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PRESYNAPTIC Α2δ SUBUNITS ARE KEY ORGANIZERS of GLUTAMATERGIC SYNAPSES Clemens L

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PRESYNAPTIC Α2δ SUBUNITS ARE KEY ORGANIZERS of GLUTAMATERGIC SYNAPSES Clemens L bioRxiv preprint doi: https://doi.org/10.1101/826016; this version posted October 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. PRESYNAPTIC α2δ SUBUNITS ARE KEY ORGANIZERS OF GLUTAMATERGIC SYNAPSES Clemens L. Schöpf1, Stefanie Geisler1, Ruslan I. Stanika1, Marta Campiglio1, Walter A. Kaufmann3, Benedikt Nimmervoll1, Bettina Schlick1, Ryuichi Shigemoto3, and Gerald J. Obermair1,2* From Division of Physiology, Medical University Innsbruck, A-6020 Innsbruck1, Division Physiology, Karl Landsteiner University of Health Sciences, A-3500 Krems2, and Institute of Science and Technology Austria, A-3400 Klosterneuburg3, Austria. Running head: Presynaptic α2δ subunits organize synapses Address correspondence to: *Gerald J. Obermair, Univ.-Prof. Dr., Phone: +43-2732-72090490, E-mail: [email protected], [email protected] In nerve cells the genes encoding for α2δ the auxiliary β and α2δ subunits. α2δ subunits, the subunits of voltage-gated calcium channels targets of the widely prescribed anti-epileptic and (VGCCs) have been linked to synaptic functions anti-allodynic drugs gabapentin and pregabalin, are and neurological disease. Here we show that α2δ membrane-anchored extracellular glycoproteins, subunits are essential for the formation and which modulate VGCC trafficking and calcium organization of glutamatergic synapses. Using a currents (Arikkath and Campbell, 2003; Dolphin, cellular α2δ subunit triple loss-of-function model, 2013; Geisler et al., 2015; Obermair et al., 2008; we demonstrate a failure in presynaptic Zamponi et al., 2015). In nerve cells α2δ subunits differentiation associated with the downscaling have been linked to neuropathic pain and epilepsy of postsynaptic AMPA receptors and the (Zamponi et al., 2015), they interact with mutant postsynaptic density. The role of α2δ isoforms as prion proteins (Senatore et al., 2012) and have been synaptic organizers is highly redundant, as each proposed to regulate synaptic release probability individual α2δ isoform can rescue presynaptic (Hoppa et al., 2012). Importantly, all α2δ isoforms calcium channel trafficking and expression of are implicated in synaptic functions. Presynaptic synaptic proteins. Mutating the MIDAS site in effects of α2δ-1, for example, may be mediated by α2δ-2 dissociates rescuing presynaptic synapsin an interaction with α-neurexins (Brockhaus et al., expression from calcium channel trafficking, 2018) or NMDARs (e.g. (Chen et al., 2018; Zhou et suggesting that the regulatory role of α2δ al., 2018)). In contrast, postsynaptic α2δ-1 acts as a subunits is independent from its role as a calcium receptor for thrombospondins (Eroglu et al., 2009) channel subunit. Our findings influence the and promotes spinogenesis via postsynaptic Rac1 current view on excitatory synapse formation. (Risher et al., 2018). α2δ-2 is necessary for normal Firstly, our study suggests that postsynaptic structure and function of auditory hair cell synapses differentiation is secondary to presynaptic (Fell et al., 2016) and has been identified as a differentiation. Secondly, the dependence of regulator of axon growth and hence a suppressor of presynaptic differentiation on α2δ implicates α2δ axonal regeneration (Tedeschi et al., 2016). subunits as potential nucleation points for the Importantly, a splice variant of α2δ-2 regulates organization of synapses. Finally, our results postsynaptic GABAA-receptor abundance and suggest that α2δ subunits act as trans-synaptic axonal wiring (Geisler et al., 2019). In invertebrates, organizers of glutamatergic synapses, thereby α2δ loss-of-function was associated with abnormal aligning the synaptic active zone with the presynaptic development in motoneurons (Caylor et postsynaptic density. al., 2013; Kurshan et al., 2009) and in mice the loss In synapses neurotransmitter release is of α2δ-3 results in aberrant synapse formation of triggered by the entry of calcium through voltage- auditory nerve fibers (Pirone et al., 2014). Finally, gated calcium channels (VGCCs). Neuronal α2δ-4 is required for the organization of rod and VGCCs consist of an ion-conducting α1 subunit and bioRxiv preprint doi: https://doi.org/10.1101/826016; this version posted October 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. cone photoreceptor synapses (Kerov et al., 2018; c57BL/6N background for more than 10 tm1Dgen Wang et al., 2017). generations. α2δ-3 