SCRAPPER Regulates the Thresholds of Long-Term Potentiation/Depression, the Bidirectional Synaptic Plasticity in Hippocampal CA3-CA1 Synapses

SCRAPPER, which is an F-box protein encoded by FBXL20, regulates the frequency of the miniature excitatory synaptic current through the ubiquitination of Rab3-interacting molecule 1. Here, we recorded the induction of long-term potentiation/depression (LTP/LTD) in CA3-CA1 synapses in E3 ubiquitin ligase SCRAPPER-deficient hippocampal slices. Compared to wild-type mice, Scrapper-knockout mice exhibited LTDs with smaller magnitudes after induction with low-frequency stimulation and LTPs with larger magnitudes after induction with tetanus stimulation. These findings suggest that SCRAPPER regulates the threshold of bidirectional synaptic plasticity and, therefore, metaplasticity

Transmembrane and Ubiquitin-Like Domain-Containing Protein 1 (Tmub1/HOPS) Facilitates Surface Expression of GluR2-Containing AMPA Receptors

Some ubiquitin-like (UBL) domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported. Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS) protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPSRNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP) were coimmunoprecipitated by the anti-Tmub1/ HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane

Visualization of acetylcholine distribution in central nervous system tissue sections by tandem imaging mass spectrometry

Metabolite distribution imaging via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundances of larger metabolite species, it is important to expand its application to smaller molecules, such as neurotransmitters. This study aimed to develop an IMS application to visualize neurotransmitter distribution in central nervous system tissue sections. Here, we raise two technical problems that must be resolved to achieve neurotransmitter imaging: (1) the lower concentrations of bioactive molecules, compared with those of membrane lipids, require higher sensitivity and/or signal-to-noise (S/N) ratios in signal detection, and (2) the molecular turnover of the neurotransmitters is rapid; thus, tissue preparation procedures should be performed carefully to minimize postmortem changes. We first evaluated intrinsic sensitivity and matrix interference using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS) to detect six neurotransmitters and chose acetylcholine (ACh) as a model for study. Next, we examined both single MS imaging and MS/MS imaging for ACh and found that via an ion transition from m/z 146 to m/z 87 in MS/MS imaging, ACh could be visualized with a high S/N ratio. Furthermore, we found that in situ freezing method of brain samples improved IMS data quality in terms of the number of effective pixels and the image contrast (i.e., the sensitivity and dynamic range). Therefore, by addressing the aforementioned problems, we demonstrated the tissue distribution of ACh, the most suitable molecular specimen for positive ion detection by IMS, to reveal its localization in central nervous system tissues

Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice

SCRAPPER, an F-box protein coded by FBXL20, is a subunit of SCF type E3 ubiquitin ligase. SCRAPPER localizes synapses and directly binds to Rab3-interacting molecule 1 (RIM1), an essential factor for synaptic vesicle release, thus it regulates neural transmission via RIM1 degradation. A defect in SCRAPPER leads to neurotransmission abnormalities, which could subsequently result in neurodegenerative phenotypes. Because it is likely that the alteration of neural transmission in Scrapper mutant mice affect their systemic condition, we have analyzed the behavioral phenotypes of mice with decreased or increased the amount of SCRAPPER. We carried out a series of behavioral test batteries for Scrapper mutant mice. Scrapper transgenic mice overexpressing SCRAPPER in the hippocampus did not show any significant difference in every test argued in this manuscript by comparison with wild-type mice. On the other hand, heterozygotes of Scrapper knockout [SCR (+/−)] mice showed significant difference in the contextual but not cued fear conditioning test. In addition, SCR (+/−) mice altered in some tests reflecting anxiety, which implies the loss of functions of SCRAPPER in the hippocampus. The behavioral phenotypes of Scrapper mutant mice suggest that molecular degradation conferred by SCRAPPER play important roles in hippocampal-dependent fear memory formation

Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission

EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells

The microtubule plus end regulator EB1 brings Sentin and possibly a microtubule polymerase to microtubule plus ends to promote microtubule dynamics

Systematic analysis of mitochondrial genes associated with hearing loss in the Japanese population: dHPLC reveals a new candidate mutation

<p>Abstract</p> <p>Background</p> <p>Variants of mitochondrial DNA (mtDNA) have been evaluated for their association with hearing loss. Although ethnic background affects the spectrum of mtDNA variants, systematic mutational analysis of mtDNA in Japanese patients with hearing loss has not been reported.</p> <p>Methods</p> <p>Using denaturing high-performance liquid chromatography combined with direct sequencing and cloning-sequencing, Japanese patients with prelingual (N = 54) or postlingual (N = 80) sensorineural hearing loss not having pathogenic mutations of m.1555A > G and m.3243A > G nor <it>GJB2 </it>were subjected to mutational analysis of mtDNA genes (<it>12S rRNA</it>, <it>tRNA</it>^{<it>Leu(UUR)</it>}, <it>tRNA</it>^{<it>Ser(UCN)</it>}, <it>tRNA</it>^<it>Lys</it>, <it>tRNA</it>^<it>His</it>, <it>tRNA</it>^{<it>Ser(AGY)</it>}, and <it>tRNA</it>^<it>Glu</it>).</p> <p>Results</p> <p>We discovered 15 variants in <it>12S rRNA </it>and one homoplasmic m.7501A > G variant in <it>tRNA</it>^{<it>Ser(UCN)</it>}; no variants were detected in the other genes. Two criteria, namely the low frequency in the controls and the high conservation among animals, selected the m.904C > T and the m.1105T > C variants in <it>12S rRNA </it>as candidate pathogenic mutations. Alterations in the secondary structures of the two variant transcripts as well as that of m.7501A > G in <it>tRNA</it>^{<it>Ser(UCN) </it>}were predicted.</p> <p>Conclusions</p> <p>The m.904C > T variant was found to be a new candidate mutation associated with hearing loss. The m.1105T > C variant is unlikely to be pathogenic. The pathogenicity of the homoplasmic m.7501T > A variant awaits further study.</p

Sulfatide species with various fatty acid chains in oligodendrocytes at different developmental stages determined by imaging mass spectrometry

HSO3‐3‐galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro‐oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4‐positive cells. In this study, we identified a sulfatide species from the pro‐oligodendroblast‐to‐myelination stage by imaging mass spectrometry. The results demonstrated that short‐chain sulfatides with 16 carbon non‐hydroxylated fatty acids (C16) and 18 carbon non‐hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18‐OH) existed in restricted regions of the early embryonic spinal cord, where pro‐oligodendroblasts initially appear, and co‐localized with Olig2‐positive pro‐oligodendroblasts. C18 and C18‐OH sulfatides also existed in isolated pro‐oligodendroblasts. C22‐OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short‐chain fatty acids in pro‐oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination