1.

Introduction

The inducible transcription factor NF-κB is involved in crucial brain functions including learning and memory formation.^1–7 The most abundant NF-κB heterodimer detected within the central nervous system is composed of p65 and p50.^3,8,9 We and others have shown that NF-κB is localized in the synapse, can be activated by glutamate at synaptic sites, and is transported back to the nucleus after its activation.^3,10–15

Axons and dendrites represent specialized neuronal cytoplasmic extensions, where movement by random diffusion alone would not permit efficient and directed delivery of proteins over long distances.^16,17 However, signals generated at synapses must be transported back to the nucleus to regulate gene expression (reviewed in Ref. 17).

Anterograde (away from nucleus) and retrograde (toward the nucleus) transports are crucial for the physiological function of neurons and are mediated by motor proteins including dynein and kinesins.^18,19 Due to their polarized nature and the relatively long distance between the nucleus and the periphery, neurons are highly dependent on intact active transport machinery (reviewed in Ref. 20). Consequently, defects in axonal transport are involved in the development of several neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease.²¹ We and others have previously demonstrated that neuronal NF-κB is actively transported toward the nucleus by the minus end-directed motor protein dynein (Refs. 11, 22, 23; Fig. 1). In contrast, diffusion seems to be sufficient for its retrograde transport in non-neuronal cells.²⁴ However, the exact biophysical parameters such as diffusion coefficients and the velocity of retrogradely transported NF-κB were unknown.

Fig. 1

Synaptic activity promotes dynein-dependent retrograde transport of NF-κB to the nucleus. Schematic presentation of retrograde transport of NF-κB in neurons. After stimulation of the neuron with an activator such as glutamate, upstream kinases induce phosphorylation of the inhibitory protein IκB stimulating its proteasomal degradation. This allows the binding of $\mathrm{p}65/\mathrm{p}50$ heterodimers to the dynein/dynactin motor proteins. After the assembly of the complex and its retrograde movement along the microtubule network, NF-κB translocates into the nucleus without disruption of the complex and induces transcription of specific target genes.

Single-particle tracking (SPT) of fluorophore-labelled receptors in the plasma membrane of a live cell provides valuable information on dynamics and interactions.²⁵ In combination with photoswitchable fluorophores,²⁶ SPT allows the observation of a large number of molecules by stochastically activating only a small subset of fluorophores at a given time and tracking them until photobleaching. This cycle of photoactivation, tracking, and photobleaching is repeated many (often a few thousand) times. Profiting from the pool of labelled biomolecules in a sample, a large number of single-molecule trajectories are recorded. SPT with photoactivated-localization microscopy (SPT-PALM²⁷) allows longer observation times, provides better statistics,^28,29 and allows high-density mapping of molecular movements.³⁰

In order to study the dynamics of retrogradely transported NF-κB in neurons at the single-molecule level, we applied SPT-PALM^27,31 and used the fluorescent protein tandem-Eos-FP (tdEos) as a reporter.^27,28 tdEos is photoconverted from a green-fluorescent to an orange-fluorescent species by irradiation with 405 nm light.³² Following this procedure, a small stochastic subset of the tdEos is transferred into the active (orange-fluorescent) state and tracked as single molecules. In the present study, we used this technique to visualize p65-tdEos (NF-κB subunit fused to tdEos) with a localization precision of 26 nm [Fig. 2(a)].

Fig. 2

SPT-PALM imaging of NF-κB p65 in hippocampal neurons. (a) The NF-κB p65 subunit was fused to the photoactivatable fluorescent protein tdEos that can be photoconverted by irradiation with UV light. Transfected neurons were identified in widefield fluorescence mode by detecting the green fluorescence signal of the tdEos in p65-tdEos. A small stochastic subset of the p65-tdEos was photoconverted from a green-fluorescent to an orange-fluorescent species and tracked as single molecules. Localization precision for tdEos was determined to 26 nm using a nearest neighbor approach, as described in Ref. 42. (b) Map of single particle trajectories (middle and lower panel) revealed highly increased mobility of p65-tdEos in neurites after glutamate stimulation compared to controls. Representative data set from a single cell for both conditions is shown. (c) Exemplary MSD plots from single-molecule trajectories of untreated and glutamate-treated p65-tdEos. The first four MSD values were considered for extracting the diffusion coefficient.

We investigated the glutamate-induced transport of NF-κB p65 in living hippocampal neurons with single-molecule resolution and determined the respective diffusion coefficients. Finally, we demonstrated that synaptic activity leads to an increased mobility of retrogradely and anterogradely transported neuronal NF-κB p65.

2.

Results and Discussion

Hippocampal neurons transfected with p65-tdEos were identified by widefield imaging detecting the green fluorescence signal from unconverted p65-tdEos. After identification of the soma (containing the nucleus), neurites of transfected cells were irradiated with low intensities of ultraviolet (UV) light and single p65-tdEos molecules were tracked by their orange fluorescence. Several thousands of trajectories per cell were recorded and used to generate a trajectory map [Fig. 2(b)].

