Tomomi Aida

Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.

Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

Up-regulation of GLT-1 severely impairs LTD at mossy fibre–CA3 synapses

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. By this action, they maintain low levels of ambient glutamate, thus preventing excitotoxic damage, and contribute to shaping synaptic currents. We show that up‐regulation of the glutamate transporter GLT‐1 by ceftriaxone severely impaired mGluR‐dependent long‐term depression (LTD), induced at rat mossy fibre (MF)–CA3 synapses by repetitive stimulation of afferent fibres. This effect involved GLT‐1, since LTD was rescued by the selective GLT‐1 antagonist dihydrokainate (DHK). DHK per se produced a modest decrease in fEPSP amplitude that rapidly regained control levels after DHK wash out. Moreover, the degree of fEPSP inhibition induced by the low‐affinity glutamate receptor antagonist γ‐DGG was similar during basal synaptic transmission but not during LTD, indicating that in ceftriaxone‐treated rats LTD induction did not alter synaptic glutamate transient concentration. Furthermore, ceftriaxone‐induced GLT‐1 up‐regulation significantly reduced the magnitude of LTP at MF–CA3 synapses but not at Schaffer collateral–CA1 synapses. Postembedding immunogold studies in rats showed an increased density of gold particles coding for GLT‐1a in astrocytic processes and in mossy fibre terminals; in the latter, gold particles were located near and within the active zones. In both CEF‐treated and untreated GLT‐1 KO mice used for verifying the specificity of immunostaining, the density of gold particles in MF terminals was comparable to background levels. The enhanced expression of GLT‐1 at release sites may prevent activation of presynaptic receptors, thus revealing a novel mechanism by which GLT‐1 regulates synaptic plasticity in the hippocampus.

Glial Dysfunction in the Mouse Habenula Causes Depressive-Like Behaviors and Sleep Disturbance

The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressive-like behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the open-field test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.

Astroglial Glutamate Transporter Deficiency Increases Synaptic Excitability and Leads to Pathological Repetitive Behaviors in Mice

An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette’s syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLASTCreERT2/+/GLT1flox/flox, iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.

Behavioral Characterization of GLT1 (+/-) Mice as a Model of Mild Glutamatergic Hyperfunction

GLT1 is one of the major transporters responsible for maintenance of glutamate homeostasis in the brain. In the present study, glutamate transporter 1-deficient GLT1 homozygous (-/-) and heterozygous (+/-) mice were investigated with the intention that they may provide a model of hyperglutamatergic state resulting in various behavioral alterations. The GLT1 (-/-) mice had lower body and brain weight, mild neuronal loss in CA1 hippocampal region as well as focal gliosis and severe focal neuronal paucity in layer II of the neocortex. The short life-span of GLT1 (-/-) precluded us from systematic behavioral studies in these mice. In contrast, GLT1 (+/-) mice exhibiting a 59% decrease in GLT1 immunoreactivity in their brain tissue, showed no apparent morphological brain abnormalities, and their life-span was not markedly different from controls. Behavior ally, GLT1 (+/-) presented moderate behavioral alterations compared to their wildtype littermates, such as: mild sensorimotor impairment, hyperlocomotion (at 3 month of age only), lower anxiety (at 6 months), better learning of cue-based fear conditioning but worse context-based fear conditioning. Our results suggest that GLT1 (+/-) mice may serve as a potentially useful model to study neurodegenerative disease conditions with mild hyperglutamatergic activity.

NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina

BackgroundLoss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A–D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.ResultsGluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.ConclusionsOur data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.

Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing.

Gene‐targeted mutant animals, such as knockout or knockin mice, have dramatically improved our understanding of the functions of genes in vivo and the genetic diversity that characterizes health and disease. However, the generation of targeted mice relies on gene targeting in embryonic stem (ES) cells, which is a time‐consuming, laborious, and expensive process. The recent groundbreaking development of several genome editing technologies has enabled the targeted alteration of almost any sequence in any cell or organism. These technologies have now been applied to mouse zygotes (in vivo genome editing), thereby providing new avenues for simple, convenient, and ultra‐rapid production of knockout or knockin mice without the need for ES cells. Here, we review recent achievements in the production of gene‐targeted mice by in vivo genome editing.

Dock3 interaction with a glutamate-receptor NR2D subunit protects neurons from excitotoxicity

BackgroundN-methyl-D-aspartate receptors (NMDARs) are critical for neuronal development and synaptic plasticity. Dysregulation of NMDARs is implicated in neuropsychiatric disorders. Native NMDARs are heteromultimeric protein complexes consisting of NR1 and NR2 subunits. NR2 subunits (NR2A–D) are the major determinants of the functional properties of NMDARs. Most research has focused on NR2A- and/or NR2B-containing receptors. A recent study demonstrated that NR2C- and/or NR2D-containing NMDARs are the primary targets of memantine, a drug that is widely prescribed to treat Alzheimer’s disease. Our laboratory demonstrated that memantine prevents the loss of retinal ganglion cells (RGCs) in GLAST glutamate transporter knockout mice, a model of normal tension glaucoma (NTG), suggesting that NR2D-containing receptors may be involved in RGC loss in NTG.ResultsHere we demonstrate that NR2D deficiency attenuates RGC loss in GLAST-deficient mice. Furthermore, Dock3, a guanine nucleotide exchange factor, binds to the NR2D C-terminal domain and reduces the surface expression of NR2D, thereby protecting RGCs from excitotoxicity.ConclusionsThese results suggest that NR2D is involved in the degeneration of RGCs induced by excitotoxicity, and that the interaction between NR2D and Dock3 may have a neuroprotective effect. These findings raise the possibility that NR2D and Dock3 might be potential therapeutic targets for treating neurodegenerative diseases such as Alzheimer’s disease and NTG.

Laminar and subcellular heterogeneity of GLAST and GLT-1 immunoreactivity in the developing postnatal mouse hippocampus

Astrocytes express two sodium‐coupled transporters, glutamate–aspartate transporter (GLAST) and glutamate transporter‐1 (GLT‐1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT‐1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high‐resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100β. In CA1, the density of GFAP‐positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT‐1 was completed only at postnatal days (P) P20–25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3–5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT‐1 exhibited a laminar expression pattern from P10–15 on, with the highest immunoreactivity in the stratum lacunosum‐moleculare. At the cellular level, GLT‐1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT‐1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT‐1 expression in the developing hippocampus. The clustering of GLT‐1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood–brain barrier during formation of the hippocampal network. J. Comp. Neurol. 522:204–224, 2014.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

BackgroundAlthough CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.ResultsHere, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.ConclusionsOur results provide a technical platform for high-throughput knock-in.