Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP
2022; Springer Nature; Volume: 23; Issue: 8 Linguagem: Inglês
10.15252/embr.202153659
ISSN1469-3178
AutoresCarolina Montenegro‐Venegas, Debarpan Guhathakurta, Eneko Pina-Fernández, Maria Andres‐Alonso, Florian Plattner, Eckart D. Gundelfinger, Anna Fejtová,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArticle29 June 2022Open Access Transparent process Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP Carolina Montenegro-Venegas Carolina Montenegro-Venegas orcid.org/0000-0002-2293-2023 Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany Institute for Pharmacology and Toxicology, Medical Faculty, Otto von Guericke University, Magdeburg, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration Search for more papers by this author Debarpan Guhathakurta Debarpan Guhathakurta orcid.org/0000-0001-6475-6366 Molecular Psychiatry, Department of Psychiatry and Psychotherapy, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation, Visualization Search for more papers by this author Eneko Pina-Fernandez Eneko Pina-Fernandez RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Contribution: Methodology Search for more papers by this author Maria Andres-Alonso Maria Andres-Alonso orcid.org/0000-0002-1585-539X RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Contribution: Methodology Search for more papers by this author Florian Plattner Florian Plattner Neuro-Research, Houston, TX, USA Contribution: Resources, Investigation Search for more papers by this author Eckart D Gundelfinger Eckart D Gundelfinger orcid.org/0000-0001-9377-7414 Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany Institute for Pharmacology and Toxicology, Medical Faculty, Otto von Guericke University, Magdeburg, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Anna Fejtova Corresponding Author Anna Fejtova [email protected] orcid.org/0000-0002-1815-4409 Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Molecular Psychiatry, Department of Psychiatry and Psychotherapy, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration Search for more papers by this author Carolina Montenegro-Venegas Carolina Montenegro-Venegas orcid.org/0000-0002-2293-2023 Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany Institute for Pharmacology and Toxicology, Medical Faculty, Otto von Guericke University, Magdeburg, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration Search for more papers by this author Debarpan Guhathakurta Debarpan Guhathakurta orcid.org/0000-0001-6475-6366 Molecular Psychiatry, Department of Psychiatry and Psychotherapy, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Contribution: Investigation, Visualization Search for more papers by this author Eneko Pina-Fernandez Eneko Pina-Fernandez RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Contribution: Methodology Search for more papers by this author Maria Andres-Alonso Maria Andres-Alonso orcid.org/0000-0002-1585-539X RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Contribution: Methodology Search for more papers by this author Florian Plattner Florian Plattner Neuro-Research, Houston, TX, USA Contribution: Resources, Investigation Search for more papers by this author Eckart D Gundelfinger Eckart D Gundelfinger orcid.org/0000-0001-9377-7414 Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany Institute for Pharmacology and Toxicology, Medical Faculty, Otto von Guericke University, Magdeburg, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Anna Fejtova Corresponding Author Anna Fejtova [email protected] orcid.org/0000-0002-1815-4409 Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Molecular Psychiatry, Department of Psychiatry and Psychotherapy, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Contribution: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration Search for more papers by this author Author Information Carolina Montenegro-Venegas1,2,3, Debarpan Guhathakurta4, Eneko Pina-Fernandez5, Maria Andres-Alonso5, Florian Plattner6, Eckart D Gundelfinger1,2,3 and Anna Fejtova *,1,4,5 1Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany 2Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany 3Institute for Pharmacology and Toxicology, Medical