Liposomal encapsulated FSC231, a PICK1 inhibitor, prevents the ischemia/reperfusion-induced degradation of GluA2-containing AMPA receptors

Article abstract of DOI:10.3390/pharmaceutics13050636

Strokes remain one of the leading causes of disability within the United States. Despite an enormous amount of research effort within the scientific community, very few therapeutics are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity. Calcium-permeable AMPA receptors (AMPARs), lacking the GluA2 subunit, contribute to calcium cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of GluA2 AMPAR subunits following oxygen-glucose deprivation/reperfusion (OGD/R) is, at least in part, mediated by protein-interacting with C kinase-1 (PICK1). The purpose of the present study is to evaluate whether treatment with a PICK1 inhibitor, FSC231, prevents the OGD/R-induced degradation of the GluA2 AMPAR subunit. Utilizing an acute rodent hippocampal slice model system, we determined that pretreatment with FSC231 prevented the OGD/R-induced association of PICK1-GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein levels. This suggests that the interaction between GluA2 and PICK1 serves as an important step in the ischemic/reperfusion-induced reduction in total GluA2 levels.

Full text of DOI:10.3390/pharmaceutics13050636

pharmaceutics
Article
Liposomal Encapsulated FSC231, a PICK1 Inhibitor, Prevents
the Ischemia/Reperfusion-Induced Degradation of
GluA2-Containing AMPA Receptors
Lindsay M. Achzet 1 , Fanny Astruc-Diaz 2 , Phillip H. Beske 2, Nicholas R. Natale 2, Travis T. Denton 1,3,4
and Darrell A. Jackson 1,
*
1
2
Department of Pharmaceutical Sciences, Washington State University Health Sciences,
Spokane, WA 99202, USA; Lindsay.achzet@wsu.edu (L.M.A.); travis.denton@wsu.edu (T.T.D.)
Department of Biomedical and Pharmaceutical Sciences, The University of Montana,
Missoula, MT 59812, USA; fanny.diaz@mso.umt.edu (F.A.-D.); phillip.beske@umontana.edu (P.H.B.);
Nicholas.natale@umontana.edu (N.R.N.)
Department of Biomedical Sciences, Elson S. Floyd, College of Medicine, Washington State University Health
Sciences, Spokane, WA 99202, USA
Steve Gleason Institute for Neuroscience, Washington State University Health Sciences,
Spokane, WA 99202, USA
3
4
*
Correspondence: darrell.jackson@wsu.edu; Tel.: +1-509-368-6542
Abstract: Strokes remain one of the leading causes of disability within the United States. Despite
an enormous amount of research effort within the scientific community, very few therapeutics
are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied
phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from
excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity.
Calcium-permeable AMPA receptors (AMPARs), lacking the GluA2 subunit, contribute to calcium
cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of
GluA2 AMPAR subunits following oxygen–glucose deprivation/reperfusion (OGD/R) is, at least
in part, mediated by protein-interacting with C kinase-1 (PICK1). The purpose of the present study
is to evaluate whether treatment with a PICK1 inhibitor, FSC231, prevents the OGD/R-induced
degradation of the GluA2 AMPAR subunit. Utilizing an acute rodent hippocampal slice model
system, we determined that pretreatment with FSC231 prevented the OGD/R-induced association
of PICK1–GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein
levels. This suggests that the interaction between GluA2 and PICK1 serves as an important step in
the ischemic/reperfusion-induced reduction in total GluA2 levels.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Achzet, L.M.; Astruc-Diaz,
F.; Beske, P.H.; Natale, N.R.; Denton,
T.T.; Jackson, D.A. Liposomal
Encapsulated FSC231, a PICK1
Inhibitor, Prevents the
Ischemia/Reperfusion-Induced
Degradation of GluA2-Containing
AMPA Receptors. Pharmaceutics 2021,
13, 636. https://doi.org/10.3390/
pharmaceutics13050636
Academic Editor: Donato Cosco
Received: 24 March 2021
Accepted: 27 April 2021
Published: 30 April 2021
Keywords: ischemic/reperfusion injury; protein interacting with C kinase 1 (PICK1); AMPA receptor;
FSC231; liposome; drug delivery system
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Stroke and ischemic heart disease were responsible for 15.2 million deaths in 2016. Is-
chemic stroke is the second leading cause of death worldwide [
prevalent form of stroke, occurs when blood flow is decreased or absent due to vessel oc-
clusion [ ]. While it is necessary to reintroduce blood flow to the infarcted area, this act also
1]. Ischemic stroke, the most
2
Copyright:
© 2021 by the authors.
results in further damage by inflammation, oxidative stress, and delayed neuronal death
(DND) within vulnerable neuronal populations, including CA1 hippocampal pyramidal
neurons [3,4]. During ischemia, the lack of energy available disrupts ATP-dependent pro-
cesses that maintain ionic gradients, which are critical to cellular function. Disrupting the
ionic balance leads to excessive release of neurotransmitters, including glutamate, which
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4.0/).
are unable to be effectively cleared from the synapse [
5]. Excessive stimulation of N-methyl-
D-aspartate receptors (NMDARs) by glutamate is a contributing factor to DND [
6–9], but
Pharmaceutics 2021, 13, 636. https://doi.org/10.3390/pharmaceutics13050636
https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 636
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multiple studies have reported that
α
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid receptors (AMPARs) also contribute to DND [10–15].
AMPARs are ionotropic glutamate receptors in the brain and mediate fast excitatory
neurotransmission. These receptors contain GluA2 subunits that render them impermeable
to Ca2+ under physiological conditions, due to a post-transcriptional modification of glu-
tamine residue 607, resulting in a charged arginine 607 residue that inhibits the passage of
Ca2+ ions through the receptor [16]. As a result of ischemia/reperfusion, AMPARs undergo
a subunit composition switch from Ca2+-impermeable, GluA2-containing AMPARs [15], to
Ca2+-permeable, GluA2-lacking AMPARs. This allows the AMPAR to conduct calcium,
which, in combination with excessive NMDAR stimulation, exacerbates cell death [17].
The internalization and subsequent degradation of the GluA2 subunit underlies
this AMPAR subunit composition switch at the cell’s plasma membrane surface. The
internalization of GluA2 subunits is, in part, mediated by protein interacting with C-
kinase 1 (PICK1). PICK1 has a post-synaptic density protein (PSD)-95/Discs-large/zonula
occludens (ZO)-1 (PDZ) binding domain which interacts with the C-terminal region of
GluA2 and regulates its recycling in the cell [18]. Previous studies have observed that
blocking the PICK1–GluA2 interaction leads to an increase in recycling of GluA2 back to
the surface [19].
Recently, the first small-molecule inhibitor of PICK1, FSC231 (Figure 1), has been
identified and shown to bind to the PDZ domain of PICK1 at an affinity similar to that
observed for endogenous c-terminus of PICK1 peptides (Ki ~ 10.1 µM) [20]. However,
the use of FSC231 as a PICK1 inhibitor has been limited due to its poor solubility in
aqueous solutions.
Figure 1. Structure of FSC231.
Liposomes are widely recognized for being superior agents for drug delivery by
solubilizing drugs and increasing their bioavailability [21]. Liposomes can also be modi-
fied for targeted delivery of drugs in a cell- or tissue-specific manner [22]. Furthermore,
extensive toxicological screenings of liposomes have shown that they are remarkably safe
for pharmaceutical use [23]. We sought to develop a drug-delivery system for FSC231 to
use in our ex vivo animal model.
To evaluate the effect of FSC231 encapsulated in liposomes on the PICK1–GluA2
interaction, we utilized adult rat acute hippocampal slices exposed to oxygen–glucose
deprivation and reperfusion (OGD/R), an in vitro model for ischemic/reperfusion injury.
Here we demonstrate that FSC231 blocks the degradation of GluA2 by inhibiting the
interaction of PICK1–GluA2.
2. Materials and Methods
2.1. Liposomal Formulation of FSC231
FSC231 was synthesized by using a previously published protocol [24]. In brief, a
cyanoacetylcarbamic acid ethyl ester intermediate was synthesized from 2-cyanoacetic acid
and urethane (ethyl carbamate), using the condensation agent phosphorous chloride oxide.
The Knoevenagel reaction between cyanoacetylcarbamic acid and 3,4-dichlorobenzaldehyde
was performed at room temperature, utilizing piperidine as a catalyst. Liposomes were
prepared with FSC231, or without FSC231 (control), by solubilization in dimethyl sulfox-
ide (DMSO, 30 mg/g, w:w; FSC231/Lipid ratio 1:10)). Then 1,2-Dimyristoyl-sn-glycero-
3-phosphocholine (DMPC; 50 mg/g in tert-butanol, w:w) and Tween-20 (1:10 tween-
20/FSC231+DMPC, w:w) were added to the solution, and tert-butanol was added to make

