (PD). Although the identification of VPS35 mutations has provided novel insight about its interactions with several PD-associated genes including leucine-rich repeat kinase 2 (LRRK2) and α-synuclein, little information is available about the molecular mechanisms of cell death downstream of VPS35 dysfunction. In this study, we showed that VPS35 has a role in the lysosomal degradation of parkin substrate aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2), of which accumulation leads to poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death. VPS35 was co-immunoprecipitated with AIMP2, as well as lysosome-associated membrane protein-2a (Lamp2a). Interestingly, this association was disrupted by PD-associated VPS35 mutant D620N. VPS35 overexpression prevented AIMP2-potentiated cell death and PARP1 activation in SH-SY5Y cells. More importantly, knockdown of VPS35 led to PARP1 activation and cell death, which was AIMP2 dependent. These findings provide new mechanistic insights into the role of VPS35 in the regulation of AIMP2 levels and cell death. As AIMP2 accumulation was reported in PD patient's brains and involved in dopaminergic cell death, identification of VPS35 as a novel regulator of AIMP2 clearance via lysosomal pathway provides alternative venue to control dopaminergic cell death in PD.

Estrogen receptor activation contributes to RNF146 expression and neuroprotection in Parkinson's disease models.

RNF146 is an E3 ubiquitin ligase that specifically recognizes and polyubiquitinates poly (ADP-ribose) (PAR)-conjugated substrates for proteasomal degradation. RNF146 has been shown to be neuroprotective against PAR polymerase-1 (PARP1)-induced cell death during stroke. Here we report that RNF146 expression and RNF146 inducers can prevent cell death elicited by Parkinson's disease (PD)-associated and PARP1-activating stimuli. In SH-SY5Y cells, RNF146 expression conferred resistance to toxic stimuli that lead to PARP1 activation. High-throughput screen using a luciferase construct harboring the RNF146 promoter identified liquiritigenin as an RNF146 inducer. We found that RNF146 expression by liquiritigenin was mediated by estrogen receptor activation and contributed to cytoprotective effect of liquiritigenin. Finally, RNF146 expression by liquiritigenin in mouse brains provided dopaminergic neuroprotection in a 6-hydroxydopamine PD mouse model. Given the presence of PARP1 activity and RNF146 deficits in PD, it could be a potential therapeutic strategy to restore RNF146 expression by natural compounds or estrogen receptor activation.

New Insights into Reelin-Mediated Signaling Pathways.

Reelin, a multifunctional extracellular protein that is important for mammalian brain development and function, is secreted by different cell types in the prenatal or postnatal brain. The spatiotemporal regulation of Reelin expression and distribution during development relates to its multifaceted function in the brain. Prenatally Reelin controls neuronal radial migration and proper positioning in cortical layers, whereas postnatally Reelin promotes neuronal maturation, synaptic formation and plasticity. The molecular mechanisms underlying the distinct biological functions of Reelin during and after brain development involve unique and overlapping signaling pathways that are activated following Reelin binding to its cell surface receptors. Distinct Reelin ligand isoforms, such as the full-length protein or fragments generated by proteolytic cleavage differentially affect the activity of downstream signaling pathways. In this review, we discuss recent advances in our understanding of the signaling transduction pathways activated by Reelin that regulate different aspects of brain development and function. A core signaling machinery, including ApoER2/VLDLR receptors, Src/Fyn kinases, and the adaptor protein Dab1, participates in all known aspects of Reelin biology. However, distinct downstream mechanisms, such as the Crk/Rap1 pathway and cell adhesion molecules, play crucial roles in the control of neuronal migration, whereas the PI3K/Akt/mTOR pathway appears to be more important for dendrite and spine development. Finally, the NMDA receptor (NMDAR) and an unidentified receptor contribute to the activation of the MEK/Erk1/2 pathway leading to the upregulation of genes involved in synaptic plasticity and learning. This knowledge may provide new insight into neurodevelopmental or neurodegenerative disorders that are associated with Reelin dysfunction.

Therapeutic Strategies for Neuropathic Pain: Potential Application of Pharmacosynthetics and Optogenetics.

Chronic pain originating from neuronal damage remains an incurable symptom debilitating patients. Proposed molecular modalities in neuropathic pain include ion channel expressions, immune reactions, and inflammatory substrate diffusions. Recent advances in RNA sequence analysis have discovered specific ion channel expressions in nociceptors such as transient receptor potential (TRP) channels, voltage-gated potassium, and sodium channels. G protein-coupled receptors (GPCRs) also play an important role in triggering surrounding immune cells. The multiple protein expressions complicate therapeutic development for neuropathic pain. Recent progress in optogenetics and pharmacogenetics may herald the development of novel therapeutics for the incurable pain. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) facilitate the artificial manipulation of intracellular signaling through excitatory or inhibitory G protein subunits activated by biologically inert synthetic ligands. Expression of excitatory channelrhodopsins and inhibitory halorhodopsins on injured neurons or surrounding cells can attenuate neuropathic pain precisely controlled by light stimulation. To achieve the discrete treatment of injured neurons, we can exploit the transcriptome database obtained by RNA sequence analysis in specific neuropathies. This can recommend the suitable promoter information to target the injury sites circumventing intact neurons. Therefore, novel strategies benefiting from pharmacogenetics, optogenetics, and RNA sequencing might be promising for neuropathic pain treatment in future.

Complex Neurological Phenotype in Mutant Mice Lacking Tsc2 in Excitatory Neurons of the Developing Forebrain(123).

Mutations in the TSC1 and TSC2 genes cause tuberous sclerosis complex (TSC), a genetic disease often associated with epilepsy, intellectual disability, and autism, and characterized by the presence of anatomical malformations in the brain as well as tumors in other organs. The TSC1 and TSC2 proteins form a complex that inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Previous animal studies demonstrated that Tsc1 or Tsc2 loss of function in the developing brain affects the intrinsic development of neural progenitor cells, neurons, or glia. However, the interplay between different cellular elements during brain development was not previously investigated. In this study, we generated a novel mutant mouse line (NEX-Tsc2) in which the Tsc2 gene is deleted specifically in postmitotic excitatory neurons of the developing forebrain. Homozygous mutant mice failed to thrive and died prematurely, whereas heterozygous mice appeared normal. Mutant mice exhibited distinct neuroanatomical abnormalities, including malpositioning of selected neuronal populations, neuronal hypertrophy, and cortical astrogliosis. Intrinsic neuronal defects correlated with increased mTORC1 signaling, whereas astrogliosis did not result from altered intrinsic signaling, since these cells were not directly affected by the gene knockout strategy. All neuronal and non-neuronal abnormalities were suppressed by continuous postnatal treatment with the mTORC1 inhibitor RAD001. The data suggest that the loss of Tsc2 and mTORC1 signaling activation in excitatory neurons not only disrupts their intrinsic development, but also disrupts the development of cortical astrocytes, likely through the mTORC1-dependent expression of abnormal signaling proteins. This work thus provides new insights into cell-autonomous and non-cell-autonomous functions of Tsc2 in brain development.