Faculty, Otto von Guericke University, Magdeburg, Germany 4Molecular Psychiatry, Department of Psychiatry and Psychotherapy, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 5RG Presynaptic Plasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany 6Neuro-Research, Houston, TX, USA *Corresponding author. Tel: +49 9131 85 46155; E-mail: [email protected] EMBO Reports (2022)23:e53659https://doi.org/10.15252/embr.202153659 (Correction added on June 28th 2022, after first online publication: figure 1D exchanged, because the color code was wrong). PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Neuronal presynaptic terminals contain hundreds of neurotransmitter-filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well-known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5-dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release. Synopsis The presynaptic protein bassoon organizes synaptic vesicles into functional pools at glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis. This study further reveals that a CDK5/PDE4/cAMP axis controls neurotransmitter release. Bassoon deletion decreases release competence of synaptic vesicles and renders more vesicles unable of release at glutamatergic synapses. Bassoon controls presynaptic CDK5/calcineurin and cAMP/PKA signalling. CDK5 regulates PDE4 activity upstream of cAMP/PKA-dependent control of synaptic vesicle release competence. Introduction Synapses are contact sites between neurons, where communication between presynaptic and postsynaptic neuron occur by means of electrochemical neurotransmission. Within the presynapse, the neurotransmitter is stored in small synaptic vesicles (SVs) and released upon their fusion with the presynaptic plasma membrane. After their fusion, the membrane and protein components of SVs are retrieved by compensatory endocytosis, and SVs are refilled with neurotransmitters and recycled (Sudhof, 2004). This SV cycle ensures the long-term structural and functional integrity of the presynaptic compartment (Chanaday et al, 2019; Bonnycastle et al, 2020). SVs, albeit structurally identical, differ in their release competence (i.e. capability to fuse with presynaptic plasma membrane upon stimulus) and display varying kinetics of release in response to stimuli, resulting in three distinct SV pools (Rizzoli & Betz, 2005; Denker & Rizzoli, 2010; Alabi & Tsien, 2012). The readily releasable pool (RRP) contains vesicles that discharge immediately upon stimulation and correspond morphologically to the docked SVs, which are in contact with the plasma membrane of the presynaptic active zone (Schikorski & Stevens, 2001). The recycling pool (RP) is a source of releasable vesicles that can replenish RRP during prolonged stimulation. Both RRP and RP together form the total recycling pool (TRP). A large proportion of SVs is unable to undergo release under physiological conditions and form the reserve pool (ResP) of SVs. The size of RRP and RP at a given synapse decisively shapes its physiological properties. RRP size determines together with release probabilities of individual SVs synaptic release probability, whereas the size of the RP influences the replenishment of SVs during stimulation trains (Alabi & Tsien, 2012). The release competence of SVs and thus their assignment to the functional pools is under the control of intracellular signalling pathways. Cyclin-dependent kinase 5 (CDK5)/calcineurin signalling plays a crucial role in the regulation of the transition of SVs between the RP and ResP (Kim & Ryan, 2010), while cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase A (PKA) signalling was linked to the control of RRP (Lonart et al, 1998; Nagy et al, 2004). Phosphoproteins of the synapsin (Syn) family are SV-associated proteins that integrate the signalling from kinases and phosphatases of multiple pathways to control the plasticity of neurotransmitter release. Syn is critical for the formation and maintenance of the SV cluster above the presynaptic active zone (Pieribone et al, 1995). Depending on its phosphorylation state, Syn oligomerises and interacts with SVs and with the actin cytoskeleton, which in turn modulates dynamic SV clustering (Chi et al, 2003; Cesca et al, 2010). Syn phosphorylation