Intracellular G-actin targeting of peripheral sensory neurons by the multifunctional engineered protein C2C confers relief from inflammatory pain

Abstract

The engineered multifunctional protein C2C was tested for control of sensory neuron activity by targeted G-actin modification. C2C consists of the heptameric oligomer, C2II-CI, and the monomeric ribosylase, C2I. C2C treatment of sensory neurons and SH-SY5Y cells in vitro remodeled actin and reduced calcium influx in a reversible manner. C2C prepared using fluorescently labeled C2I showed selective in vitro C2I delivery to primary sensory neurons but not motor neurons. Delivery was dependent on presence of both C2C subunits and blocked by receptor competition. Immunohistochemistry of mice treated subcutaneously with C2C showed colocalization of subunit C2I with CGRP-positive sensory neurons and fibers but not with ChAT-positive motor neurons and fibers. The significance of sensory neuron targeting was pursued subsequently by testing C2C activity in the formalin inflammatory mouse pain model. Subcutaneous C2C administration reduced pain-like behaviors by 90% relative to untreated controls 6 h post treatment and similarly to the opioid buprenorphene. C2C effects were dose dependent, equally potent in female and male animals and did not change gross motor function. One dose was effective in 2 h and lasted 1 week. Administration of C2I without C2II-CI did not reduce pain-like behavior indicating its intracellular delivery was required for behavioral effect.

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
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Intracellular G-actin targeting
of peripheral sensory neurons
by the multifunctional engineered
protein C2C confers relief
from inammatory pain
Derek Allen1, You Zhou2, Audrey Wilhelm1 & Paul Blum1*
The engineered multifunctional protein C2C was tested for control of sensory neuron activity by
targeted G-actin modication. C2C consists of the heptameric oligomer, C2II-CI, and the monomeric
ribosylase, C2I. C2C treatment of sensory neurons and SH-SY5Y cells in vitro remodeled actin
and reduced calcium inux in a reversible manner. C2C prepared using uorescently labeled
C2I showed selective in vitro C2I delivery to primary sensory neurons but not motor neurons.
Delivery was dependent on presence of both C2C subunits and blocked by receptor competition.
Immunohistochemistry of mice treated subcutaneously with C2C showed colocalization of subunit
C2I with CGRP-positive sensory neurons and bers but not with ChAT-positive motor neurons and
bers. The signicance of sensory neuron targeting was pursued subsequently by testing C2C activity
in the formalin inammatory mouse pain model. Subcutaneous C2C administration reduced pain-
like behaviors by 90% relative to untreated controls 6 h post treatment and similarly to the opioid
buprenorphene. C2C eects were dose dependent, equally potent in female and male animals and did
not change gross motor function. One dose was eective in 2 h and lasted 1 week. Administration of
C2I without C2II-CI did not reduce pain-like behavior indicating its intracellular delivery was required
for behavioral eect.
Acute peripheral pain can be treated with anesthetics which inhibit ion channels1 and thereby formation or
propagation of an action potential required for neuronal signaling. For example, lidocaine (xylocaine)2,3 targets
sodium channels NaV1.34 and NaV 1.7-NaV1.94,5 that are implicated in pain. Calcium channels play an additional
role in pain perception because action potentials promote membrane depolarization leading to calcium inux6.
In neurons, calcium channel depolarization opens other channels, releases neurotransmitters7, and acts as a
second messenger in neuronal signaling8. ese channels include high and low voltage activated families. High
voltage families consist of ve dierent subclasses of channels: L, N, P, Q, and R. e N-type are found throughout
the body and play a major role in neurotransmitter release in neuronal cells9. ey transmit pain signals from
peripheral neurons through secondary neurons to the central nervous system. For this reason calcium channels
are a target for pain therapeutics10.
Neuronal signaling also depends on the cytoskeletal protein actin11. Actin occurs as monomeric subunits
(G-actin), or in polymerized form (F-actin) in the neuron cell body, axon and dendrite. Treadmilling is the
reversible process that maintains and remodels F-actin through addition and removal of G-actin12. Inhibition of
this process using actin inhibitors can modulate formation of an action potential through reduction of calcium
inux13. In addition, the modulation of actin polymerization has been shown to inhibit sodium inux14. Inhibi-
tion of an action potential oers a route for pain reduction because it can block peripheral neuronal signaling15.
However, all known actin inhibitors are small untargeted and membrane soluble molecules that lack cell type
specicity16,17. Clinical use was abandoned because of reliance on excessive dosage to overcome untargeted dif-
fusion thereby promoting irreversible inhibition of actin polymerization18.
OPEN
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 *
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C2C is a recombinant protein-based actin inhibitor19. It was derived from the C. botulinum serotype C toxin
called C2 with a retargeted cell specicity through C-terminal replacement of its native binding domain with
that of another C. botulinum serotype C protein called C119. is conferred on the modied protein the ability
to bind the GT1b subclass of gangliosides20 that occur on animal neurons21 in a protein receptor-independent
manner unlike Botox22. e native form of C2 is composed of two non-covalently associated proteins called
C2I and C2II. C2I is a G-actin ADP-ribosyltransferase that inhibits F-actin formation by blocking polymeriza-
tion. C2II is a heptameric oligomer that binds target cells and translocates C2I into the cytoplasm. C2II with its
engineered C-terminus called C2II-C1, spontaneously oligomerizes, associates with C2I and then binds cells in
a GT1b-dependent manner19. e C2I/C2II-C1 complex is then internalized by clathrin and Rho-dependent
mechanisms within endosomes. Acidication of the endosome causes membrane pore formation by C2II-C1
oligomers followed by C2I dissociation and diusion into the cytoplasm. Interestingly, unlike other actin inhibi-
tors, C2I controls actin remodeling eects without killing mammalian neurons (no activation of apoptosis)23. In
addition, and unlike C1, C2 is not a neurotoxin and is not associated with botulism therefore C2C is not a toxin.
Here the eects of C2C on neurons was studied invitro and invivo. Its apparent eects on calcium channels
and specicity towards neuronal subclass, prompted an assessment of its eects on pain-like behaviors using a
conventional animal pain model.
Results
Inhibition of actin polymerization. e native C2 subunit C2I inhibits F-actin polymerization through
ADP-ribosylation of G-actin leading to actin remodeling24. To conrm the occurrence of similar activity by
C2C, its eect on actin polymerization was tested. Primary chicken sensory neurons were cultured as described
previously25, followed by treatment with 60nM C2C or latrunculin A and staining of treated cells with the
F-actin stain phalloidin26. Microscopic analysis showed a reduction in F-actin staining for both C2C and latrun-
culin A treated cells compared to an untreated control cell line though the eect of latrunculin A appeared
more signicant at this dose (Fig.1a). To quantitate the eect of C2C treatment and better compare its eects to
latrunculin A, the assay was repeated using SH-SY5Y human neuroblastoma cells (Fig.1b). SH-SY5Y cells were
treated with a range of doses of C2C and latrunculin A from 6 to 600nM for 2h, followed by staining with phal-
Figure1. Inhibition and reversibility of actin polymerization. Polymerized F-actin was measured using an
alexa uor conjugated phalloidin stain and imaged by confocal microscopy or quantitated using a microtiter
plate reader. e eect of C2C treatment compared to latrunculin A was tested using primary chicken sensory
neurons using uorescent confocal microscopy (a). e eect of C2C treatment relative to latrunculin A was
quantitated for the percentage of F-actin inhibition and reversibility using GT1b-positive SH-SY5Y cells (b).
Error bars represent the standard deviation between replicates.
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loidin. is resulted in a dose dependent inhibition of polymerized actin by both agents. As small molecule actin
inhibitors like latrunculins have been described as irreversible27, the reversibility of inhibition of actin polymeri-
zation was tested. SH-SY5Y cells were treated with C2C or latrunculin for 2h, followed by treatment removal,
media replacement and a recovery period of 48h (Fig.1b). e eect of C2C treatment was more reversible than
latrunculin at particular doses. While 88% and 89% respectively of actin polymerization was observed using
C2C doses of 6 and 60nM, only 66% and 40% respectively was observed with latrunculin A at identical doses.
is suggests the eect of C2C was more reversible than latrunculin A.
Inhibition and reversibility of calcium inux. Neuronal inux of calcium ions is required to create or
propagate an action potential for signaling28. C2II-C1 delivers the G-actin ribosylase, C2I, to the cytoplasm19
where it inhibits F-actin formation through post translational modication of G-actin29. Because depolymeri-
zation of F-actin mediated by latrunculin A has been shown to disrupt calcium inux30, the ability of C2C to
inhibit calcium inux was examined. Primary chicken sensory cells prepared as described25 were treated with
C2C and then with the calcium uorescent dye, Fluo-431. C2C treatment reduced calcium inux as indicated by
a reduction in uorescence at a range of concentrations of C2C (Fig.2a). To expand on these results using a more
rapid and quantitative test, the eect of C2C on calcium inux was examined using the GT1b-positive human
neuroblastoma cells, SH-SY5Y32 (Fig.2b). SH-SY5Y cells were plated on black sided 96 well plates, then treated
with C2C at doses ranging from 0.2 to 60nM, or latrunculin A, at a dose of 500nM. Calcium inux was again
reduced by C2C treatment exhibiting a maximum eect of 61% reduction of intracellular calcium compared to
an untreated control at 60nM C2C. e minimum eective dose that reduced calcium inux was 0.2nM C2C
with a 2% reduction compared to an untreated control. e eect of C2C treatment at dierent doses were sig-
moidal in pattern indicating saturation of the GT1b receptor and or the G-actin target. However, neither C2C
or latrunculin A were fully eective at blocking calcium inux indicating the activity of other nonresponsive
ion ux channels in this cell line33. In addition, sodium inux was measured using SBFI sodium indicator and a
uorescent microtiter plate reader with SH-SY5Y cells (Fig S1). SH-SY5Y cells were treated with the indicated
doses of C2C for 2h prior to analysis. No signicant reduction in sodium inux was apparent with C2C while
latrunculin A elicited a 40% reduction.
Because small molecule actin depolymerizing agents have been shown to have an irreversible eect on calcium
inux27,34 the reversibility of C2C treatment on calcium inux was examined. Primary chicken sensory neurons
were treated with C2C or latrunculin A for 2h followed by their removal (Fig.2c). Removal of C2C led to the
gradual recovery of calcium inux reaching a capacity of 95% aer 48h aer C2C removal. SH-SY5Y cells were
treated with C2C or latrunculin A for 2h followed by either their removal or in separate tests, their continued
presence. Removal of C2C led to a gradual recovery of calcium inux capacity reaching 90% of pretreatment
Figure2. Inhibition and reversibility of calcium inux. Calcium inux was measured using Fluo 4 calcium
indicator and a uorescent microtiter plate reader. e eect of C2C treatment relative to latrunculin A was
examined using primary chicken sensory neurons (a). e eect of C2C treatment relative to latrunculin A was
tested in GT1b-positive SH-SY5Y cells (b). e eect of removal of C2C and latrunculin was also examined
with primary chicken sensory neurons (c) along with the eect of removal from SH-SY5Y cells (d). Error bars
represent the standard deviation between replicates.
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levels in 48h while continued presence of C2C blocked recovery of calcium inux capacity (Fig.2d). In con-
trast, SH-SY5Y cells did not recover calcium inux capacity with either the removal or the continued presence
of latrunculin A. e reversibility of the eect of C2C treatment on calcium inux capacity indicates C2C is
temporary in action and may undergo intracellular titration, degradation or enzymatic alteration35,36.
Targeting specicity of neuronal subclass. In prior studies C2C was shown to target immortal neu-
ronal and nonneuronal cell lines in a GT1b-stimulated manner19. To assess its targeting specicity with pri-
mary neurons, sensory and motor chicken neuronal lines were established as described25,37. ese cells were
then probed with C2C prepared with uorescently derivatized C2I, the G-actin ribosylase component of C2C.
Neuronal subclass identity was determined using established neuronal markers. Sensory neurons were identi-
ed using antibodies against calcitonin gene-related peptide (CGRP)38. Motor neurons were identied using
antibodies against choline acetyltransferase (ChAT)39. Nuclei and mitochondria were identied using DAPI40.
Primary neuron cultures were established using dorsal root ganglia (DRGs) for sensory neuron cultures and
spinal cord neurons for motor neuron cultures using at least three chicks for each experiment. e occurrence
of GT1b was also demonstrated with chicken sensory neurons (Fig. S2). C2C colocalized with CGRP-positive
cells but not with ChAT-positive motor cells (Fig.3a). When sensory neurons were treated with the uorescently
labeled enzymatic component, C2I, but without the binding component, C2II-C1, there was no colocalization
of C2I with CGRP. When sensory cells were pretreated with antibodies against the ganglioside GT1b, there was
no colocalization of C2I with CGRP (Fig.3a) and higher magnication images (Fig. S3). ese data indicate that
C2C preferentially targets primary sensory neurons and that C2I delivery is dependent on C2II-CI.
