Synaptopodin regulates denervation-induced plasticity at hippocampal mossy fiber synapses

Neurological diseases can lead to the denervation of brain regions caused by demyelination, traumatic injury or cell death. The molecular and structural mechanisms underlying lesion-induced reorganization of denervated brain regions, however, are a matter of ongoing investigation. In order to address this issue, we performed an entorhinal cortex lesion (ECL) in mouse organotypic entorhino-hippocampal tissue cultures of both sexes and studied denervation-induced plasticity of mossy fiber synapses, which connect dentate granule cells (dGCs) with CA3 pyramidal cells (CA3-PCs) and play important roles in learning and memory formation. Partial denervation caused a strengthening of excitatory neurotransmission in dGCs, CA3-PCs and their direct synaptic connections, as revealed by paired recordings (dGC-to-CA3-PC). These functional changes were accompanied by ultrastructural reorganization of mossy fiber synapses, which regularly contain the plasticity-regulating protein synaptopodin and the spine apparatus organelle. We demonstrate that the spine apparatus organelle and synaptopodin are related to ribosomes in close proximity to synaptic sites and unravel a synaptopodin-related transcriptome. Notably, synaptopodin-deficient tissue preparations that lack the spine apparatus organelle failed to express lesion-induced synaptic adjustments. Hence, synaptopodin and the spine apparatus organelle play a crucial role in regulating lesion-induced synaptic plasticity at hippocampal mossy fiber synapses.

Methods
Ethics statement

Mice were maintained in a 12 h light/dark cycle with food and water available ad libitum. Every effort was made to minimize distress and pain of animals. All experimental procedures were performed according to German animal welfare legislation and approved by the appropriate animal welfare committee and the animal welfare officer of Albert-Ludwigs-University Freiburg, Faculty of Medicine (X-17/07K, X-17/09K, X-21/01B) and Hannover Medical School (2022/308).

Preparation of tissue cultures

Entorhino-hippocampal tissue cultures were prepared at postnatal day 3-5 from C57BL/6J (wildtype, Synpo+/+) and B6.129-Synpotm1Mndl/Dllr (Synpo-/-) mice of either sex. Synpo-/- strain was back crossed for at least 10 times to establish C57BL/6J genetic background. Cultivation medium contained 50% (v/v) MEM, 25% (v/v) basal medium eagle, 25% (v/v) heat-inactivated normal horse serum, 25 mM HEPES buffer solution, 0.15% (w/v) bicarbonate, 0.65% (w/v) glucose, 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamax. The pH was adjusted to 7.3. All tissue cultures were allowed to mature for at least 18 days in a humidified atmosphere with 5% CO2 at 35°C. Cultivation medium was replaced 3 times per week.

Entorhinal cortex lesion (ECL)

The entorhinal cortex of mature slice cultures (≥18 days in vitro) was transected with a sterile scalpel from the rhinal to the hippocampal fissure to achieve a complete lesion of the perforant path. Except for the lesion-induced partial denervation of the hippocampus, cytoarchitecture of both the hippocampus and the entorhinal cortex remained unchanged.

Whole-cell patch-clamp recordings

Whole-cell patch-clamp recordings were carried out at 35°C (3-6 neurons per culture). The bath solution contained (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, and 10 glucose (aCSF) and was oxygenated continuously (5% CO2 / 95% O2). Patch pipettes contained (in mM) 126 K-gluconate, 10 HEPES, 4 KCl, 4 ATP-Mg, 0.3 GTP-Na2, 10 PO-Creatine, 0.3% (w/v) biocytin (pH 7.25 with KOH, 290 mOsm with sucrose), having a tip resistance of 4-6 MΩ. Neurons were visually identified using a LN-Scope (Luigs & Neumann) equipped with an infrared dot-contrast and a 40x water-immersion objective (NA 0.8; Olympus). Spontaneous excitatory postsynaptic currents (sEPSCs) of both dGCs and CA3-PCs were recorded in voltage-clamp mode at a holding potential of −70 mV. In the dentate gyrus, recordings were performed in granule cells at the outer parts of the granule cell layer in the suprapyramidal blade with maximum distance to the subgranular zone. Moreover, the resting membrane potential was estimated before our recordings to exclude immature granule cells. In CA3, recordings were performed in pyramidal cells close to the dentate gyrus in proximity to str. lucidum. Series resistance was monitored before and after each recording and recordings were discarded if the series resistance reached ≥ 30 MΩ. For recording of intrinsic cellular properties in current-clamp mode, pipette capacitance of 2.0 pF was corrected and series resistance was compensated using the automated bridge balance tool of the MultiClamp commander. I-V-curves were generated by injecting 1 s square pulse currents starting at −100 pA and increasing in 10 pA steps until +500 pA current injection was reached (sweep duration: 2 s).

