Kinesin Family of Proteins Kif11 and Kif21B Act as Inhibitory Constraints of Excitatory Synaptic Transmission Through Distinct Mechanisms

Abstract

Despite our understanding of the functions of the kinesin family of motor proteins (Kifs) in neurons, their specific roles in neuronal communication are less understood. To address this, by carrying out RNAi-mediated loss of function studies, we assessed the necessity of 18 Kifs in excitatory synaptic transmission in mouse primary hippocampal neurons prepared from both sexes. Our measurements of excitatory post-synaptic currents (EPSCs) have identified 7 Kifs that were found to be not critical and 11 Kifs that are essential for synaptic transmission by impacting either frequency or amplitude or both components of EPSCs. Intriguingly we found that knockdown of mitotic Kif4A and Kif11 and post-mitotic Kif21B resulted in an increase in EPSCs suggesting that they function as inhibitory constraints on synaptic transmission. Furthermore, Kifs (11, 21B, 13B) with distinct effects on synaptic transmission are expressed in the same hippocampal neuron. Mechanistically, unlike Kif21B, Kif11 requires the activity of pre-synaptic NMDARs. In addition, we find that Kif11 knockdown enhanced dendritic arborization, synapse number, expression of synaptic vesicle proteins synaptophysin and active zone protein Piccolo. Moreover, expression of Piccolo constrained Kif11 function in synaptic transmission. Together these results suggest that neurons are able to utilize specific Kifs as tools for calibrating synaptic function. These studies bring novel insights into the biology of Kifs and functioning of neural circuits.

Molecular Motor KIF3B Acts as a Key Regulator of Dendritic Architecture in Cortical Neurons

“…To address this question, we knocked down KIF3B in cortical cultures at DIV 13-14 for 72 h and imaged the dendritic architecture by confocal imaging (Figures 3A,B). Sholl analysis (Swarnkar et al, 2018) on the resultant confocal images show a significant enhancement in dendritic branching with KIF3B knockdown. From distances of 20 to 70 µm, knockdown of KIF3B dramatically increases the dendritic arborization of cortical pyramidal neurons ( Figure 3C; two-way ANOVA with Sidak post hoc test, F (1,37) = 34.46, * * * * p < 0.0001).…”

“…We first studied the effect of KIF3B knockdown on spine density and morphology. ShKIF3B-A plasmid was transfected into cortical neurons at DIV 13-14 ( Figure 2A) and allowed to express for 72 h following an established protocol in the laboratory (Swarnkar et al, 2018). These cultures were used for live confocal imaging focusing on the dendritic spines of pyramidal neurons since they are the primary sites of input for glutamatergic synapses (Figure 2B).…”

“…These cultures were used for live confocal imaging focusing on the dendritic spines of pyramidal neurons since they are the primary sites of input for glutamatergic synapses (Figure 2B). Using a customwritten MATLAB pipeline (Swarnkar et al, 2018), we were able to measure the total spine number per 100 µm as well as determine the morphology of the spines. As depicted in the bar graph ( Figure 2C), 72 h of KIF3B knockdown significantly increased the total spine density of pyramidal neurons (shKIF3B 51.56 ± 2.7 and shScrambled 33.69 ± 2.9, Student's t-test, t (56) = 4.546, p < 0.0001).…”

“…These mitotic KIFs have recently been shown to have a post-mitotic role. Work described in Swarnkar et al (2018) described that KIF11 inhibits synaptic transmission in a pre-synaptic manner via the regulation of the presynaptic protein, piccolo.…”

A cAMP/PKA-dependent synaptically targeted lncRNA mediates structural plasticity in hippocampal neurons by functionally interacting with the Spectrin/Ankyrin Network

“…Given that Kif2a is part of the Kinesin family of motor proteins responsible for anterograde transport, we hypothesized that Kif2a mediates ADEPTR transport. To test this hypothesis, we knocked down Kif2a, or 4 other Kinesins, using siRNAs that were previously validated in hippocampal neurons for knockdown efficiency [26]. Following 72 hours of knockdown, we treated neurons with Forskolin prior to synaptoneurosome fractionation or ADEPTR FISH (Fig.…”