e magnitude of this specicity was determined by cell counting. Primary sensory and motor neuron
cells were quantitated for the number of cells that stained positively for DAPI, for the neuron subtype marker,
and for uorescent C2I. Five elds of view were quantitated in two biological replicates for motor and sensory
neurons to reach a minimum of 500 cells for each neuronal subclass treatment. Counted cells were placed into
the following categories: positive for DAPI and negative for neuron subtype marker and C2C, positive for DAPI
and neuron subtype marker and negative for C2C, positive for DAPI and C2C and negative for neuron subtype
marker, and positive for DAPI, neuron subtype marker, and C2C (Fig.3b). Consistent with prior reports, these
primary neuron subclass enrichments contained a diversity of cells as reected in the respective fractions for
DAPI and dierent subtype markers25,37,41,42. Motor and sensory neuron cultures were positive for DAPI only
in 24.8% and 26.6% of cells respectively25,41,43. Motor and sensory neuron cultures were positive for DAPI and
C2C only at 3.4% and 3.9% of cells respectively. Motor and sensory neuron cultures were positive for DAPI and
neuron subtype marker in 67.5% and 25.7% of cells respectively. Finally, motor and sensory neuron cultures were
positive for DAPI, neuron subtype marker, and C2C in 4.3% and 43.8% of cells respectively.
In vivo targeting of peripheral mouse sensory neurons and nerve bers. To assess the specicity
of C2C targeting in animals, its localization with peripheral neurons and nerve bers was examined in mouse
tissue using immunouorescence microscopy. C2C was prepared using uorescent C2I and exhibited identical
potency as C2C prepared using non uorescent C2I. It was then injected subcutaneously in the back of mice as
described44. Aer 6h, animals were euthanized and both fresh and xed tissues from the latissimus dorsi muscle
were removed, and either cryosectioned before imaging (Fig.4a) or examined as whole mounts (Fig.4b). Images
shown are representative of at least three mice for all markers. Cell type identity was determined using uores-
cently labeled antibodies specic for the sensory neuron marker, calcitonin gene-related peptide (CGRP)38, the
motor neuron marker, choline acetyltransferase (ChAT)39, and Neurolament (NeuF) a general neuronal marker
along with DAPI for nuclear and mitochondrial DNA. C2I co-localized with nerve bers detected by NeuF but
only those that were CGRP-positive (Fig.4a, top row). In this nerve ber, uorescent C2I was seen co-localizing
with some sections that were CGRP-positive producing a cyan color that results from the combination of the
green CGRP and the blue C2I (Fig.4a, top row, inset). C2I did not colocalize with ChAT positive bers (Fig.4a,
bottom row and inset). C2I also colocalized with CGRP-positive aerent sensory nerve bers in 1.2µm optical
slices of whole mount tissues (Fig.4b) where C2I is green and CGRP is blue. In addition, C2I (green) colocal-
ized with CGRP-positive (blue) aerent nerve ber in compiled optical slices of whole mount tissue (Fig S4).
Fluorescent C2I (green) was also observed throughout the length of axons of sensory neurons (Fig. S5). is
may indicate a propensity to undergo intracellular diusion. While C2I colocalized with CGRP, this association
occurred in only a subset of CGRP-positive locations. is is consistent with limited distribution of the GT1b
receptor combined with intracellular diusion of C2I. Overall, these results indicate C2C targets sensory nerve
bers not motor nerve bers invivo and are consistent with its apparent invitro specicity targeting primary
sensory not motor neurons.
Eect of C2C on nociceptive pain in BALB/c mice. Because C2C reduced calcium inux and preferen-
tially targeted sensory neurons and nerve bers invitro and invivo, it was predicted it would inuence pain-like
behaviors in an animal model for peripheral nociceptive pain. To test this possibility, the eect of C2C admin-
istration was examined in the inammatory formalin test45. BALB/C female mice were treated either with C2C,
each of the subunits (C2II-C1 and C2I) individually, and with controls by subcutaneous (SC) injection into the
lateral, dorsal surface of the hind paw. e response to a range of C2C doses and the duration of the response to a
single dose were examined. e total duration of all behaviors along with the total number and duration of each
behavior were recorded over a 30-min window following an initial 5min acute phase as described45. Statistical
and power analyses of the experimental design and results used SAS 9.3 statistical soware and G* power 3.1.
Six h aer administration, a C2C dose of 2.4pmol blocked 90% of pain-like behaviors relative to the carrier only
(PBS). e eect of C2C closely approximated the amount of reduction in pain-like behaviors observed with the
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opioid, buprenorphine, administered identically but at a higher dose 12.82nmol (Fig.5a). A two fold increase in
C2C dose (4.8pmol) conferred no additional benet. Reduced amounts of C2C conferred correspondingly less
reduction in pain-like behavior with a minimum eective dose of 0.6pmol. Similarly, administration of either
C2C protein subunits, C2II-CI or C21, produced no reduction in pain-like behavior. is indicates that delivery
Figure3. In vitro specicity of C2C. a Primary sensory and motor neurons were cultured from chicken
embryos and treated with 120nM of uorescently derivatized C2C (green). Sensory neuron cultures were
stained with sensory neuron marker anti-calcitonin gene related peptide (CGRP, red) and DAPI DNA stain
(blue). Motor neuron cultures were stained with motor neuron marker anti-choline acetyltransferase (ChAT,
red) and DAPI (blue). b Presence of C2C in neuron subtype positive cells was quantitated and compared
between motor and sensory neurons. Cell counts were taken in eight elds of views in two biological replicates.
