Javier Díez-García

Ankyrin repeat domain-containing protein 55 (ANKRD55) is genetically associated with multiple sclerosis, but its cellular function remains incompletely defined. To investigate its potential role in neuroinflammatory pathways, we mapped ANKRD55-interacting proteins in neuroblastoma, astrocytic, microglial, and monocytic cell lines by affinity purification–mass spectrometry. Across all four cell lines, 14-3-3 isoforms were the most reproducible partners, whereas the IMhu-M microglial interactome uniquely contained eight intraflagellar transport complex B (IFT-B) components. In visible immunoprecipitation assays, IFT46–IFT56 was the smallest tested IFT-B subassembly sufficient to support detectable association with ANKRD55. Confocal microscopy localized endogenous ANKRD55 to pericentriolar material and basal bodies, but not to primary cilia, and identified partial association with P-body markers. These data distinguish a conserved 14-3-3 interaction core from cell-restricted centrosomal/IFT-B and RNP-associated ANKRD55 pools.

There is an actual need of advanced materials for the emerging field of bioelectronics. One commonly used material is the conducting polymer poly(3,4‐ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) due to its general use in organic electronics. However, depending on the application in bioelectronics, PEDOT:PSS is not fully biocompatible due to the high acidity of the residual sulfonate protons of PSS. In this paper, the synthesis and biocompatibility properties of new poly(3,4‐ethylenedioxythiophene):GlycosAminoGlycan (PEDOT:GAG) aqueous dispersions and its resulting films are shown. Thus, negatively charged GAGs as an alternative to PSS are presented. Three different commercially available GAGs, hyaluronic acid, heparin, and chondroitin sulfate are used. Indeed, PEDOT:GAGs dispersions are prepared through an oxidative chemical polymerization in water. Biocompatibility assays of the PEDOT:GAGs coatings are performed using SH‐SY5Y and CCF‐STTG1 cell lines and with ATP and Ca²⁺. Results show full biocompatibility and a pronounced anti‐inflammatory effect. This last characteristic becomes crucial if implanted in the body. These materials can be used for in vivo applications, as transistor or electrode for electrical recording and for all the possible situations when there is contact between electronic circuits and living tissues. image

Knowledge on the normal structure and molecular composition of the peripheral nerves is essential to understand their pathophysiology and to select the regeneration strategies after injury. However, the precise lipid composition of the normal peripheral nerve is still poorly known. Here, we present the first study of distribution of individual lipids in the mature sciatic nerve of rats by imaging mass spectrometry. Both positive and negative ion modes were used to detect, identify and in situ map 160 molecular species of mainly glycerophospholipids, sphingomyelins, sulfatides, and diacyl and triacylglycerols. In parallel, lipid extracts were analyzed by LC-MS/MS to verify and complement the identification of lipids directly from the whole tissue. Three anatomical regions were clearly identified by its differential lipid composition: the nerve fibers, the connective tissue and the adipose tissue that surrounds the nerve. Unexpectedly, very little variety of phosphatidylcholine (PC) species was found, being by far PC 34:1 the most abundant species. Also, a rich composition on sulfatides was detected in fibers, probably due to the important role they play in the myelin cover around axons, as well as an abundance of storage lipids in the adipose and connective tissues. The database of lipids here presented for each region and for the whole sciatic nerve is a first step towards understanding the variety of the peripheral nerves’ lipidome and its changes associated with different diseases and mechanical injuries.

Genetically encoded fluorescent Ca²⁺ indicator proteins (FCIPs) are promising tools to study Ca²⁺ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well‐defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5′ regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca²⁺ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation‐induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse‐induced presynaptic Ca²⁺ transients of parallel fibers are not affected by blockade of N‐methyl‐d‐aspartate receptors.

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca²⁺, Cl^–, or transmembrane voltage have been developed. These sensors are based on variants of green‐fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow‐fluorescent protein (EYFP), a yellow‐green pH‐ and Cl^– sensitive variant of GFP, under control of the Kv3.1 K⁺ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl^– concentration using imaging techniques and conditions analogous to those used with conventional ion‐sensitive dyes. We conclude that transgenic mice expressing GFP‐derived sensors under the control of cell‐type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.