Paired whole-cell patch-clamp recordings of hippocampal mossy fiber synapses

Action potentials were generated in the presynaptic dGC by 5 ms square current pulses (1 nA). For connectivity analysis, 5 action potentials were applied at 20 Hz respectively (inter-sweep-interval: 10 s; at least 30 repetitions), while recording unitary excitatory postsynaptic currents (uEPSCs) from CA3-PCs. Neurons were considered to be connected if > 5 % of action potentials evoked time-locked inward uEPSCs. Recordings were performed in the presence of (-)-bicuculline-methiodide (10 µM, Abcam, #ab120108) to prevent inhibitory synapse recruitment. Moreover, glutamine (200 µM, Gibco, #11539876) was added to the recording medium to avoid synaptic depletion.

Post-hoc staining and imaging

Tissue cultures were fixed overnight in a solution of 4% (w/v) paraformaldehyde (PFA; in PBS (0.1M, pH 7.4) with 4% (w/v) sucrose). After fixation, cultures were washed in PBS (0.1 M, pH 7.4) and consecutively incubated for 1 h with 10% (v/v) normal goat serum (NGS) in 0.5% (v/v) Triton X-100 containing PBS to reduce nonspecific staining. For post-hoc visualization of patched dGCs and CA3-PCs, Streptavidin Alexa Fluor 488 (Streptavidin A488, 1:1000; Invitrogen, #S32354) was added to the incubation solution. DAPI nuclear stain (1:5000 in PBS for 10 min; Thermo Scientific, #62248) was used to visualize cytoarchitecture. Cultures were washed, transferred onto glass slides and mounted for visualization with DAKO anti-fading mounting medium (Agilent, #S302380-2).

Serial block-face scanning electron microscopy (SBF-SEM)

For volume electron microscopic evaluation, samples were fixed in 1.5 % glutaraldehyde and 1.5 % paraformaldehyde buffered in 0.15 M HEPES, pH = 7.35. Further processing was based on accessible protocols (https://ncmir.ucsd.edu/sbem-protocol). In brief, for postfixation and staining reduced osmium tetroxide, thiocarbohydrazide and osmium tetroxide (rOTO), followed by uranyl acetate and lead aspartate were applied. After dehydration in an ascending acetone series, samples were embedded in epoxy resin, Araldite CY212 premix kit (Agar Scientific, Stansted Essex, UK).

Target regions were identified on semithin sections (1 µm, stained with toluidine blue). Accordingly, resin blocks were trimmed, mounted with conductive epoxy glue (Chemtronics, CircuitWorks, Kennesaw, USA) on sample pin stubs (Micro to Nano, Haarlem, Netherlands), precisely trimmed again and sputter coated with a gold layer.

Image stacks were acquired with a Zeiss Merlin VP Compact SEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a Gatan 3View2XP system (Gatan Inc., Pleasanton, CA, USA). Every second block-face (section thickness 50 nm) was scanned with 3.5 kV acceleration voltage and 100 % focal charge compensation (10 nm pixel size, 3 µs dwell time) by using the Multi-ROI mode to record multiple areas on each surface.

Transmission electron microscopy – mossy fiber synapse visualization

Wildtype (Synpo+/+) and Synpo-/- tissue cultures were fixed in 4 % paraformaldehyde (w/v) and 2 % glutaraldehyde (w/v) in 0.1 M phosphate buffer (PB) overnight and washed for 1 hour in 0.1 M PB. After fixation, tissue cultures were sliced with a vibratome and the slices were incubated with 1 % osmium tetroxide for 20 min in 5 % (w/v) sucrose containing 0.1 M PB. The slices were washed 5 times for 10 min in 0.1 M PB and washed in graded ethanol [10 min in 10 % (v/v) and 10 min in 20 % (v/v)]. The slices were then incubated with uranyl acetate [1 % (w/v) in 70 % (v/v) ethanol] overnight and were subsequently dehydrated in graded ethanol 80 % (v/v), 90 % (v/v) and 98 % (v/v) for 10 min. Finally, slices were incubated with 100 % (v/v) ethanol twice for 15 min followed by two 15 min washes with propylene oxide. The slices were then transferred for 30 min in a 1:1 mixture of propylene oxide with durcupan and then for 1 hour in durcupan. The durcupan was exchanged with fresh durcupan and the slices were transferred to 4 °C overnight. The slices were then embedded between liquid release-coated slides and coverslips. Cultures were re-embedded in blocks and ultrathin sections were collected on copper grids, at which point an additional Pb-citrate contrasting step was performed (3 min). Electron microscopy was performed at a LEO 906E microscope (Zeiss) at 4646x magnification.