Error bars represent standard deviation between all elds of view.
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of C2I by C2II-C1 is a requirement for reduction in pain-like behavior by C2C. Pain-like behaviors were binned
in 5-min increments and then summed for each treatment for dose response to distinguish between phase I and
phase II response periods (Fig. S6). At all doses tested, the eect of C2C on pain-like behavior achieved a power
of 0.8. C2C was equally eective in reducing pain-like behavior in both female and male animals using a dose
of 2.4pmol 6h post treatment (Fig S7). No change in normal motor function was evident in all treated animals
as indicated by the digit abduction test46 conducted on all tested animals. Similarly, no change was observed in
any C2C treated animal in normal grooming, eating, other behaviors, or in weight gain during the period of
evaluation.
Local anesthetics typically target ion channels thereby inhibiting formation or propagation of an action poten-
tial. ey are uniformly short acting in duration largely because they diuse away from the site of action and are
subsequently taken up by non-neuronal tissues47. e longest acting form, liposomal bupivacaine, extends this
duration to 3days48. Because C2C is a protein and cannot readily diuse away from the site of action and also
acts intracellularly through import by receptor mediated endocytosis, it was of interest to measure its duration
of action. is was done using the formalin mouse pain model by varying the period of incubation following a
single dose (2.2pmol) of C2C before formalin treatment (Fig.5b). Pain-like behaviors were also binned in 5-min
increments and then summed for each treatment for duration (Fig. S8). Incubation periods of 2h, 6h, 174h
(7.2days) and 246h (10.2 days) were tested. Inhibition of pain-like behavior was evident within 2h of treatment
and lasted throughout the 174h (7.25day) period. ere was a gradual decrease in the amount of reduction of
pain behavior aer this period, until there was no reduction in pain-like behaviors at 246h (10.2days). Again,
Figure4. In vivo targeting of mouse sensory nerve bers by C2C. Latissimus dorsi samples were obtained aer
subcutaneous administration of uorescently derivatized C2C followed by treatment with antibody reporters.
a Cryosections. Top row; DAPI DNA stain (white), sensory neuron marker anti-calcitonin gene related peptide
(CGRP, green), general nerve ber marker anti-neurolament (NF, red), C2C (blue). Bottom row; as for top
row but anti-choline acetyltransferase (ChAT) replaces NeuF (red). Scale bar is 20µm, inset scale bar is 5µm. b
Whole mounts with 1.2µm optical slices; anti-calcitonin gene related peptide (CGRP, blue), C2C (green). Scale
bar is 30µm.
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statistical and power analysis of these data used SAS 9.3 and G* power 3.1 and achieved a power of 0.8. Similarly,
no change in normal motor function was evident as indicated by the digit abduction test46 and no change was
observed in grooming, eating, other behaviors, or weight gain during the period of evaluation.
Discussion
C2C is an engineered protein designed to reduce pain signaling through delivery of an actin remodeling enzyme
to peripheral sensory neurons by GT1b receptor targeting. C2C was shown to inhibit pain-like behaviors in a
mouse formalin pain model in a manner similar to local anesthetics but with novel features. While C2C imparted
pain relief slowly, having full activity 2h post administration, this eect continued for one week. is exceeds
the duration of action of local anesthetics including those designed for extended release49,50.
