Rem2 is important for excitatory synapse formation in primary hippocampal neurons.

<p><b>A</b>) Representative images of excitatory synapse staining for the presynaptic marker, synapsin I (blue) and the postsynaptic marker PSD-95 (red) in neurons co-transfected with GFP and either: Mock (empty pSuper vector), overexpression with the Rem2 RNAi-resistant cDNA (OE), RNAi with the Rem2 short hairpin 1 (RNAi 1), RNAi with a second Rem2 short hairpin (RNAi 2), or rescue of RNAi 1 (RE 1), or RNAi 2 (RE 2) with the Rem2 RNAi-resistant cDNA. Scale bar indicates 5 µm. <b>B</b>) Synapse density measured by the co-localization of synapsin I and PSD-95 on a GFP positive stretch of dendrite. n > 40 neurons per condition. * p ≤ 0.05 compared to mock or ^# p ≤ 0.05 compared to each appropriate RNAi condition (i.e. RE1 vs. RNAi 1) using a two-way between-effect ANOVA followed by a Tukey’s post hoc test.</p

Rem2 is a negative regulator of dendritic complexity.

<p><b>A</b>) Representative images of Mock, RNAi 1, RNAi 2, OE, RE 1, and RE 2 transfected neurons. Scale bar = 50 µM. <b>B</b>) Quantification of dendritic complexity for each condition using Sholl analysis. n > 40 neurons per condition. * p ≤ 0.05 compared to mock determined by multivariate ANOVA with a Tukey’s post hoc test.</p

Rem2 is important for functional excitatory synapse formation.

<p><b>A</b>) Representative 20 s miniature excitatory postsynaptic current (mEPSC) trace selected for each experimental condition: Mock, OE, RNAi 1, RE 1, RNAi 2, and RE 2, recorded from primary hippocampal neurons at 14 DIV. <b>B</b> and <b>C</b>) Average mEPSC frequency (<b>B</b>) and amplitude (<b>C</b>) measured for each condition for a total of 300 s. <b>B</b> and <b>C</b>) n ≥ 15 neurons per condition (3 separate experiments). <b>D</b>) Cumulative distribution of mEPSC amplitudes for Mock, OE, RNAi 1, RE 1, RNAi 2, and RE 2 generated from 100 random mEPSC per cell (n=15 cells per condition). RNAi 1 and RNAi 2 mEPSC amplitude plots were significantly different from Mock (p < 0.05, Kolmogorov-Smirnov test). Additionally, the leftward shift following RNAi 1 and RNAi 2 was significantly different from their respective rescue conditions (RE 1 and RE 2, respectively; p < 0.05). OE, RE 1, and RE 2 were not significantly different from Mock. (<b>B</b> and <b>C</b>) * p ≤ 0.05 compared to mock or ^# p ≤ 0.05 compared to the appropriate RNAi condition using a one-way ANOVA followed by a student’s independent <i>t</i>-test.</p

Rem2 expression in hippocampal neurons.

<p><b>A</b>) Western blot with anti-Rem2 antibody of HEK 293T cell lysates. anti-β-actin serves as a loading control. HEK 293T cells were transfected with either myc-Rem2 or RNAi-resistant myc-Rem2 and either the Rem2 short hairpin 1 (RNAi 1) or the Rem2 short hairpin 2 (RNAi 2) to confirm Rem2 knockdown and rescue. <b>B</b>) Representative images of Rem2 (red) and MAP2 (blue) staining in neurons transfected with GFP and either Mock (empty pSuper vector), overexpression with the Rem2 RNAi-resistant cDNA (OE), RNAi with the Rem2 short hairpin 1 (RNAi 1), RNAi with a second Rem2 short hairpin (RNAi 2), or rescue of RNAi 1 (RE 1), or RNAi 2 (RE 2) with the Rem2 RNAi-resistant cDNA. Scale bar = 5 µM. <b>C</b>) Average Rem2 staining intensity (top left) or MAP2 intensity (bottom left) measured in arbitrary units for each condition (Right). Average Rem2 immunostaining intensity normalized to MAP2 intensity in hippocampal neurons. n > 40 neurons per condition. * p ≤ 0.05 compared to mock or ^# p ≤ 0.05 compared to the appropriate RNAi condition (i.e. RE1 vs. RNAi 1) using a two-way ANOVA followed by a Tukey’s post hoc test.</p

Rem2 overexpression significantly reduces voltage-gated calcium current.

<p><b>A</b>) Cells were given a series of voltage steps (-90 to +40 mV) from a holding potential of -70 mV (inset). Representative isolated calcium current, following offline subtraction of a recording in the presence of nifedipine (10 µM). <b>B</b>) Current-voltage (I–V) relationship of calcium current density (pA/pF) following Rem2 knockdown, rescue, or overexpression. <b>C</b>) Peak calcium current amplitude was determined following a ramp step from -80 mV to +40 mV (duration 500 ms). n ≥ 10 cells per condition (3 separate experiments). * p ≤ 0.05 compared to mock or ^# p ≤ 0.05 compared to each appropriate RNAi condition determined using a one-way ANOVA followed by a student’s independent <i>t</i>-test.</p

The Kinesin KIF21B Regulates Microtubule Dynamics and Is Essential for Neuronal Morphology, Synapse Function, and Learning and Memory

The kinesin KIF21B is implicated in several human neurological disorders, including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological functions. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor protein and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, Kif21b-null mice exhibit behavioral changes involving learning and memory deficits. Our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation