Epilepsy-related CDKL5 deficiency slows synaptic vesicle endocytosis in central nerve terminals

Abstract

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippocampal neurons generated from a Cdkl5 knockout rat model. Using a genetically-encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in CDKL5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of amphiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD.

The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis

“…Our data support previous observations that the phospho-null mutant S293A associates with endophilin A1, but the phosphomimetic mutant S293E does not (Sekiguchi et al 2013). Critically, this residue is the prominent in vivo phosphorylation site on Amph1 (Craft et al 2008), and is subject to activity-dependent dephosphorylation by the calcium-dependent protein phosphatase calcineurin (Craft et al 2008; Murakami et al 2006; Kontaxi et al 2022). Therefore, the Amph1-endophilin A1 interaction is coupled to neuronal activity via the action of calcineurin, similar to other key endocytosis molecules that form the dephosphin group of proteins (Anggono et al 2006; Cousin & Robinson 2001; Lee et al 2004; Lee et al 2005).…”

“…Point mutations were introduced into Amph1 using standard site-directed mutagenesis protocols with the following primers: 5’-GAGAACATCATCAATT C CT C T AG GGACAACTTTGTACCAG-3’ (sense) and 5’-CTGGTACAAAGTTGTCC CT A G AG G AATTGATGATGTTCTC (antisense) for 323 FFE 325 to 323 SSR 325 , 5’-GGAGACTTTG C TGCATT C G CG CTTTGACCCTTTCAAAC-3’ (sense) and 5’-GTTTGAAAGGGTCAAAG CG C G AAT G CAGCAAAGTCTCC-3’ (antisense) for 353 DLD 355 to 353 HSR 355 , 5’-CAGACATTGCCCT C G CG CTTATGGACGACAAG-3’(sense) and 5’-CTTGTCGTCCATAA GC GCGAGGGCAATGTCTG-3’ (antisense) for 382 WD 383 to 382 SR 383 , 5’-GCCACATACAAA C GCCTCTTTCTAGAGAACTTCACAC-3’ (sense) and 5’-GTGTGAAGTTCTCT A GAAAGAGGC G TTTGTATGTGGC-3’ (antisense) for 672 GLFP 675 to 672 RLFL 675 , 5’-GCATCTCCTGCC G CAGTGCGA G CCAGATCACCTTCAC-3’ (sense) and 5’-GTGAAGGTGATCTGG C TCGCACTG C GGCAGGAGATGC-3’ (antisense) for 288 PVRP 291 to 288 AVRA 291 , 5’-CAAGGAAAGGG G CT G CTGTCGCAGCTCT G CCTAAAG-3’ (sense) 5’-and CTTTAGGCAGAG C TG C GACAG C AG C CCCTTTCCTTG-3’ (antisense) for 301 PPVPP 305 to 301 A 1 A 2 VA 3 A 4 305 , 5’-GACAAGGAAAGGGGCTGCTGTCCCACCTCTGC-3’ (sense) and 5’-GCAGAGGTGGGACAGCAGCCCCTTTCCTTGTC-3’ (antisense) for 301 PP 302 to 301 A1A2 302 , and 5’-GAAAGGGCCTCCTGTC G CA G CTCTGCCTAAAGTC-3’ (sense) and 5’-GACTTTAGGCAGAG C TGCGACAGGAGGCCCTTTC-3’ (antisense) for 304 PP 305 to 304 A3A4 305 with modified sites underlined. The Amph1 phosphomutants, S293A and S293E, were generated as previously described (Kontaxi et al 2022). GST-Amph1 constructs were generated by subcloning Amph1 constructs into a pGEX-KG vector (obtained from Dr. C. Rickman, Heriot-Watt University, Edinburgh, UK) using the primers 5’-CATCAT GAATTC TAGGAGCTCCCAGTGATTCGGGTC-3’ (sense; ΔNBAR), 5’-ATGATG CTCGAG CTATTCTAGGTGTCGTGTGAAGTTCTC-3’ (antisense), and 5’-ATGAT GCTCGAG CTAAGGAGGCAGTTCCTGAGCGG-3’ (antisense; ΔSH3) with restriction sites underlined.…”

“…Scientific, cat no. HG73010)) staining prior to immunoblot analysis as previously described (Kontaxi et al 2022). The primary antibodies used in this study are as follows: goat endophilin A1 (1:1000; Santa Cruz Biotechnology, cat no.…”

“…Amph1 is a phosphoprotein with most of its potential phosphorylation sites located within the PRD, including S293, which is its most abundant in vivo phosphosite (Craft et al 2008). We and others have shown that Amph1-S293 undergoes calcineurinmediated dephosphorylation coupled to neuronal activity, indicating its physiological importance (Craft et al 2008;Murakami et al 2006;Kontaxi et al 2022). Importantly, the phosphorylation status of S293 is critical in controlling the formation of the Amph1endophilin A1 complex (Sekiguchi et al 2013;Murakami et al 2006).…”

Epilepsy-linked kinase CDKL5 phosphorylates voltage-gated calcium channel Cav2.3, altering inactivation kinetics and neuronal excitability

“…Phosphorylation targets include microtubule binding proteins EB2 and MAP1S 6,20 and transcriptional regulators ELOA 21 and Sox9 22 , showing its diverse cellular roles. CDKL5 knockout (KO) neurons have deficits in synaptic transmission, dendritic spine development, neurite outgrowth, cilia elongation and microtubule dynamics 23,24,25,26,27,28,29 . However, molecular mechanisms linking CDKL5 loss to neuronal hyperexcitability remain unknown.…”