In vitro, C2C reduces calcium inux in primary chick sensory neurons and immortal human neuroblastoma
cells. Anesthetics, which are uniformly small molecules such as lidocaine, also reduce ion ux and thereby limit
formation or propagation of an action potential51. Invivo, these eects reduce neuronal pain signaling. Despite
its molecular complexity, C2C exhibits a greater molar potency than local anesthetics52. C2C had a maximum
eective dose of 2.4pmol in the animal pain test model. is eect plateaued at higher doses suggesting occur-
rence of target saturation, but it remains to be seen if this results from titration of receptor binding or limited
recycling, G-actin ribosylation or calcium channel disruption. Additional invitro studies using cultured primary
chick neurons and uorescence microscopy demonstrated that C2C is specic for sensory neurons and not
motor neurons. is targeting enables preferential intracellular delivery of the G-actin ribosylase and arises from
specicity of the C2C oligomeric subunit, C2II-C1, for the GT1b ganglioside receptor19. Unlike other clostridial
proteins such as Botox, GT1b appears to be necessary and sucient for C2C binding and is not co-dependent on
protein receptors22. Multiple types of calcium channels are known to be dependent on actin polymerization in
neuronal cells53. e reduction of calcium inux does result in a reduction of action potential initiation54. Actin
dependence of calcium inux is thought to be through uptake of calcium because of recruitment done by calcium
channel tracking, that has been shown to be inhibited by other actin polymerization inhibitors55. is could
be a similar model for the action of C2C in inhibiting calcium inux. Such specicity provides a mechanism to
perform actin remodeling in only a subset of neurons using an intracellular mechanism.
Figure5. Eect of C2C on pain-like behaviors in BALB/c mice. e formalin nociceptive pain test was
utilized to evaluate the dose and duration of responses to C2C administration in BALB/c mice. Shown are
data for female mice ages 8–10weeks. a Dose response of C2C (black bars) relative to buprenorphene (grey
bar, 12.82nmol), carrier (open bar, PBS), C2C subunits C2I and C2II-CI (light and dark grey respectively,
1.2pmol). Numbers of animals used are indicated over each bar (n). b Duration of C2C eect. A single dose of
C2C (2.4pmol) was administered followed by formalin aer 2h, 6h, 174h (7.2days), and 246h (10.2days).
Statistical signicance is indicated by *(p < 0.05), or **(p < 0.01).
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Histologic studies of cryosectioned and whole tissue mounts examined using confocal laser microscopy of
uorescent C2C and various reporters of neuron subclasses, demonstrated specicity of C2C towards sensory
neurons and sensory bers in mouse peripheral tissue. Interestingly C2C was well distributed along sensory
neuron axons that innervated muscle tissue. is may indicate the C2I protein undergoes intracellular diusion
as has been observed with other neuron targeting proteins56 and it suggests that C2C could inuence calcium
channel activity at locations distal to its site of injection. Alternatively, the apparent distribution of C2C is also
consistent with GT1b receptor location along sensory neuron axons in mouse tissue. Most importantly, C2C
localized to aerent sensory nerve bers but did not colocalize with eerent motor nerve bers. GT1bis present
on > 90% of peripheral sensory neurons in mice57 and to some extent on motor neurons but to a lesser extent57.
It also has been reported that there is a higher amount ofGT1bfound on sensory neurons than motor neurons
in tissue collected from human cadavers58. In addition, it has been shown that injuries to the peripheral nervous
system results in increased expression of ganglioside GT1b59. It may be possible that C2C binds some motor
neurons but a combination of reduced uptake and insucient GT1b-positive motor neurons leads to a dierence
in C2C activity in dierent neuron subtypes. Additional mechanisms are also plausible. It is known that GT1b is
present on most mammal species that have been tested for its presence60–62. is specicity was consistent with
the lack of an apparent eect on motor function in animal tests.
C2C disrupts calcium inux in primary sensory neurons and immortal SH-Sy5Y cells in a dose dependent
manner. e utility of the SH-SY5Y response could allow for more robust and quantitative studies on the eect
of C2C on calcium channel inhibition. For example, C2C removal following primary sensory neuron treatment
invitro, restored calcium inux capacity. e mechanism underlying this restoration could be examined for
kinetic and dose response relationships. is reversibility also is consistent with the behavioral data indicating
that signal dose administration produces a transitory eect ending one week post administration.
Materials and methods
Preparation of C2C. C2C was prepared as described previously19. Briey, plasmid encoded expression con-
structs for C2I and C2II-C1 were expressed in E. coli BL21. Cell lines were grown in LB medium, with ampicil-
lin (100µg/mL) at 37°C, and induced at an optical density of ~ 0.6 at 600nm wavelength with 0.5mM IPTG.
A french pressure cell was used to lyse cell paste with 10,000 psi pressure. Glutathione resin (Genscript) was
used for anity purication. GST fusion tags were removed using thrombin (ermo Fisher). C2II-C1 was
further activated using trypsin by incubation at 37°C for 30min at a 1:5 enzyme to substrate ratio as previously
described63.
Cell culture. e SH-SY5Y cell line was used for some ion ux analysis. It is an immortal human neuron-like
cell line derived from glioblastoma64. Cells were cultured at 37°C with 5% CO2, as described65. Chicken sensory
neurons were cultured from chicken embryo dorsal root ganglia as described25 using embryos at Hamburger
stages 31–3666. Chicken sensory neurons were cultured in Ham’s F12 media. Nerve growth factor (1.25ng/mL)
and Neurotrophin 3 (1.25ng/mL) were added for neuronal dierentiation. Chicken motor neurons were cul-
tured from chicken embryo spinal cords as described37 using embryos at Hamburger stages 29–3466.
Actin uorescence assays. Primary chicken sensory neurons and GT1b containing SH-SY5Y cells were
tested for staining using Alex Fluor 488 phalloidin (Invitrogen) with modication67. Sensory neurons were cul-
tured on acid washed, round coverslips in black sided 24 well plates (Corning) or on glass coverslips. Primary
cells were allowed to incubate at 37°C for 48h in hormone containing media. Following incubation, cells were
treated with latrunculin A (Sigma) and C2C at the doses indicated for 2h. Aer incubation, the medium was
removed from the wells and cells were washed with PBS pH 7.2. Cells were xed with 4% paraformaldehyde for
30min at room temperature. Cells were washed 2 times with PBS then permeabilized with 0.5% (v/v) Triton
X-100 for 10min, then washed with PBS 2 times. Cells were stained with phalloidin-488 for 30min at room
temperature. Microscopy used a Nikon A1R confocal microscope. Imaging soware was Nikon Instruments
(NIS). A uorescent plate reader was used to measure 30–40,000 cells per well and an average of three wells for
each dose.
Immunocytochemistry. Coverslips containing primary sensory and motor neurons were probed with
C2C prepared using uorescently derivatized C2I. Derivatization used an Alexa uor micro labelling kit as
described by the supplier (ermo Fisher). Cells were incubated with uorescent C2C for 2h at 37°C in a CO2
incubator. Cells were xed with a fresh 4% (w/v) paraformaldehyde solution at room temperature for 15min.
Coverslips were blocked before antibody addition with 5% (v/v) normal goat serum (Sigma) for 1h at room
temperature. Sensory neurons were identied using antibodies against calcitonin gene-related peptide (CGRP,
Abcam, Cat # ab26001) with a 1:300 dilution38. Motor neurons were identied using antibodies against choline
acetyltransferase (ChAT, Abcam, Cat # ab18736) with a 1:300 dilution39. Coverslips were then mounted on slides
using uorogel mounting solution (ermo Fisher). A Nikon A1R confocal microscope was used to examine
cultured cells. Imaging soware was Nikon Instruments (NIS) and Image J.
Whole-mount and cryosection preparations. C2C was prepared using uorescently derivatized C2I
then injected subcutaneously into the latissimus dorsi of 8–10week old female BALB/c mice. Following C2C
administration and 6h subsequent incubation, mice were euthanized and perfused with 30mL of 0.9% saline
solution, followed by 30mL of 4% paraformaldehyde solution. Tissue preparations were dissected from the
latissimus dorsi and prepared as cryosections and whole-mount samples. Tissue was xed with 4% paraformal-
dehyde diluted in phosphate buered-saline at 25°C for 1h. In addition, whole-mount tissue samples were xed
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with 4% paraformaldehyde overnight at 4°C. Following xation whole-mount samples were washed with phos-
phate-buered saline, pH 7.2 (PBS) twice to remove xative. Samples of latissimus dorsi were frozen for cryo-
sectioning in OCT medium (ermo Fisher) on dry ice. Frozen tissue was sectioned using a cryostat at a width
of 10–20µm. Sections were mounted onto polyethyleneimine coated slides. Cryosections were blocked with
5% (v/v) normal goat serum (Sigma) and washed with Tween 20 (0.1% v/v) in PBS three times for 10min each.
Whole-mount tissue was permeabilized with 0.05% Triton X-100 TBS solution for 3h at 4°C. Whole-mount
tissue was blocked with 5% (v/v) normal goat serum for 3h at 4°C. Primary and secondary antibody incuba-
tions were performed for 1h for cryosections and 16h for whole-mount preparations. Sensory neurons were
identied using antibodies against calcitonin gene-related peptide (CGRP, Abcam, Cat # ab26001) with a 1:300
dilution38. Motor neurons were identied using antibodies against choline acetyltransferase (ChAT, Abcam, Cat
# ab18736) with a 1:300 dilution39. Neurolament (NeuF, Abcam, Cat # ab204893) was used to identify general
nerve bers. Nuclei and mitochondria were identied using DAPI (Sigma)40. Coverslips were then mounted on
slides using uorogel mounting solution (ermo Fisher). A Nikon A1R confocal microscope was used to exam-
ine cryosections and whole-mount tissue. Whole-mount images were taken with a z-series of 60–200 um depth,
with individual images of 1–2µm optical slices. Imaging soware was Nikon Instruments (NIS).