knockout (Cacna2d3 ) Despite these important functions, knockout strains generated by Deltagen (San Mateo, CA, du du/du mice for α2δ-1 and α2δ-3 show only mild USA) and ducky (Cacna2d2 ; α2δ-2 ) mice were neurological phenotypes (Fuller-Bicer et al., 2009; obtained from The Jackson Laboratory (Bar Harbor, Geisler et al., 2015; Landmann et al., 2019; ME, USA). One week before delivery, male mice Landmann et al., 2018; Neely et al., 2010; Zhou et were separated from the breeding cages and al., 2018). In contrast, mutant mice for α2δ-2 BALB/c foster mothers were included. Mice were (ducky) display impaired gait, ataxia, and epileptic bred and maintained at the central laboratory and seizures (Barclay et al., 2001), all phenotypes animal facility of the Medical University Innsbruck consistent with a cerebellar dysfunction due to the according to national and EU regulations and predominant expression of α2δ-2 in the cerebellum. conforming to the Austrian guidelines on animal Hence, in contrast to the specific functions of α2δ welfare and experimentation. Animal protocols, isoforms (see above) the phenotypes of the available including breeding of single- and double-knockout knockout or mutant mouse models suggest a partial mice, were approved by the Austrian Federal functional redundancy in central neurons. Ministry of Science, Research and Economy Moreover, detailed mechanistic insights into the (BMWFW-66.011/0113-WF/V/3b/2014 and putative synaptic functions of α2δ subunits are BMWFW-66.011/0114-WF/V/3b/2014). Survival complicated by the simultaneous and strong rate of different α2δ double-knockout pups was expression of three isoforms (α2δ-1 to -3) in neurons continuously monitored over 3 years, whereby the of the central nervous system (Schlick et al., 2010). guidelines for identifying humane endpoint criteria In this study, by transfecting cultured were strictly applied. The number of animals used for this project was annually reported to the hippocampal neurons from α2δ-2/-3 double- Austrian Federal Ministry of Science, Research and knockout mice with shRNA against α2δ-1, we Economy (bmwfw). developed a cellular α2δ subunit triple loss-of- function model. Excitatory synapses from these Tattooing and genotyping of potential double- cultures show a severe failure of synaptic vesicle knockout mice. To identify mice for genotyping, recycling associated with loss of presynaptic newborn pups were tattooed on the paws using a calcium channels and presynaptic vesicle- sterile needle with green tattoo paste associated proteins as well as a reduced size of the (ketschum.mfg.co). presynaptic active zone. Lack of presynaptic α2δ Genotyping. DNA was extracted by incubating subunits also induces a failure of postsynaptic PSD- a tail biopsy of ~1-2 mm length in 100 µl of 25 mM 95 and AMPA receptor localization and a thinning NaOH at 95 °C for 30 min followed by cooling to 4 of the postsynaptic density (PSD). Each individual °C and the addition of 100 µl of 40 mM Tris-HCl α2δ isoform (α2δ-1 to -3) could rescue the severe neutralization buffer. The PCR reaction buffer phenotype revealing the highly redundant role of further contained 1.25 mM MgCl, 0.125 mM presynaptic α2δ isoforms in glutamatergic synapse dNTP’s, 1 mM 5x Green GoTaq Flexi Buffer, 0.5 formation and differentiation. Together our results mM Green GoTaq Polymerase (Promega) and 2 µl show that α2δ subunits regulate presynaptic of genomic DNA. Probes were analyzed using differentiation as well as the trans-synaptic standard PCR conditions and gel electrophoresis. tm1Dgen alignment of postsynaptic receptors and are thus Cacna2d3 (α2δ-3 knockout mice): The critical organizers of glutamatergic synapses. following primers were used for detecting the wildtype allele (F1-R, 183 bp fragment) and the Experimental Procedures knockout allele (F2-R, 331 bp fragment): F1:5’- TAGAAAAGATGCACTGGTCACCAGG-3’; F2: Breeding of α2δ-2/-3 double-knockout mice. 5’-GGGCCAGCTCATTCCTCCCACTCAT-3’, R: Double-knockout mice and littermate controls were 5’-GCAGAAGGCACATTGCCATACTCAC-3’. du du/du obtained by crossbreeding double heterozygous Cacna2d2 (α2δ-2 , ducky mice): The evaluation +/- +/du α2δ-3 , α2δ-2 mice, both backcrossed into a of the ducky mutation was performed as previously 2 bioRxiv preprint doi: https://doi.org/10.1101/826016; this version posted October 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. described (Brodbeck et al., 2002). PCR with the curve method as previously described (Schlick et primers du-F, 5’- al., 2010). TaqMan gene expression assays specific ACCTATCAGGCAAAAGGACG-3’
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