Shouta Sugio

Expanding the repertoire of optogenetically targeted cells with an enhanced gene expression system.

Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.

Flexible Accelerated STOP Tetracycline Operator-Knockin (FAST): A Versatile and Efficient New Gene Modulating System

We created the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields two distinct knockin mice-STOP-tetO and tetO knockin-that permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue, 3) tetracycline-controlled transcriptional activator (tTA)-mediated misexpression, 4) tetracycline-controlled transcriptional activator (tTA)-mediated overexpression, and 5) tetracycline-controlled transcriptional silencer (tTS)-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-of-function and loss-of-function strains can therefore be generated on a time scale not previously achievable. These strains can then be screened for clinically relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor.

Influence of Inhibitory Serotonergic Inputs to Orexin/Hypocretin Neurons on the Diurnal Rhythm of Sleep and Wakefulness

STUDY OBJECTIVE Serotonergic (5HT) neurons of the dorsal raphe nuclei receive excitatory input from hypothalamic orexin (hypocretin) neurons and reciprocally inhibit orexin neurons through the 5HT1A receptor. However, the physiological significance of this negative feedback circuit for sleep/wakefulness regulation is little understood. DESIGN 5HT1A receptor expression level was specifically and reversibly controlled in the orexin neurons using the Tet-off system. The responsiveness of orexin neurons to 5HT in vitro and the sleep/wakefulness patterns were compared between 5HT1A-overexpressing and control mice. MEASUREMENTS AND RESULTS When the 5HT1A receptor was overexpressed in orexin neurons of Orexin-EGFP; orexin-tTA; TetO Htr1a mice, 5HT-induced inhibition of orexin neurons was prolonged. In the absence of doxycycline, Orexin-tTA; TetO Htr1a mice exhibited severe fragmentation of sleep/wakefulness during the first half of the dark period-the time of maximal activity in nocturnal rodents-without affecting sleep/wakefulness during the light period when sleep time is maximal. However, when the 5HT1A receptor in orexin neurons was reduced to basal expression levels in the presence of doxycycline, sleep/wakefulness patterns in Orexin-tTA; TetO Htr1a mice during the early active period were indistinguishable from those of littermate TetO Htr1a mice. These results strongly suggest that enhancement of inhibitory serotonergic input to orexin neurons caused fragmentation of wakefulness. In contrast, sleep/wakefulness architecture in the light period was unaffected by 5HT1A receptor overexpression in the orexin neurons. CONCLUSION Inhibitory serotonergic input likely functions as negative feedback to orexin neurons in the early dark period and helps stabilize wakefulness bouts, thereby contributing to the diurnal rhythm of sleep and wakefulness.

Gene induction in mature oligodendrocytes with a PLP-tTA mouse line

Mature oligodendrocytes are critical for myelin maintenance. To understand the molecular basis for this, genetic manipulation of mature oligodendrocytes is needed. Here we generated a mature oligodendrocyte tTA (tetracycline‐controlled transcriptional activator) mouse line which, in combination with a tTA‐dependent promoter line driving the expression of the desired transgene, can be used for gain‐of‐function studies. We used an oligodendrocyte promoter, the mouse proteolipid protein (PLP) promoter, to express mammalianized tTA, and generated a PLP‐mtTA mouse line. In adults, mtTA mRNA was predominantly detected in brain white matter where it co‐localized with PLP mRNA. mtTA‐mediated gene induction was confirmed by crossing to mice with a tTA‐dependent promoter driving expression of yellow fluorescent protein (tetO‐YFP mice). YFP induction in PLP‐mtTA::tetO‐YFP mice was consistent with PLP expression in adult mature oligodendrocytes and premyelinating‐stage myelinating oligodendrocytes. This PLP‐mtTA mouse line is the first to enable gain‐of‐function studies in mature oligodendrocytes with the tet system. genesis 50:424–428, 2012.

Astrocyte-mediated infantile-onset leukoencephalopathy mouse model.

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile‐onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte‐specific Mlc1 overexpression causes infantile‐onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na+/K+ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na+/K+ pump. In contrast, no changes in Na+/K+ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na+/K+ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile‐onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile‐onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150–168

Transient receptor potential vanilloid 2 activation by focal mechanical stimulation requires interaction with the actin cytoskeleton and enhances growth cone motility.

We have previously reported that transient receptor potential vanilloid 2 (TRPV2) can be activated by mechanical stimulation, which enhances axonal outgrowth in developing neurons; however, the molecular mechanisms that govern the contribution of TRPV2 activation to axonal outgrowth remain unclear. In the present study, we examined this mechanism by using PC12 cells as a neuronal model. Overexpression of TRPV2 enhanced axonal outgrowth in a mechanical stimulus‐dependent manner. Accumulation of TRPV2 at the cell surface was 4‐fold greater in the growth cone compared with the soma. In the growth cone, TRPV2 is not static, but dynamically accumulates (within ∼100 ms) to the site of mechanical stimulation. The dynamic and acute clustering of TRPV2 can enhance very weak mechanical stimuli via focal accumulation of TRPV2. Focal application of mechanical stimuli dramatically increased growth cone motility and caused actin reorganization via activation of TRPV2. We also found that TRPV2 physically interacts with actin and that changes in the actin cytoskeleton are required for its activation. Here, we demonstrated for the first time to our knowledge that TRPV2 clustering is induced by mechanical stimulation generated by axonal outgrowth and that TRPV2 activation is triggered by actin rearrangements that result from mechanical stimulation. Moreover, TRPV2 activation enhances growth cone motility and actin accumulation to promote axonal outgrowth. —Sugio, S., Nagasawa, M., Kojima, I., Ishizaki, Y., Shibasaki, K. Transient receptor potential vanilloid 2 activation by focal mechanical stimulation requires interaction with the actin cytoskeleton and enhances growth cone motility. FASEB J. 31, 1368–1381 (2017) www.fasebj.org