Synaptopodin co-Immunoprecipitation (SP-coIP), RNA-Seq analysis and negative contrast electron microscopy

Six tissue cultures per biological replicate were washed in coIP-solution and dounce homogenized on ice in a solution containing (150 µl per sample, coIP-solution, in mM): 10 HEPES, 120 NaCl, 0.05% (v/v) NP-40 or IGEPAL CA-630 (Sigma Aldrich, #I8896), cOmplete protease inhibitor (Roche, according to manufacturer’s instructions), murine RNase inhibitor (1:1000, New England Biolabs, #M0314) and cycloheximide (0.5 mg/ml, Sigma Aldrich, #C-4859). Input was precleared twice with protein A beads (10 µl per 100 µl sample/supernatant) to reduce non-specific binding. Protein A beads (10 µl, New England Biolabs, #S1425) were washed twice in coIP-solution and consecutively incubated for 1 hour at 4°C with an anti-synaptopodin antibody (rabbit anti-synaptopodin, Synaptic Systems, #163002). Beads were carefully washed twice to remove unbound antibodies and incubated for 1 hour with the precleared supernatant (‘input’) at 4 °C with head-over-tail rotation. 10 µl of the precleared supernatant were kept as input control. After incubation, beads were washed twice with coIP-solution and finally dissolved in RNA protection buffer (New England Biolabs).

In another round of experiments, hippocampal tissue from adult animals was used for co-immunoprecipitation experiments (antibody: monoclonal rabbit anti-synaptopodin, Abcam, #ab259976 Abcam) with subsequent negative contrast electron microscopy. Beads (30 µl) were adsorbed on carbon-coated films stabilized by a copper grid and stained with 3% uranyl acetate (Serva, Heidelberg, Germany). Samples were immediately examined with a transmission electron microscope (Morgagni 268, 80 kV, FEI, Eindhoven, The Netherlands).

RNA was released from the beads through protein K lysis (Monarch® Total RNA Miniprep Kit; New England Biolabs, #T2010S). After lysis, beads were removed from the solution in a magnetic rack and the RNA containing supernatant was transferred to fresh tubes. RNA isolation on coIP- and input-samples was then performed according to the manufacturer’s instructions. RNA content was determined using the Agilent RNA 6000 Pico Kit (Agilent, #5067-1513) with a 2100 Bioanalyzer (Agilent, #G2939BA). RNA Library preparations for Synpo-related transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 Sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 150 bp read length. Data analysis was performed at the Galaxy platform (usegalaxy.eu). All files contained more than 10 M high-quality reads (after mapping to the reference genome; mm10) having at least a phred quality of 30 (> 90 % of total reads).

Regional mRNA library preparations and transcriptome analysis

RNA Library preparations for transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. Briefly, isolation of the dentate gyrus and CA3 area from individual tissue cultures was performed using a scalpel. One isolated dentate gyrus or CA3 area respectively was transferred to 7.5 µl lysis buffer (supplemented with murine RNase inhibitor) and homogenized using a pestle. Samples were centrifuged for 30 s at 10,000 g and 5 µl of supernatant were collected from individual samples and further processed. After cDNA synthesis, cDNA amplification was performed according to the manufacturer's protocol with 12 PCR cycles. cDNA yield was consecutively analyzed by a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). cDNA amount was adjusted to 10 ng for further downstream applications. After fragmentation and adaptor ligation, dual index primers (New England Biolabs, #E7600S) were ligated in a library amplification step using 10 PCR cycles. Libraries were finally cleaned up with 0.8X SPRI beads following a standard bead purification protocol. Library purity and size distribution was assessed with a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 Sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 100 bp read length. Data analysis was performed at the Galaxy platform (usegalaxy.eu). All files contained more than 10 M high-quality reads (after mapping to the reference genome; mm10) having at least a phred quality of 30 (>90% of total reads).