Download 689. BDNF-Mimetic Peptide Amphiphiles for Neural Regeneration A

BDNF-Mimetic Peptide Amphiphiles for Neural Regeneration
Alexandra Edelbrock1, Zaida Alvarez-Pinto2 & Samuel Stupp1,2,3
1
Department of Biomedical Engineering, Northwestern University, Evanston, IL
2
Simpson-Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL
3
Department of Chemistry and Department of Materials Science and Engineering, Northwestern University, Feinberg School
of Medicine, Chicago, IL, USA
Statement of Purpose: Neurotrophins are proteins of
great clinical interest due to their ability to modulate
development, survival, and function of neuronal cells.
Brain-derived neurotrophic factor (BDNF), which binds
specifically to the tropomyosin-related kinase B (TrkB)
receptor, has been shown to promote neuronal survival,
differentiation, maturation, re-myelination, and synaptic
plasticity [1]. Unfortunately, BDNF protein based
therapies have had little success in clinical settings, due to
the short half-life (<5 min) of native BDNF protein in
serum [2]. Nanofiber scaffolds displaying BDNF-mimetic
moieties could provide a support structure and
simultaneously promote cell survival at the site of injury
over a longer period. The Stupp laboratory has developed
a novel class of biomaterials known as peptide
amphiphiles (PAs), which self-assemble into nano-fibrous
gels upon exposure to physiological divalent ions [4]. PAs
are an ideal scaffold for neural regeneration because of
their ability to form aligned gels in vivo that mimic the
extra cellular matrix structure of the central nervous
system (CNS). Here, we present a PA containing a cyclic
peptide mimetic (CPM) BDNF sequence, which forms a
fibrous scaffold and activates Trk-B in vitro.
Methods:
All
PAs
were
synthesized
by
fluorenylmethoxycarbonyl (Fmoc) chemistry using solidphase peptide synthesis. CPM PA (10 mol%) was coassembled with 90mol% of a non-bioactive filler PA. PA
solutions were annealed to reach a thermodynamically
stable state prior to use. After annealing, 1 wt% PAs were
gelled with a solution containing 25 mM CaCl2. Gelled
PA materials were imaged using scanning electron
microscopy (SEM) to assess their fiber alignment and
ability to form fibrous scaffolds. For in vitro studies,
primary cortical neurons were dissected from mice at
embryonic day 16 and cultured on poly-D-lysine (PDL)
coated wells for 14 days before PA treatments were
added. Solutions of CPM PA and the cyclic mimetic
peptide unattached to PA were diluted in serum-free
media to obtain 0.5 µM final concentrations of epitope.
The cells were incubated with PA for 1, 2, 4, 6 and 24
hours and the protein was collected to assess TrkB
phosphorylation using Western blot. Confocal microscopy
was used to analyze changes in cell morphology. Cell
survival was assessed using flow-cytometry.
Results: SEM showed that when CPM PAs are coassembled with the filler PA, annealed, and gelled with
calcium, they form nanofiber networks (Figure 1A). In
some cases, these networks become twisted and bundled
in the presence of salts. Treatments were dissolved in
media and incubated with cells for 1, 3 and 7 days. Cell
survival assays showed greater than 90% survival for all
conditions at all time points. Confocal microscopy
showed that cells took on a normal neuronal morphology
(Figure 1B). Western blot (Figure 1C & 1D) showed rapid
activation of TrkB with BDNF and no activation with
starvation media at time zero. CPM showed delayed
activation of the TrkB receptor, but a similar amount of
phosphorylation compared to BDNF at 4 and 6 hours.
There is a cross-over point where the activation of TrkB
was higher at 6 hours with the CPM PA material than
with the native BDNF. Activation of TrkB eventually
decreases to zero after 24 hours in both conditions.
Figure 1: A) SEM image of CPM PA after gelation.
Inset: schematic molecular-graphics representation of
CPM nanofiber. B) Confocal images of primary cortical
neurons exposed to treatments for 24 hours. DAPI =
nuclear stain, MAP2 = maturation marker, SMI312 =
neurofilament marker. C) Western blot showing amount
of TrkB phosphorylation overtime in comparison to
native BDNF protein. D) Graphical representation of 1C phosphorylation over time, normalized to total TrkB.
Conclusions: The functionalization of PAs with BDNF
mimetic cyclic peptides does not inhibit their selfassembly into nanofibers. When added to primary neurons
in vitro, this material activates the TrkB receptor after 4
hours of exposure. We hypothesize that this delay is due
to the PA material floating in media after addition, and it
does not contact the cells right away. Future
investigations will identify the optimal concentration and
exposure time of the CPM material. Based on these
results, we conclude that BDNF-mimetic promotes the
survival and neuronal outgrowth after 24 hours in vitro
through the activation of the Trk-B signaling pathway.
References:
[1] Lu B. Nat Rev Neurosci. 2013; 6: 401-416.
[2] Bradley WG. Neurology. 1999; 7: 1427-1433.
[3] Silva GA. Science. 2004; 5662: 1352-1355.