Ion inux assays. Primary chicken sensory neurons and non-stimulated SH-SY5Y neuroblastoma cells
were tested for calcium inux or sodium inux using uor-4 (ermo Fisher) or SBFI sodium indicator (ermo
Fisher) respectfully as described65,68 with modication. Sensory neurons were cultured on acid washed, round
coverslips in black sided 24 well plates (Corning). Cells were allowed to incubate at 37°C for 48h in hormone
containing media. Following incubation, cells were treated with latrunculin A (3µM, Sigma) and C2C at the
doses indicated for 2h. Aer incubation, the medium was removed from the wells and cells were washed with
PBS pH 7.2. Fluo-4 calcium indicator in kit assay buer containing 1.26mM calcium or SBFI sodium indicator
in an assay buer containing 1.1mM sodium was added into each well and incubated for 30min at 37°C, fol-
lowed by a 30min incubation at room temperature. A uorescent plate reader was used to measure the ux of
ion indicators using 30–40,000 cells per well and an average of three wells for each indicator.
Animal behavior tests. Animal experimental procedures were performed in accordance with the National
Institutes of Health Guide for Care and Use of Laboratory animals. Experimental procedures involving animals
were approved by the Institutional Animal Care & Use Committee of University of Nebraska-Lincoln. e ani-
mal behavior test for acute pain was done using the formalin test45,69. Mice were anesthetized using isourane
prior to all injections. In dose response experiments and aer anesthesia, mice were subcutaneously injected
with 20µL of sterile ltered C2C in the amounts indicated ranging from 0.6 to 4.8nmol into the dorsal, lateral
surface of the le hind paw. In duration studies, mice were anesthetized with isourane, then subcutaneously
injected with 2.4pmol of C2C and incubated prior to formalin challenge in times ranging from 2, to 174h
(7.25days). In all procedures, mice were treated with PBS as a negative pain relief injection control or with
12.82nmol buprenorphine as a positive pain relief control. Additional mice were treated with 1.2pmol of each
individual component of C2C, C2I and C2II-C1, to test if they produced any reduction in pain-like behavior.
Following initial treatments, mice were placed in housing cages for 6h. Following the incubation period, mice
were moved to viewing containers and allowed to equilibrate to the new environment for 15min. Mice were then
anesthetized and subcutaneously injected with 20µL of 5% (v/v) sterile ltered formalin into the dorsal, medial
surface of the le hind paw. Mice were then moved back to the viewing containers and a video recorder was
used to monitor behavior of mice for 30min. Pain-like behavior was recorded as “hind paw licking behavior”
indicated as licking of the dorsal surface of the le hind paw. Video recordings were then evaluated during the
30min recording by counting the total duration of behavior, the number of behaviors, and the duration of each
individual behavior. Blinding was performed in all animal behavior studies. A third party not present at the time
of animal treatments recorded the pain-like behaviors in each video recording. Preliminary animal behavior
studies used the formalin test to obtain pilot data for a power analysis necessary to determine the number of
experimental animals needed to achieve statistical signicance. e formalin test45,69 was used at 11 dierent
C2C duration time-points and 5 dierent doses.
Statistics. Formalin test statistical analyses were completed using the SAS 9.3 statistical soware and G*
power 3.1 e pilot data was treated as a factorial split plot, divided into 3 dierent levels of variables: (i) dura-
tion or dosage, (ii) treatment (C2C, untreated, buprenorphine control), and (iii) formalin test phase (phase 1 &
2). For the pilot data, a gamma distribution was selected based on the student residuals test. e PROC GLIM-
MIX procedure and LSMEANS were used to produce standard error means. e LSMEANS and standard error
means were utilized to determine an eect size for each duration and dosage experiment. e eect size for each
duration and dosage was used to determine the probability of correctly nding a dierence within a given phase
and duration or dosage. Using the eect sizes, and a cuto of 80% power, and an alpha of 0.05, a power analysis
was performed using G* power.
Received: 15 April 2020; Accepted: 15 July 2020
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Acknowledgements
e eorts of Jaydeep Kolape for image analysis are acknowledged. ese studies were supported by funds from
the University of Nebraska Cell Development Facility.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-69612 -9.
Correspondence and requests for materials should be addressed to P.B.
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