Next, we compared the mobility of NF-κB p65 in unstimulated and glutamate-treated neurons. We observed that glutamate treatment led to an increased mobility of p65-tdEos particles compared to the baseline control [Fig. 2(b)]. This increase in mobility is in general accordance with the reports on rapid retrograde transport of NF-κB in neurons after glutamate treatment.^11,22

We then calculated the mean diffusion coefficient ($D_{\text{mean}}$) of p65-tdEos molecules from the SPT-PALM data (Fig. 3). In the absence of stimulation (baseline), p65-tdEos molecules showed a $D_{\text{mean}}$ of $0.12\pm 0.05{\mu \mathrm{m}}^2/\mathrm{s}$ [Fig. 3(a)]. Stimulation with glutamate resulted in a higher occurrence of fast molecules ($D_{\text{mean}}$ of $0.61\pm 0.03{\mu \mathrm{m}}^2/\mathrm{s}$) compared to unstimulated controls [Figs. 3(a) and 3(b)] and narrowed the distribution of single-molecule diffusion coefficients [Figs. 3(a) and 3(b)]. Notably, the $D_{\text{mean}}$ measured for p65-tdEos without stimulation is in a similar range to the diffusion coefficient reported for the cytoplasmic HIV Gag-Eos fusion ($0.11\pm 0.08{\mu \mathrm{m}}^2/\mathrm{s}$).²⁷ After stimulation with glutamate, $D_{\text{mean}}$ of p65-tdEos is similar to the mobile fraction of membrane residing $\alpha$-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors with a diffusion coefficient of $>0.5{\mu \mathrm{m}}^2/\mathrm{s}$.³³ We further followed how the mobility of p65-tdEos developed with time and found that the glutamate-dependent increase in $D_{\text{mean}}$ persists for at least 400 s [Fig. 3(d)].

Fig. 3

(a)–(b) Effect of glutamate on the distribution of diffusion coefficients of NF-κB in hippocampal neurons. Note that glutamate treatment narrows the distribution of single-molecule diffusion coefficients compared to the control. (c) Average diffusion coefficient $D_{\text{mean}}$ of p65-tdEos under baseline conditions and after treatment with glutamate monitored over time (a representative data set from a single cell each is shown). Glutamate treatment leads to significantly increased $D_{\text{mean}}$. Without stimulation p65 molecules showed $D_{\text{mean}}$ of $0.12\pm 0.05{\mu \mathrm{m}}^2/\mathrm{s}$. Stimulation with glutamate resulted in a strongly increased occurrence of fast particles and $D_{\text{mean}}$ of p65 to $0.61\pm 0.03{\mu \mathrm{m}}^2/\mathrm{s}$. (d) $D_{\text{mean}}$ of p65-tdEos under control conditions and after treatment with glutamate monitored over time (a representative data set from a single cell each is shown). Error bars: SEM.

Next, we determined the extent to which glutamate affects the immobile fraction as well as retrogradely and anterogradely transported p65-tdEos particles. In glutamate-stimulated neurons, we recorded a lower occurrence of immobile molecules in neurites that was accompanied by a significant increase in retrogradely transported p65-tdEos [Fig. 4(a)]. Further, although not significant, a slight increase of anterogradely transported molecules was measured. Finally, we determined the velocities of single transported p65-tdEos particles. Although glutamate treatment resulted in heterogeneous velocity distribution for both retrograde and anterograde transport, a significantly increased mean velocity was assessed in both directions [Fig. 4(b)]. Specifically, glutamate treatment resulted in an increase of the mean retrograde velocity from $10.9\pm 1.9$ to $15\pm 4.9\mu \mathrm{m}/\mathrm{s}$, whereas a mean velocity increase from $9\pm 1.3$ to $14\pm 3\mu \mathrm{m}/\mathrm{s}$ was observed for the anterograde transport. Notably, the mean velocities calculated for p65-tdEos are in the same range reported for the transport of NGF in neurites of rat sympathetic neurons ($\sim 3$ to $6\mu \mathrm{m}/\mathrm{s}$) as measured in compartmented cultures after applying radioactive ${}^{125}\mathrm{I}$-NGF.³⁴ In neuronal cells, the transport of mitochondria is accomplished by microtubule-based motors (kinesins and dynein) with velocities ranging from $\sim 5$ to $30\mu \mathrm{m}/\mathrm{s}$.³⁵ Moreover, flagellar dyneins achieve a velocity of up to $19\mu \mathrm{m}/\mathrm{s}$ (reviewed in Refs. 36, 37), which is again in general accordance with the mean velocity of $15\mu \mathrm{m}/\mathrm{s}$ for p65-tdEos after glutamate treatment (this study).

Fig. 4

(a) Effect of glutamate on velocity of retrogradely and anterogradely transported p65-tdEos particles. Single-molecule data were used to calculate the occurrence of immobile and retro- and anterogradely transported p65. Treatment of hippocampal neurons with glutamate resulted in significantly decreased amount of immobile particles and strongly increased retrograde transport. (b) Glutamate treatment increases the mean velocity of retrogradely and anterogradely transported p65-tdEos.

In summary, we report that glutamate stimulation promotes an increase in mobility of the NF-kB subunit p65 in living hippocampal neurons. Exposure of neurons to glutamate leads to an increased mean diffusion coefficient of p65-tdEos and an increase in the velocity of both retrogradely and anterogradely transported NF-κB p65 in neurites.

3.

Methods