by cAMP/PKA signalling increases the SV mobilisation. It allows the transition of vesicles from RP to the RRP but also promotes an increased exchange of SVs between adjacent synaptic varicosities along axons (Menegon et al, 2006; Valente et al, 2012; Patzke et al, 2019; Chenouard et al, 2020). Phosphorylation of Syn by CDK5 enhances its association with actin filaments and mediates a shift of SVs from RP to ResP (Verstegen et al, 2014). Interference with phosphorylation of Syn by PKA and CDK5, respectively, affects the presynaptic short-term and homeostatic plasticity highlighting the importance of dynamic phosphorylation at presynapse for physiological regulation of neurotransmission (Menegon et al, 2006; Valente et al, 2012; Verstegen et al, 2014). Bassoon (Bsn) is a large scaffolding protein localised exclusively at the active zone of neurotransmitter release (Dieck et al, 1998). It plays a key role in the organisation and maintenance of the presynaptic release apparatus (Gundelfinger et al, 2015). Mice with a deletion of the central part of Bsn show generalised seizures and impaired synaptic structure and function (Altrock et al, 2003; Dick et al, 2003). Bsn was shown to coordinate the recruitment of specific presynaptic voltage-gated calcium channels to the active zone via its interaction with RIM-binding proteins (Davydova et al, 2014). Therefore, the Cav2.1 and Cav1.3 channels, respectively, are less abundant in cortico-hippocampal glutamatergic synapses or cochlear and retinal ribbon synapses of Bsn mutants (Frank et al, 2010; Davydova et al, 2014; Babai et al, 2019; Ryl et al, 2021). Investigations of mouse strains expressing shortened or null Bsn alleles detected reduced RRP and slower vesicular replenishment in glutamatergic synapses of cultured hippocampal neurons, cerebellar mossy fibres, photoreceptors, cochlear inner hair cells and auditory nerve fibres (Altrock et al, 2003; Khimich et al, 2005; Frank et al, 2010; Hallermann et al, 2010; Jing et al, 2013; Mendoza Schulz et al, 2014; Babai et al, 2019, 2021; Ryl et al, 2021). The morphological analyses also pointed towards a role of Bsn (and its paralogue Piccolo) in the SV clustering and RRP regulation (Altrock et al, 2003; Mukherjee et al, 2010; Mendoza Schulz et al, 2014; Ackermann et al, 2019; Hoffmann-Conaway et al, 2020). However, the molecular basis for the role of Bsn in the regulation of RRP size and/or release site replenishment remained unclear. In this study, we investigated the cellular signalling that drives the dynamic regulation of SV recycling using in vivo imaging of hippocampal neurons derived from Bsn mutant mice (BsnGT). We observed a reduced size of RRP and TRP and a significant drop in the overall release competence of SVs in glutamatergic synapses lacking Bsn. These phenotypes were associated with a dysregulation of CDK5/calcineurin balance and cAMP/PKA presynaptic signalling. Acute pharmacological treatment that restored physiological signalling fully normalised the SV release competence in BsnGT neurons indicating a causal role of aberrant signalling in the loss of release competence in the absence of Bsn. Finally, our data uncovered a key role of CDK5-dependent regulation of PDE4 in the control of SV pools upstream of cAMP/PKA signalling. Results Deletion of Bsn restricts recycling competence of SVs Previous studies indicated changes in the size of RRP in the absence of Bsn in various types of synapses. However, to date, the mechanistic understanding of this phenomenon is missing. To monitor SV exocytosis in living neurons, we expressed the pH-sensitive probe, synaptophluorin-tdimer2 (sypHy), using lentiviral vectors in cultured hippocampal neurons that were derived from BsnGT animals, where Bsn expression was abolished by insertion of a gene-trap cassette (Hallermann et al, 2010) and from their WT littermates. In sypHy, pH-sensitive GFP is inserted in the lumenal domain of the integral SV protein synaptophysin. The fluorescence of sypHy is quenched by the acidic milieu of SVs, it increases after SV fusion and exposure to the neutral media and decreases again upon SVs endocytosis and re-acidification, which allows monitoring of SV recycling. The red fluorescent tdimer2 inserted within the