General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process.

Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.

Methodological approaches to understand the molecular mechanism of structural plasticity of dendritic spines.

Dendritic spines are tiny protrusions emanating from the neuronal dendrites, typically housing single excitatory postsynapses. Structural plasticity of dendritic spines is considered to be essential for synaptic functional plasticity and also reorganization of neural circuits during learning and memory. Structural plasticity of spines is mediated by complex biochemical signaling with various spatial and temporal scales. A variety of methods based on pharmacological, genetic, molecular, imaging and optical approaches has been developed and applied to dissect the complex signal transduction pathways. In this review, we overview both conventional and new methodological approaches to identify, monitor and manipulate key molecules for structural plasticity of dendritic spines, ultimately aiming to understand the molecular mechanism of learning and memory in behaving animals.

Genome editing-based approaches for imaging protein localization and dynamics in the mammalian brain.

A neuron contains thousands of proteins, each of which mediates neuronal processes at distinct subcellular compartments. Thus, precise mapping of each protein with subcellular resolution in the brain is essential to understand neuronal processes at the molecular level. However, no conventional methods have provided rapid, generalizable, and high-throughput readouts for the subcellular localization of endogenous proteins in the mammalian brain. Recently, new methods based on in vivo genome editing have been developed for high-throughput determination of the protein localization with high specificity, resolution and contrast in mammalian brain tissue. In this review, I first describe the merits and demerits of each conventional method for imaging the protein localization in the brain. I then introduce the new genome editing-based methods to discuss their advantages, limitations and future potential in molecular and cellular neuroscience research.

Retrograde Signaling from Progranulin to Sort1 Counteracts Synapse Elimination in the Developing Cerebellum.

Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.

Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain.

Precise genome editing via homology-directed repair (HDR) in targeted cells, particularly in vivo, provides an invaluable tool for biomedical research. However, HDR has been considered to be largely restricted to dividing cells, making it challenging to apply the technique in postmitotic neurons. Here we show that precise genome editing via HDR is possible in mature postmitotic neurons as well as mitotic cells in mice brain by combining CRISPR-Cas9-mediated DNA cleavage and the efficient delivery of donor template with adeno-associated virus (AAV). Using this strategy, we achieved efficient tagging of endogenous proteins in primary and organotypic cultures in vitro and developing, adult, aged, and pathological brains in vivo. Thus, AAV- and CRISPR-Cas9-mediated HDR will be broadly useful for precise genome editing in basic and translational neuroscience.

Alternative splicing in the C-terminal tail of Cav2.1 is essential for preventing a neurological disease in mice.

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1aCtmKO/CtmKO mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cavß4 and Rimbp2 were significantly reduced while those with GABAB2 were enhanced in the cerebellum of Cacna1aCtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Cacna1aCtmKO/CtmKO mice. These results suggest that the carboxyl-terminal domain of Cav2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Cav2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.

High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.

A scalable and high-throughput method to identify precise subcellular localization of endogenous proteins is essential for integrative understanding of a cell at the molecular level. Here, we developed a simple and generalizable technique to image endogenous proteins with high specificity, resolution, and contrast in single cells in mammalian brain tissue. The technique, single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (SLENDR), uses in vivo genome editing to insert a sequence encoding an epitope tag or a fluorescent protein to a gene of interest by CRISPR-Cas9-mediated homology-directed repair (HDR). Single-cell, HDR-mediated genome editing was achieved by delivering the editing machinery to dividing neuronal progenitors through in utero electroporation. We demonstrate that SLENDR allows rapid determination of the localization and dynamics of many endogenous proteins in various cell types, regions, and ages in the brain. Thus, SLENDR provides a high-throughput platform to map the subcellular localization of endogenous proteins with the resolution of micro- to nanometers in the brain.

Retrograde signaling for climbing fiber synapse elimination.

Neurons form exuberant synapses with target cells early in development. Then, necessary synapses are selectively strengthened whereas unnecessary connections are weakened and eventually eliminated during postnatal development. This process is known as synapse elimination and is a crucial step for shaping immature neural circuits into functionally mature versions. Accumulating evidence suggests that retrograde signaling from postsynaptic cells regulates synapse elimination, but the underlying mechanisms remain unknown. Here, we show that semaphorin3A (Sema3A) and semaphorin7A (Sema7A) mediate retrograde signals for elimination of redundant climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum, a representative model of synapse elimination in the central nervous system. We picked up candidate retrograde signaling molecules that are expressed in PCs during the period of CF synapse elimination and the receptors of these candidate molecules that are present in CFs. We then assessed the effects of lentivirus-mediated RNAi-knockdown of these molecules on CF synapse elimination. By this systematic screening, we found that knockdown of Sema3A in PCs or its co-receptor, plexinA4 (PlxnA4), in CFs accelerated CF synapse elimination and decreased CF-mediated synaptic inputs. Conversely, knockdown of Sema7A in PCs or either of the two receptors for Sema7A, plexinC1 (PlxnC1) and integrinB1 (ItgB1), in CFs impaired CF synapse elimination. Importantly, the effect of Sema7A involves signaling by type 1 metabotropic glutamate receptor (mGluR1), a canonical pathway in PCs for the final stage of CF synapse elimination. These results demonstrate that specific semaphorins act as retrograde signaling molecules and regulate distinct processes of CF synapse elimination during postnatal cerebellar development.

Global scaling down of excitatory postsynaptic responses in cerebellar Purkinje cells impairs developmental synapse elimination.

