Abl2 Kinase Differentially Regulates iGluRs Current Activity and Synaptic Localization

Abstract

Abelson non-receptor tyrosine kinases (Abl1 and Abl2) are established cellular signaling proteins, implicated in cytoskeletal reorganization essential for modulation of cell morphology and motility. During development of the central nervous system, Abl kinases play fundamental roles in neurulation and neurite outgrowth, relaying information from axon guidance cues and growth factor receptors to promote cytoskeletal rearrangements. In mature neurons, Abl kinases localize to pre- and postsynaptic compartments and are involved in regulation of synaptic stability and plasticity. Although emerging evidence indicates interchangeability of these isoforms in managing of cellular functions, in healthy adult neurons, Abl1 contribution is less elucidated, while Abl2 is required for optimal synaptic functioning. Our previous study demonstrated compartmentalization of Abl1 to the presynapse and Abl2 to the postsynapse and characterized their modulatory effect on spontaneous excitatory synaptic transmission. Here, we further delineate the role of Abl2 on regulation of the postsynaptic component of miniature excitatory postsynaptic current (mEPSC). Our findings show that both acute and prolonged activation of Abl2, in line with reduction of mEPSC amplitude, also decrease AMPA and NMDA current amplitudes. In contrast with the current-detrimental effect, prolonged Abl2 activity stabilizes spines, particularly contributing to maintenance of active synapses at distal (perhaps apical) segments of dendrites. Hence, we propose that attenuation of ion currents via ionotropic glutamatergic receptors by Abl2 kinase derives from either reduction of the receptor sensitivity for glutamate or is due to alteration of channel gating mechanisms. Abl2 and excitatory postsynapses: Abl2 expression level affects active excitatory synapse density on distal dendrites, while Abl2 activity impacts current density through AMPA and NMDA receptors.

Keywords: AMPA; Abelson non-receptor tyrosine kinase (Abl); Miniature excitatory post synaptic currents (mEPSC); NMDA; Spontaneous synaptic activity.

Fig. 1

Effect of Abl kinase activation and inhibition on mEPSC rise time and decay constant. A Representative averaged traces with ± standard deviation trace range of mEPSC per neuronal recording of control (DMSO solution 1:2000 dilution), 10 µM DPH, and 10 µM imatinib treatment traces with fit lines of rising and decay phases generated by rise-time analysis in the range of 10–90% of peak and decay one-exponential fitting using equation I = I₀e^−t/τ + C. B Assessment of rise time slopes (I/t) average differences between control, DPH-, and imatinib-treated groups. C Assessment of mEPSC decay time constant extracted from equation in A for control, DPH-, and imatinib-treated neurons. Data presented as mean (± SD). ***p < 0.001

Fig. 2

Activation of Abl kinase reduces AMPA and NMDA currents. A, C Representative AMPA current traces recorded during DPH (A) and imatinib (C) application; B, D Average effects of DPH (B) or imatinib (D) on AMPA currents normalized to control; E, G Representative NMDA current traces recorded during DPH (E) and imatinib (G) application; F, H Average effects of DPH (F) or imatinib (H) on NMDA currents normalized to control; Data represent means (± SD). *p < 0.05; ***p < 0.001; N = 5, n(control) = 26, n(DPH) = 26, n(washout) = 18 for A, B; N = 4, n(cont, ima, washout) = 23 for C, D; N = 3 and n(cont, DPH) = 17, n(washout) = 16 for E, F; n = 14 for E–H. N number of independent experiments, n number of recordings

Fig. 3

Abl2-knockdown increases AMPA and NMDA current densities. A Representative current traces of mEPSCs elicited in siControl, siAbl1-, and siAbl2-transfected cultured hippocampal neurons. B Averaged amplitudes of mEPSCs elicited as in A [N = 4, n_sicont = 20, n_siAbl1 = 21, n_siAbl2 = 22—representing the number of experiments (N) and of analyzed cells (n)]. C, E Representative AMPA (C) and NMDA (E) traces recorded from different cells transfected with siRNAs; D, F Average effects of Abl kinase knockdown on AMPA (D) and NMDA (F) current densities. N = 5, n_sicont = 25, n_siAbl1 = 28, n_siAbl2 = 35 for C, and D; N = 3, n_{sicont, siAbl1, siAbl2} = 26. G Representative traces of mEPSCs elicited in siControl and siAbl2-transfected cultured hippocampal neurons treated with DPH. H Averaged amplitudes of mEPSCs elicited as in G. I Immunofluorescence staining and colocalization analysis of vGlut (green) and GluA1 (red) proteins after siAbl2 or siControl transfection. 70,000 neurons per 12 mm coverslip were plated and transfected at DIV18; cells were fixed with 4% PFA at DIV21 and stained with vGlut (1:200) and GluA1 (1:200) antibodies. Scale bar 10 μm. Proximal (50 μm) and distal (20 μm) parts of dendrites were analyzed by Coloc2 plugin for ImageJ. J Summarized collocalization assessment by Pearson’s correlation coefficient between GluA1 and vGlut1 in siAbl2 transfected (n = 33) cultures compared to the neurons from the same cultures, transfected with siControl (n = 23). Data represent means (± SD). **p < 0.01, ***p < 0.005

Fig. 4

Abl2 overexpression reduces spontaneous synaptic activity. A Representative mEPSC traces of hippocampal neurons, transduced with lentiviruses encoding mCherry alone (EV = Empty vector), and Abl2-WT (WT); B, D average effect of Abl2-WT overexpression on mEPSC inter-event intervals (D) and amplitudes (B) over three independent experiments (n = 13 and 17 for EV and Abl2-WT, respectively); C, E Representative cumulative distribution plots of mEPSC inter-event intervals (E) and amplitudes (C). F, G Effect of Abl2 overexpression on AMPA current. F Representative current traces; G Average effect of Abl2-WT overexpression on AMPA current density over four independent experiments (n = 9 and 11 for EV and Abl2-WT, respectively). H Representative immunoblot images of GluA1, GluA2, and γ-actin expression; I Averaged fold change of protein expression assessed by immunoblot in three independent experiments. Data represent means (± SD); *p < 0.05; **p < 0.01