KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons

Abstract

Neurons rely heavily on axonal transport to deliver materials from the sites of synthesis to the axon terminals over distances that can be many centimetres long. KIF1A is the neuron-specific kinesin with the fastest reported anterograde motor activity. Previous studies have shown that KIF1A transports a subset of synaptic proteins, neurofilaments and dense-core vesicles. Using two-colour live imaging, we showed that beta-secretase 1 (BACE1)-mCherry moves together with KIF1A-GFP in both the anterograde and retrograde directions in superior cervical ganglions (SCG) neurons. We confirmed that KIF1A is functionally required for BACE1 transport by using KIF1A siRNA and a KIF1A mutant construct (KIF1A-T312M) to impair its motor activity. We further identified several cargoes that have little or no co-migration with KIF1A-GFP and also move independently from BACE1-mCherry. Together, these findings support a primary role for KIF1A in the anterograde transport of BACE1 and suggest that axonally transported cargoes are sorted into different classes of carrier vesicles in the cell body and are transported by cargo-specific motor proteins through the axon.

Keywords: BACE1; KIF1A; axonal transport; live-cell imaging.

Figure 1

(A) Schematic diagram for axonal transport experiments. Dissociated superior cervical ganglions (SCG) neurons were microinjected with GFP‐tagged KIF1A with different cargo candidates constructs, vesicle transport in axons was imaged live after 6‐8 hours, and resultant kymographs were analyzed. (B) Average speed of moving lysosomes (µm/seconds) in cells injected with a GFP construct (n = 19 neurites) and uninjected cells (n = 11 neurites). Error bars indicate standard error of the mean (SEM). (C) Quantification of the number of moving lysosomes in injected (n = 19 neurites) cells and uninjected cells (n = 11 neurites). Error bars indicate SEM. The direction, velocity and number of moving lysosomes in both the anterograde and retrograde directions are similar in the cells that have been injected as in uninjected cells.

Figure 2

(A) Co‐migration with KIF1A‐GFP was analyzed using kymographs (time‐distance graphs) obtained from live imaging of axons from dissociated superior cervical ganglions (SCG) neurons. Representative kymographs from axons co‐labelled with KIF1A‐GFP and (a) amyloid precursor protein (APP)‐mCherry (b) beta‐secretase 1 (BACE1)‐mCherry (c) BDNF‐mCherry (d) NMNAT2‐mCherry (e) mitochondria‐RFP (f) SCG10‐mCherry. (B) Quantification of co‐migration with KIF1A‐GFP. The quantification shown for each construct represents the percentage of moving vesicles that co‐migrate with KIF1A‐GFP. [amyloid precursor protein (APP): n = 18 neurites, beta‐secretase 1 (BACE1): n = 22 neurites, BDNF: n = 21 neurites, MITO: n = 21 neurites, nicotinamide nucleotide adenylyltransferase 2 (NMNAT2): n = 10 neurites, SCG10: n = 14 neurites). Error bars indicate standard error of the mean (SEM)].

Figure 3

(A) KIF1A‐T312M‐GFP reduces the transport of beta‐secretase 1 (BACE1) vesicles significantly in axons of dissociated superior cervical ganglions (SCG) neurons. Representative kymographs comparing transport of BACE1‐mCherry between (a) KIF1A‐WT‐GFP and (b) KIF1A‐T312M‐GFP expressing neurons. Representative kymographs comparing transport of mito‐RFP between (c) KIF1A‐WT‐GFP and (d) KIF1A‐T312M‐GFP expressing neurons. (B) Quantification of the number of moving BACE1‐mCherry particles (alone: n = 18, with KIF1A‐WT‐GFP: n = 17, with KIF1A‐T312M‐GFP: n = 23, ****P ≤ .0001, t‐test) (bottom left panel). Quantification of the number of moving mitochondria‐RFP particles (alone: n = 10, with KIF1A‐WT‐GFP: n = 28, with KIF1A‐T312M‐GFP: n = 32) (bottom right). Error bars indicate standard error of the mean (SEM).

Figure 4

KIF1A‐T312M‐GFP impairs axonal transport of endogenous beta‐secretase 1 (BACE1) but not amyloid precursor protein (APP). KIF1A‐T312M‐GFP expressing neurons or control were immunostained for (A) BACE1 or (B) APP. Representative cells are shown with merge (n = 5, scale bar = 10 µm).

Figure 5

(A) Knockdown of KIF1A inhibits beta‐secretase 1 (BACE1) but not mitochondria movement in axons of dissociated superior cervical ganglions (SCG) neurons. Representative kymographs comparing transport of BACE1‐mCherry between (a) control and (b) non‐targeting siRNA and (c) KIF1A siRNA neurons (upper panel). Representative kymographs comparing transport of mito‐RFP between (a) control and (b) non‐targeting siRNA and (c) KIF1A siRNA neurons (bottom panel). (B) Quantification of the number of moving BACE1‐mCherry particles (control: n = 18, non‐targeting siRNA: n = 11, KIF1A siRNA: n = 11, P < .001, t‐test) (left panel). Quantification of the number of moving mitochondria‐RFP particles (control: n = 12, non‐targeting siRNA: n = 10, KIF1A siRNA: n = 10) (right panel). Error bars indicate standard error of the mean (SEM). (C) Representative immunoblots performed on cell lysates to show knockdown of KIF1A. Quantification of the knockdown of KIF1A is expressed as a ratio of KIF1A to Tuj1 expression. (n = 3). Error bars indicate standard error of the mean (SEM).

Figure 6

Two‐colour microscopy shows that beta‐secretase 1 (BACE1)‐mCherry and NMNAT2‐GFP are transported in different carriers in co‐injected superior cervical ganglions (SCG) axons. (A) Representative kymographs of (a) NMNAT2‐GFP, (b) BACE1‐mCherry and (c) Overlay of (a) and (b). As shown in the kymographs, the localization of mobile BACE1/NMNAT2 was mostly non‐overlapping. (B) Five frames from the time‐lapse images are shown, with each channel (GFP and RFP) displayed separately. Yellow arrows mark positions of a vesicle containing only NMNAT2‐GFP. Grey arrows mark positions of a vesicle containing only BACE1‐mCherry.

Figure 7

Two‐colour microscopy shows that beta‐secretase 1 (BACE1)‐GFP and mitochondria‐RFP are transported in different carriers in co‐injected SCG axons. (A) Representative kymographs of (a) BACE1‐GFP, (b) mitochondria‐RFP and (c) Overlay of (a) and (b). As shown in the kymographs, the localization of mobile BACE1/NMNAT2 was mostly non‐overlapping. (B) Five frames from the time‐lapse images are shown, with each channel (GFP and RFP) displayed separately. Yellow arrows mark positions of a vesicle containing only BACE1‐GFP. Grey arrows mark positions of a vesicle containing only mitochondria‐RFP. (C) Quantification of co‐migration of (a) BACE1 with NMNAT2 or mitochondria (b) NMNAT2 with BACE1 or mitochondria (c) Mitochondria with BACE1 or NMNAT2. Error bars indicate standard error of the mean (SEM).

Figure 8

Beta‐secretase 1 (BACE1), nicotinamide nucleotide adenylyltransferase 2 (NMNAT2) and mitochondria vesicles have subtle differences in both anterograde and retrograde transport kinetics. (A) Overlay of the velocity frequency histograms describe the velocity distribution of mobile BACE1 (red bars, n = 24, 905 vesicles), NMNAT2 (yellow bars, n = 21, 408 vesicles) and mitochondria (green bars, n = 29, 443 vesicles).