Increase in α-tubulin modifications in the neuronal processes of hippocampal neurons in both kainic acid-induced epileptic seizure and Alzheimer's disease

Neurodegeneration includes acute changes and slow-developing alterations, both of which partly involve common cellular machinery. During neurodegeneration, neuronal processes are impaired along with dysregulated post-translational modifications (PTMs) of cytoskeletal proteins. In neuronal processes, tubulin undergoes unique PTMs including a branched form of modification called glutamylation and loss of the C-terminal tyrosine residue and the penultimate glutamic acid residue forming Δ2-tubulin. Here, we investigated the state of two PTMs, glutamylation and Δ2 form, in both acute and slow-developing neurodegenerations, using a newly generated monoclonal antibody, DTE41, which had 2-fold higher affinity to glutamylated Δ2-tubulin, than to unmodified Δ2-tubulin. DTE41 recognised glutamylated Δ2-tubulin preferentially in immunostaining than in enzyme-linked immunosorbent assay and immunoblotting. In normal mouse brain, DTE41 stained molecular layer of the cerebellum as well as synapse-rich regions in pyramidal neurons of the cerebral cortex. In kainic acid-induced epileptic seizure, DTE41-labelled signals were increased in the hippocampal CA3 region, especially in the stratum lucidum. In the hippocampi of post-mortem patients with Alzheimer’s disease, intensities of DTE41 staining were increased in mossy fibres in the CA3 region as well as in apical dendrites of the pyramidal neurons. Our findings indicate that glutamylation on Δ2-tubulin is increased in both acute and slow-developing neurodegeneration.This work was supported in part by grants-in-aid for Challenging Exploratory Research (26670091) and for Scientific Research on Innovative Areas (23117517) to K.I., and by grants-in-aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network, 221S0003) and for Platform of Supporting Cohort Study and Biospecimen Analysis (JSPS KAKENHI JP 16H06277). H.T.V. is receiving a scholarship from MEXT and formerly from Shizuoka Bank

KIFC3, a microtubule minus end–directed motor for the apical transport of annexin XIIIb–associated Triton-insoluble membranes

We have identified and characterized a COOH-terminal motor domain–type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end–directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100–insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413–1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb–associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells

Increased phosphatidylcholine (16:0/16:0) in the folliculus lymphaticus of Warthin tumor

Warthin tumor (War-T), the second most common benign salivary gland tumor, consists mainly of neoplastic epithelium and lymphoid stroma. Some proteins and genes thought to be involved in War-T were evaluated by molecular biology and immunology. However, lipids as an important component of many tumor cells have not been well studied in War-T. To elucidate the molecular biology and pathogenesis of War-T, we investigated the visualized distribution of phosphatidylcholines (PCs) by imaging mass spectrometry (IMS). In our IMS analysis of a typical case, 10 signals were significantly different in intensity (p < 0.01) between the War-T and non-tumor (Non-T) regions. Five specific PCs were frequently found in the War-T regions of all of the samples: [PC (16:0/16:0) + K](+) (m/z 772.5), [PC (16:0/20:4) + K](+) (m/z 820.5), [PC (16:0/20:3) + K](+) (m/z 822.5), [PC (18:2/20:4) + K](+) (m/z 844.5), and [PC (18:0/20:5) + K](+) (m/z 846.5). PC (16:0/16:0) was increased specifically in the folliculus lymphaticus of War-T lymphoid stroma, suggesting a different metabolism. Localization of PC (16:0/16:0) might reflect inflammation activity participating in the pathogenesis of War-T. Thus, our IMS analysis revealed the profile of PCs specific to the War-T region. The molecules identified in our study provide important information for further studies of War-T pathogenesis

Submolecular-scale imaging of α-helices and C-terminal domains of tubulins by frequency modulation atomic force microscopy in liquid

In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins. © 2011 Biophysical Society

Plastic brain structure changes associated with the division of labour and ageing in termites

Division of labour is a prominent feature of social insect societies, where different castes engage in different specialised tasks. As brain differences are associated with behavioural differences, brain anatomy may be linked to caste polymorphism. Here, we show that termite brain morphology changes markedly with caste differentiation and age in the termite, Reticulitermes speratus. Brain morphology was shown to be associated with reproductive division of labour, with reproductive individuals (alates and neotenic reproductives) having larger brains than non-reproductives (workers and soldiers). Micro-computed tomography (CT) imaging and dissection observations showed that the king's brain morphology changed markedly with shrinkage of the optic lobes during their long life in the dark. Behavioural experiments showed that mature primary kings lose visual function as a result of optic lobe shrinkage. These results suggested that termites restructure their nervous systems to perform necessary tasks as they undergo caste differentiation, and that they also show flexible changes in brain morphology even after the final moult. This study showed that brain morphology in social insects is linked to caste and ageing, and that the evolution of the division of labour is underpinned by the development of diverse neural systems for specialised tasks. This article is protected by copyright. All rights reserved

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

Arachidonic acid containing phosphatidylcholine increases due to microglial activation in ipsilateral spinal dorsal horn following spared sciatic nerve injury

Peripheral nerve injury induces substantial molecular changes in the somatosensory system that leads to maladaptive plasticity and cause neuropathic pain. Understanding the molecular pathways responsible for the development of neuropathic pain is essential to the development of novel rationally designed therapeutics. Although lipids make up to half of the dry weight of the spinal cord, their relation with the development of neuropathic pain is poorly understood. We aimed to elucidate the regulation of spinal lipids in response to neuropathic peripheral nerve injury in mice by utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry, which allows visualization of lipid distribution within the cord. We found that arachidonic acid (AA) containing [PC(diacyl-16:0/20:4)+K]+ was increased temporarily at superficial ipsilateral dorsal horn seven days after spared nerve injury (SNI). The spatiotemporal changes in lipid concentration resembled microglia activation as defined by ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemistry. Suppression of microglial function through minocycline administration resulted in attenuation of hypersensitivity and reduces [PC(diacyl-16:0/20:4)+K]+ elevation in the spinal dorsal horn. These data suggested that AA containing [PC(diacyl-16:0/20:4)+K]+ is related to hypersensitivity evoked by SNI and implicate microglial cell activation in this lipid production

The royal food of termites shows king and queen specificity

シロアリの王と女王の特別食を世界初解明 --王と女王の繁殖と長寿を支えるロイヤルフード--. 京都大学プレスリリース. 2023-07-13.Society in eusocial insects is based on the reproductive division of labor, with a small number of reproductive individuals supported by a large number of non-reproductive individuals. Because inclusive fitness of all colony members depends on the survival and fertility of reproductive members, sterile members provide royals with special treatment. Here we show that termite kings and queens each receive special food of a different composition from workers. Sequential analysis of feeding processes demonstrated that workers exhibit discriminative trophallaxis, indicating their decision-making capacity in allocating food to the kings and queens. LC-MS/MS analyses of the stomodeal food and midgut contents revealed king- and queen-specific compounds including diacylglycerols and short-chain peptides. DESI-MSI analyses of ¹³C-labelled termites identified phosphatidylinositol and acetyl-L-carnitine in the royal food. Comparison of the digestive tract structure showed remarkable differences in the volume ratio of the midgut-to-hindgut among castes, indicating that digestive division of labor underlies reproductive division of labor. Our demonstration of king- and queen-specific food in termites provides insight into the nutritional system that underpins the extraordinary reproduction and longevity of royals in eusocial insects

Imaging mass spectrometry analysis reveals an altered lipid distribution pattern in the tubular areas of hyper-IgA murine kidneys.

Immunoglobulin A (IgA) nephropathy is the most common glomerular disease worldwide. To investigate the pathogenesis of this renal disease, we used animal models that spontaneously develop mesangioproliferative lesions with IgA deposition, which closely resemble the disease in humans. We analyzed the molecular distribution of lipids in hyper-IgA (HIGA) murine kidneys using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF)-based imaging mass spectrometry (IMS), which supplies both spatial distribution of the detected molecules and allows identification of their structures by their molecular mass signature. For both HIGA and control (Balb/c) mice, we found two phosphatidylcholines, PC(16:0/22:6) and PC(18:2/22:6), primarily located in the cortex area and two triacylglycerols, TAG(16:0/18:2/18:1) and TAG(18:1/18:2/18:1), primarily located in the hilum area. However, several other molecules were specifically seen in the HIGA kidneys, particularly in the tubular areas. Two HIGA-specific molecules were O-phosphatidylcholines, PC(O-16:0/22:6) and PC(O-18:1/22:6). Interestingly, common phosphatidylcholines and these HIGA-specific ones possess 22:6 lipid side chains, suggesting that these molecules have a novel, unidentified renal function. Although the primary structure of the HIGA-specific molecules corresponding to m/z 854.6, 856.6, 880.6, and 882.6 remained undetermined, they shared similar fragmentation patterns, indicating their relatedness. We also showed that all the HIGA-specific molecules were derived from urine, and that artificial urinary stagnation-due to unilateral urethral obstruction-caused HIGA-specific distribution of lipids in the tubular area

Investigation by Imaging Mass Spectrometry of Biomarker Candidates for Aging in the Hair Cortex

BACKGROUND: Human hair is one of the essential components that define appearance and is a useful source of samples for non-invasive biomonitoring. We describe a novel application of imaging mass spectrometry (IMS) of hair biomolecules for advanced molecular characterization and a better understanding of hair aging. As a cosmetic and biomedical application, molecules whose levels in hair altered with aging were comprehensively investigated. METHODS: Human hair was collected from 15 young (20±5 years old) and 15 older (50±5 years old) volunteers. Matrix-free laser desorption/ionization IMS was used to visualize molecular distribution in the hair sections. Hair-specific ions displaying a significant difference in the intensities between the 2 age groups were extracted as candidate markers for aging. Tissue localization of the molecules and alterations in their levels in the cortex and medulla in the young and old groups were determined. RESULTS: Among the 31 molecules detected specifically in hair sections, 2--one at m/z 153.00, tentatively assigned to be dihydrouracil, and the other at m/z 207.04, identified to be 3,4-dihydroxymandelic acid (DHMA)--exhibited a higher signal intensity in the young group than in the old, and 1 molecule at m/z 164.00, presumed to be O-phosphoethanolamine, displayed a higher intensity in the old group. Among the 3, putative O-phosphoethanolamine showed a cortex-specific distribution. The 3 molecules in cortex presented the same pattern of alteration in signal intensity with aging, whereas those in medulla did not exhibit significant alteration. CONCLUSION: Three molecules whose levels in hair altered with age were extracted. While they are all possible markers for aging, putative dihydrouracil and DHMA, are also suspected to play a role in maintaining hair properties and could be targets for cosmetic supplementation. Mapping of ion localization in hair by IMS is a powerful method to extract biomolecules in specified regions and determine their tissue distribution