cytoplasmic part of synaptophysin permits the identification of sypHy-expressing cells at rest (Rose et al, 2013). We utilised an established stimulation protocol consisting of 40 action potentials (AP) at 20 Hz to drive the fusion of docked SVs (corresponding to RRP) and 900 AP at 20 Hz to release all release-competent SVs (TRP) (Burrone et al, 2006). To prevent re-acidification of endocytosed SVs that occurs concomitantly with SV exocytosis during stimulation experiments were performed in the presence of bafilomycin A, a vesicular proton pump inhibitor. Under these conditions, all vesicles that once underwent exocytosis remain visible (Fig 1A and B). We detected significantly lower RRP and TRP in BsnGT compared with WT neurons (Fig 1A–C and E; RRP: WT 0.13 ± 0.01, BsnGT 0.08 ± 0.01; TRP: WT 0.41 ± 0.01, BsnGT 0.29 ± 0.01). Application of ammonium chloride, which alkalizes the SV and un-quenches all sypHy-expressing SVs that do not recycle, revealed a rise in the ResP in BsnGT (Fig 1A and B; ResP: WT 0.60 ± 0.013, BsnGT 0.71 ± 0.02). Quantification of the total synaptic intensity of sypHy upon unquenching also confirmed no changes in its synaptic expression levels between BsnGT and WT neurons (Fig 1A; WT 1,061 ± 143.8 AU, BsnGT 1,041 ± 90.2 AU). A clear-cut left shift in the bell-shaped frequency distribution of the response amplitude was evident in BsnGT upon 40 APs (Fig 1D) and 900APs (Fig 1F) confirming overall lower response magnitude in the BsnGT neurons compared with WT. This argues against functional inactivation of a fraction of synapses upon deletion of Bsn reported in earlier studies and for overall impairment (Altrock et al, 2003). Collectively, these experiments indicate a role of Bsn in the regulation of release competence of SV and their distribution to RRP and RP. Figure 1. Bassoon deletion affects the release competence of SV at glutamatergic synapse A. Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20 Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation. B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy in WT (black) and BsnGT (red) neurons as described in (A). Intensities were normalised to the peak of NH4Cl response. C. Quantifications of mean RRP fraction in WT and BsnGT. D. Frequency distribution histograms of the response amplitudes of 1,050 individual synaptic puncta (from six independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT. E. Quantifications of mean TRP fraction in WT and BsnGT. F. Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes. G. Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and BsnGT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels). H. Quantification of normalised IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in (G). I. Quantification of the fraction of active (Syt1Ab-labelled) excitatory and inhibitory synapses on experiments illustrated in (G). J. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in (G). K. Quantification of normalised IF of Syt1Ab uptake on experiments from (J). L. Quantification of the fraction of active excitatory and inhibitory synapses in (J). Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The dashed lines in (H) and (K) depict IF in WT that were used for normalisation. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) or to the number of analysed images (G, J). In (D) and (F), the data of 1,050 synapses per genotype were processed. Data is obtained from 6 (A–F) or 3 (G–L) independent culture preparations. Significance was assessed with the Student's t-test; ***P < 0.001. Scale bar is 2 μm in (A) and 5 μm in (G) and (J). Download figure Download PowerPoint The hippocampal neuron cultures used in these experiments contain predominantly excitatory pyramidal cells (Kaech et al, 2012). To assess the SV recycling specifically in excitatory and inhibitory neurons we combined labelling of active synapses in living neurons using a fluorophore-coupled anti-synaptotagmin1 antibody (Syt1Ab) with postfixation synapse type-specific immunostaining. This Syt1Ab antibody recognises the luminal domain of the integral SV protein Syt1. If added to the media of living cells, it can access its epitope only after fusion of SV with the plasma membrane, when the extracellular media contacts the lumen of SV (Kraszewski et al, 1995; Lazarevic et al, 2011, 2017). First, we performed Syt1Ab labelling in living neurons without any further manipulations to monitor SV recycling driven by endogenous neuronal activity (Fig 1G). The excitatory synapses were assessed as Syt1Ab-labelled puncta positive for synapsin and vesicular glutamate transporter 1 (VGLUT1) immunoreactivity and inhibitory synapses as puncta co-labelled for synapsin and vesicular GABA transporter (VGAT) (Fig 1G). Syt1Ab uptake driven by basal network activity was significantly lower in BsnGT excitatory presynapses, and no significant difference was seen in inhibitory boutons (Fig 1G and H; exc: WT 1.00 ± 0.08, BsnGT 0.49 ± 0.02; inh: WT 1.00 ± 0.07, BsnGT 0.84 ± 0.08). To assess the total recycling pool in both synapse types, we performed Syt1Ab labelling during depolarization induced by a brief application of 50 mM KCl, which leads to the release of the total recycling pool of SV (TRP) (Harata et al, 2001). Depolarization-induced Syt1Ab uptake was significantly decreased in the excitatory but not in the inhibitory synapses in neurons derived from BsnGT animals (Fig 1J and K; exc: WT 1.00 ± 0.07, BsnGT 0.69 ± 0.04; inh: WT 1.00 ± 0.06, BsnGT 0.86 ± 0.07). Postfixation labelling of neurons with a general synaptic marker after Syt1Ab uptake also enabled us to quantify the number of active vs. silent presynapses, i.e. presynapses competent for SV release and recycling under basal network activity or upon chemical depolarization (Moulder et al, 2010). The fraction of active glutamatergic synapses, defined as VGLUT1-positive puncta with detectable immunofluorescence (IF) of Syt1Ab uptake, was reduced by 40% in BsnGT neurons under basal conditions, indicating a higher proportion of silent (i.e. release incompetent) presynapses (Fig 1I; WT 0.54 ± 0.05, BsnGT 0.33 ± 0.03). The number of glutamatergic synapses capable of release upon KCl-induced depolarization was also decreased in BsnGT neurons compared with the WT (Fig 1L; WT 0.75 ± 0.05, BsnGT 0.45 ± 0.05). No significant differences in number of release-competent inhibitory, i.e. VGAT and synapsin-positive, synapses were detected (Fig 1I and L; basal: WT 0.55 ± 0.05, BsnGT 0.50 ± 0.04; KCl: WT 0.53 ± 0.03, BsnGT 0.46 ± 0.04). These analyses indicate that loss of Bsn affects the SV release competence predominantly at glutamatergic synapses. One plausible explanation for the lower release competence of SV could be a change in the expression of synaptic proteins linked to the regulation of synaptic release competence in the absence of Bsn. Indeed, previous studies reported a lower synaptic abundance of RIM or Munc13 and an increased abundance of piccolo (Pclo) upon deletion of Bsn (Altrock et al, 2003; Davydova et al, 2014; Montenegro-Venegas et al, 2021). Therefore, we quantified the synaptic abundance of Munc13 and Pclo in WT and BsnGT excitatory and inhibitory synapses (Fig EV1A and C). The quantification revealed significant decrease and increase, of immunoreactivity for Munc13-1 and Pclo, respectively, in both excitatory and inhibitory synapses (Fig EV1B and D; Munc13: exc: WT 1.00 ± 0.03, BsnGT 0.77 ± 0.02; inh: WT 1.00 ± 0.04, BsnGT 0.79 ± 0.03; Pclo: exc: WT 1.00 ± 0.03, BsnGT 1.48 ± 0.04; inh: WT 1.00 ± 0.04 BsnGT 1.24 ± 0.04). Thus, the synapse type-specific defect in SV release competence cannot be explained just by changes in abundance of Munc13-1 or Pclo upon deletion of Bsn. Click here to expand this figure. Figure EV1. Synaptic expression of Munc13 and Pclo are changed independently of synapse type in BsnGT neurons A, B. Representative images of 20 DIV cultured hippocampal neurons from WT and BsnGT immunolabelled for Munc13-1 (A) and Pclo (B) together with VGLUT1 or VGAT to mark excitatory and inhibitory synapses, respectively. C, D. Quantification of IF intensity for Munc13-1 (C) and for Pclo (D) in excitatory and inhibitory synapses from WT and BsnGT reveals a decreased IF for Munc13-1 and an increased IF for Pclo in both synapse types in BsnGT neurons. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to quantified independent visual fields obtained from three and four independent culture preparations (C, D). Statistical significance was assessed using the Student's t-test and is given as: ***P < 0.0001 for (C) in excitatory and (D) in excitatory and inhibitory puncta and ***P = 0.0003 in inhibitory synapses. Scale bar is 5 μm. Download figure Download PowerPoint CDK5 activity is increased in BsnGT and uncoupled from regulation by neuronal activity CDK5 plays an important role in the regulation of release competence of SVs, especially via dynamic recruitment of SVs from the RP and to the ResP (Kim & Ryan, 2010, 2013; Verstegen et al, 2014). To assess the potential involvement of CDK5 in the shift of SV from recycling to resting pool that we observed in BsnGT neurons, we pharmacologically interfered with the activity of this enzyme in WT and BsnGT neurons. In accordance with the reported role of CDK5, we observed a significant increase in the fraction of RRP and TRP in WT neurons after acute inhibition of CDK5 with roscovitine (100 μM, 30 min). The analyses of data revealed that roscovitine increased RRP by 68% and TRP by 57% in BsnGT but only by 38% (RRP) and 29% (TRP) in WT (Fig 2A, B and E; WT vs. BsnGT; RRP 138 ± 9 vs. 168 ± 10%; TRP: 129 ± 5 vs. 157 ± 7%). Importantly, the roscovitine treatment restored RRP and TRP fractions in BsnGT neurons to levels similar to treated WT neurons (Fig 2B–D; RRP: WT 0.13 ± 0.01, WT + roscovitine 0.18 ± 0.01, BsnGT 0.09 ± 0.01, BsnGT + roscovitine 0.15 ± 0.01; TRP: WT 0.40 ± 0.01, WT + roscovitine 0.51 ± 0.02, BsnGT 0.29 ± 0.02, BsnGT + roscovitine 0.45 ± 0.02). This indicates that an elevated activity of CDK5 possibly contributes to the changes in SV pools in BsnGT neurons. Figure 2. Inhibition of CDK5 activity normalises SV pools in BsnGT neurons A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100 μM, grey and pink trace) or vehicle (black and red trace) for 30 min before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared with WT. Data information: n corresponding to the number of imaging experiments done on four independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with the Tukey's multiple comparison test (C, D) and by the Student's t-test (E) *P ≤ 0.05, **P < 0.01, ***P < 0.001. Scale bar is 2 μm. Download figure Download PowerPoint An important substrate of CDK5 in the context of regulation of SV recycling is the Ser551 of SV-associated protein synapsin1 (Syn1) (Verstegen et al, 2014). Phosphorylation at Ser551 of Syn1 (pSer551Syn1) enhances the binding of Syn1 to F-actin, promotes a shift of SVs to the ResP, and thereby, it restricts SV recycling (Fig 3D) (Verstegen et al, 2014). To address whether deregulation of CDK5-dependent pSer551Syn1 underlies the changes in SV pools in BsnGT neurons we utilised the previously published phospho-specific antibody (Verstegen et al, 2014). We detected significantly higher levels of pSer551Syn1 in P2 fractions of hippocampal lysates from BsnGT mice than in WT, while the total expression levels of Syn1 were unchanged (Fig 3A–C; pSer551Syn1/Syn1: WT 1.00 ± 0.14, BsnGT 1.32 ± 0.12; Syn1: WT 1.00 ± 0.14, BsnGT 1.16 ± 0.14). Next, we used this antibody to visualise the abundance of pSer551Syn1 in individual synapses in cultured WT and BsnGT neurons. We detected a significantly higher pSer551Syn1 IF intensity in synapses of neurons from BsnGT compared with the WT, while the total expression of Syn1 did not differ between genotypes (Fig 3E and F; WT 1.00 ± 0.06, BsnGT 1.55 ± 0.08). Importantly, the pSer551Syn1 immunoreactivity was dramatically decreased in WT terminals upon treatment with roscovitine, confirming the specificity of the antibody (Fig 3E and F; 0.43 ± 0.03). In the next step, we quantified the abundance of total Syn1 and pSer551Syn1 in excitatory synapses labelled against VGLUT1 and in inhibitory synapses labelled against VGAT (Fig EV2A). Expression of Syn1 did not differ between BsnGT and WT in neither excitatory nor inhibitory synapses (Fig EV2B). However, the situation was different for pSer551Syn1. While IF intensity of pSer551Syn1 labelling was significantly increased in excitatory synapses, it was unchanged in inhibitory ones (Fig EV2C; exc: WT 1.00 ± 0.03, BsnGT 1.40 ± 0.04; inh: WT 1.00 ± 0.05, BsnGT 1.09 ± 0.03. This result indicates increased CDK5-mediated phosphorylation of Se
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