Synapse elimination is crucial for precise neural circuit formation during postnatal development. We examined how relative differences in synaptic strengths among competing inputs and/or absolute synaptic strengths contribute to climbing fiber (CF) to Purkinje cell (PC) synapse elimination in the cerebellum. We generated mice with PC-selective deletion of stargazin (TARP γ-2), the major AMPA receptor auxiliary subunit in PCs (γ-2 PC-KO mice). Whereas relative differences between "strong" and "weak" CF-mediated postsynaptic response are preserved, absolute strengths of CF inputs are scaled down globally in PCs of γ-2 PC-KO mice. Although the early phase of CF elimination is normal, dendritic translocation of the strongest CF and the late phase of CF elimination that requires Ca(2+)-dependent activation of Arc/Arg3.1 in PCs are both impaired in γ-2 PC-KO mice. We conclude that, although relative differences in CF synaptic inputs are initially essential, proper synaptic scaling is crucial for accomplishing CF synapse elimination.

Retrograde semaphorin signaling regulates synapse elimination in the developing mouse brain.

Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.

Arc/Arg3.1 is a postsynaptic mediator of activity-dependent synapse elimination in the developing cerebellum.

Neural circuits are shaped by activity-dependent elimination of redundant synapses during postnatal development. In many systems, postsynaptic activity is known to be crucial, but the precise mechanisms remain elusive. Here, we report that the immediate early gene Arc/Arg3.1 mediates elimination of surplus climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum. CF synapse elimination was accelerated when activity of channelrhodopsin-2-expressing PCs was elevated by 2-day photostimulation. This acceleration was suppressed by PC-specific knockdown of either the P/Q-type voltage-dependent Ca(2+) channels (VDCCs) or Arc. PC-specific Arc knockdown had no appreciable effect until around postnatal day 11 but significantly impaired CF synapse elimination thereafter, leaving redundant CF terminals on PC somata. The effect of Arc knockdown was occluded by simultaneous knockdown of P/Q-type VDCCs in PCs. We conclude that Arc mediates the final stage of CF synapse elimination downstream of P/Q-type VDCCs by removing CF synapses from PC somata.

Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.

We developed an organotypic coculture preparation allowing fast and efficient identification of molecules that regulate developmental synapse elimination in the mammalian brain. This coculture consists of a cerebellar slice obtained from rat or mouse at postnatal day 9 (P9) or P10 and a medullary explant containing the inferior olive dissected from rat at embryonic day 15. We verified that climbing fibers (CFs), the axons of inferior olivary neurons, formed functional synapses onto Purkinje cells (PCs) in the cerebellum of cocultures. PCs were initially reinnervated by multiple CFs with similar strengths. Surplus CFs were eliminated subsequently, and the remaining CFs became stronger. These changes are similar to those occurring in developing cerebellum in vivo. Importantly, the changes in CF innervations in cocultures involved the same molecules required for CF synapse elimination in vivo, including NMDA receptor, type 1 metabotropic glutamate receptor and glutamate receptor δ2 (GluRδ2). We demonstrate that gain- and loss-of-function analyses can be efficiently performed by lentiviral-mediated overexpression and RNAi-induced knockdown of GluRδ2. Using this approach, we identified neuroligin-2 as a novel molecule that promotes CF synapse elimination in postsynaptic PCs. Thus, our coculture preparation will greatly facilitate the elucidation of molecular mechanisms of synapse elimination.

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression.

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2-AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG-mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG-mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.

Initial and long-term effects of cloxazolam with intractable epilepsy.

Cloxazolam has been used mainly as an anxiolytic agent. The present study was designed to evaluate the effectiveness of cloxazolam as an add-on antiepileptic drug in patients with intractable epilepsy. A total of 32 patients with intractable epilepsy were treated with cloxazolam: 13 with generalized epilepsy, 15 with focal epilepsy, and 4 with undetermined type of epilepsy. The initial effects were evaluated at 1 month after reaching a maintenance dose (0.3-0.5 mg/kg). The long-term effects were investigated at 2 years after reaching a maintenance dose. With cloxazolam, seizure frequency was reduced by ≥ 50% in 19/32 patients (59%) during initial therapy and in 6/23 patients (26%) during long-term therapy. Two became seizure free throughout the cloxazolam therapy. During initial therapy, 8/32 patients (25%) developed 11 episodes of adverse events during the initial therapy, including 5 with drowsiness, 3 with hyperactivity, 2 with irritability, and 1 with loss of appetite. During long-term therapy, 2/23 (9%) developed drowsiness. The mean dose of cloxazolam in patients with an effective response was 0.30 ± 0.18 mg/kg for initial therapy and 0.26 ± 0.20 mg/kg for long-term therapy. Seven of the 19 effective responders developed tolerance (37%). Cloxazolam is an effective and safe antiepileptic drug for intractable epilepsy.

[Sulthiame treatment for patients with intractable epilepsy].

We examined the antiepileptic effect and side effects of sulthiame in 28 patients with intractable epilepsy. The patients' ages ranged from 6 months to 34 years (mean: 8 years 7 months), and 26 of them were under 18-years-old. Nineteen patients had severe physical and mental disabilities. Sixteen patients had generalized seizures, and 12 had partial seizures. Sulthiame was administered at the dose of 50-300 mg/day (4-14 mg/kg body weight) as add-on therapy in all except one patient. Among the 28 patients, two with complex partial seizures (7%) became seizure-free. Eight patients (29%) (6 patients with generalized seizures and 2 patients with partial seizures) showed seizure reduction by > 50%. Among these 10 patients who showed positive responses, six developed tolerance within 2-5 months. Side-effects were observed in 5 patients, including enuresis, drowsiness, and drooling, none of which caused discontinuation of treatment. Therefore, we conclude that sulthiame is an effective and safe antiepileptic drug for the treatment of intractable epilepsy.