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this hypothesis, we recently developed a strategy to manually manipulate
signaling endosomes in growth cones. We approached this problem by load-
ing active signaling endosomes with 50 nm superparamagnetic nanoparticles
(MNPs) functionalized with a TrkB agonist antibody (fMNPs) (
Qian et al.,
2006
) and then manipulating the localization of these fMNP-loaded signal-
ing endosomes with a focal magnetic field (
Steketee et al., 2011
). These
fMNPs enhanced RGC survival and neurite growth similar to BDNF
and unbound agonist antibody and were sensitive to Trk receptor inhibition
by K252a (
Hu, Cho, & Goldberg, 2010
), which inhibits BDNF-induced
neurite growth in RGCs (
Goldberg et al., 2002
). FMNPs endocytosed
by RGCs were transported in RGC axons both
in vivo
and
in vitro
(
Fig. 2.5A and B
).
In vitro
, cultured RGCs endocytosed fMNPs in cell bod-
ies, neurites, and particularly the central domain of growth cones, consistent
with selective central domain endocytosis of activated TrkB receptors (
Tani
et al., 2005
). RGCs responded rapidly to fMNPs by increasing lamellipodial
protrusion, similar to that reported for BDNF (
Myers & Gomez, 2011
) and
NGF (
Tani et al., 2005
). Endocytosed fMNPs increased and colocalized
with phospho-TrkB (p-Trk), and increased both phospho-ERK1/2 and
phospho-Akt similar to BDNF or agonist antibody alone (
Hu et al.,
2010; Qian et al., 2006
). Magnetic recovery of endocytosed fMNPs
pulled down activated phospho-TrkB, indicating functionalization
maintains agonist activity. Finally, fMNPs altered the transcription of
genes known to promote neurite growth and to be upregulated by
BDNF, again in a K252a-sensitive manner (
Cazzin, Mion, Caldara,
Rimland, & Domenici, 2011; Gokce, Runne, Kuhn, & Luthi-Carter,
2009
). Together,
these results
indicate that
fMNPs are targeted to
functional TrkB signaling endosomes.
4.2. Nanoparticle-mediated changes in transport
Manipulating TrkB/fMNP signaling endosomes biases transport. Time-
lapse microscopy revealed that fMNP signaling endosomes were transported
bidirectionally in RGC neurites (
Fig. 2.5B
) at rates similar to those reported
for Trk signaling endosomes in PC-12 cells (
Jullien et al., 2003
) and cortical
neurons (
Gomes, Hampton, El-Sabeawy, Sabo, & McAllister, 2006
).
Applying a 15 pN magnetic force halted anterograde transport, biasing
fMNP signaling endosome transport retrogradely out of distal neurites
and away from growth cones into proximal neurites and somas
(
Fig. 2.5C
). This net retrograde transport was unaccompanied by process
retraction or evacuation of other vesicles, mitochondria, or vacuoles, which