Retinal detachment-induced Müller glial cell swelling activates TRPV4 ion channels and triggers photoreceptor death at body temperature

Using region-specific injection of hyaluronic acid, we developed a mouse model of acute retinal detachment (RD) to investigate molecular mechanisms of photoreceptor cell death triggered by RD. We focused on the transient receptor potential vanilloid 4 (TRPV4) ion channel, which functions as a thermosensor, osmosensor, and/or mechanosensor. After RD, the number of apoptotic photoreceptors was reduced by ∼50% in TRPV4KO mice relative to wild-type mice, indicating the possible involvement of TRPV4 activation in RD-induced photoreceptor cell death. Furthermore, TRPV4 expressed in Müller glial cells can be activated by mechanical stimuli caused by RD-induced swelling of these cells, resulting in release of the cytokine MCP-1, which is reported as a mediator of Müller glia-derived strong mediator for RD-induced photoreceptor death. We also found that the TRPV4 activation by the Müller glial swelling was potentiated by body temperature. Together, our results suggest that RD adversely impacts photoreceptor viability via TRPV4-dependent cytokine release from Müller glial cells and that TRPV4 is part of a novel molecular pathway that could exacerbate the effects of hypoxia on photoreceptor survival after RD. SIGNIFICANCE STATEMENT Identification of the mechanisms of photoreceptor death in retinal detachment is required for establishment of therapeutic targets for preventing loss of visual acuity. In this study, we found that TRPV4 expressed in Müller glial cells can be activated by mechanical stimuli caused by RD-induced swelling of these cells, resulting in release of the cytokine MCP-1, which is reported as a mediator of Müller glia-derived strong mediator for RD-induced photoreceptor death. We also found that the TRPV4 activation by the Müller glial swelling was potentiated by body temperature. Hence, TRPV4 inhibition could suppress cell death in RD pathological conditions and suggests that TRPV4 in Müller glial cells might be a novel therapeutic target for preventing photoreceptor cell death after RD.

Ectopic positioning of Bergmann glia and impaired cerebellar wiring in Mlc1-over-expressing mice

Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts, and is expressed in astrocytes. Mlc1‐over‐expressing mice represent an animal model of early‐onset leukoencephalopathy, which manifests as astrocytic swelling followed by myelin membrane splitting in the white matter. It has been previously reported that Mlc1 is highly expressed in Bergmann glia, while the cerebellar phenotypes of Mlc1‐over‐expressing mouse have not been characterized. Here, we examined the cerebellum of Mlc1‐over‐expressing mouse and found that the distribution of Bergmann glia (BG) was normally compacted along the Purkinje cell (PC) layer until postnatal day 10 (P10), while most BG were dispersed throughout the molecular layer by P28. Ectopic BG were poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter glutamate‐aspartate transporter. Extraordinarily slow and small climbing fiber (CF)‐mediated excitatory post‐synaptic currents, which are known to be elicited under accelerated glutamate spillover, emerged at P20‐P28 when BG ectopia was severe, but not at P9‐P12 when ectopia was mild. Furthermore, maturation of CF wiring, which translocates the site of innervation from somata to proximal dendrites, was also impaired. Manipulations that restricted the Mlc1‐over‐expressing period successfully generated mice with and without BG ectopia, depending on the over‐expressing period. Together, these findings suggest that there is a critical time window for mechanisms that promote the positioning of BG in the PC layer. Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs.

The role of Bergmann glia in cerebellar development

KIAA0319 gene is associated with the onset of dyslexia. KIAA0319 encodes a transmembrane protein that is reported to be involved in the regulation of neuronal migration in the developing brain. Because KIAA0319 mRNA is also expressed in the adult brain, it may have other functions than the regulation of neuronal migration. In this study we examined the function of KIAA0319 on neuronal morphology. We found that primary cultured neurons expressing full-length KIAA0319 protein formed complex neurites and fine branches. On the other hand, expression of a mutant KIAA0319 protein that lacked the intracellular region had less effect. It was thus likely that KIAA0319 was involved in the regulation of neuronal morphology, presumably by interacting with a certain intracellular molecule(s). We next performed yeast two-hybrid screening and identified radixin, a member of the ERM (ezrin, radixin, moesin) protein family, as a potential binding partner of KIAA0319. ERM proteins are adapter molecules that link membrane proteins to actin cytoskeletone and involved in the regulation of cellular morphology and movement. Pull-down experiments indicated that radixin specifically bound to the intracellular region of KIAA0319. We are now trying to clarify the physiological significance of the interaction in primary cultured neurons, which may lead to better understanding of the molecular mechanisms involved in dyslexia.