Pharmaceutics 2021, 13, 636
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a total volume of 5 mL. The solution was frozen at
−
80 ◦C and then lyophilized over a
period of 2 days. The thin film produced was a hygroscopic brittle glass. These physical
properties indicate the complete removal of all solvents (water, tert-butanol, and DMSO).
On the day of the experiment, the FSC231-lipid thin film was reconstituted in 5 mL of
artificial cerebrospinal fluid (aCSF) or glucose-free aCSF by spinning the film in the pres-
ence of the solvent, in a 100 mL round-bottomed flask, connected to a rotary evaporator
at atmospheric pressure (no vacuum), frozen with liquid nitrogen, reconnected to the
rotary evaporator, and thawed to room temperature, with spinning, for ten cycles. The
liposomal suspensions were diluted to a final concentration of 100
µ
M (based on mmol of
◦
FSC231) and kept at 4 C until use in OGD/R experiments. Liposomes were characterized
by liquid chromatography/mass spectrometry (LC/MS), dynamic light scattering (DLS),
and transmission electron microscopy (TEM) and evaluated for encapsulation efficiency.
2.2. LC/MS
A total of 20 µL of FSC231 liposome suspension was added to 200 µL of ethanol. The
instrument used was a HPLC/MS, Micromass LCT mass spectrophotometer composed of
a Waters Alliance e2695 HPLC system that has a Waters 2998 photodiode array detector
and orthogonal acceleration of TOF detection. A reverse-phase Phenomenex HPLC column
(3
µ
m NX-C18 110 Å, LC Column 50
×
4.6 mm, Waters, Milford, MA, USA) was used for
the LC/MS analysis. Limit of detection (LOD) and limit of quantification (LOQ) were 0.1
and 0.3 mg/mL for DMPC, respectively. Baseline resolution of FSC231 was performed
by a gradient elution at a flow of 0.6 mL/min with a gradient mobile phase composed
of “mobile phase A” composed of water (0.8% trifluoroacetic acid) and “mobile phase
B” composed of acetonitrile, ramped over a 15 min period, with an injection volume of
10 µL, monitored at 254 nm, using the following conditions: In the first three minutes, an
isocratic run by 80% (v/v) mobile phase A; from 3.0 to 3.5 min, a linear gradient from 80%
(v/v) mobile phase A to 55% (v/v) mobile phase A; from 3.5 to 5.0 min, a linear gradient
from 55% (v/v) mobile phase A to 30% (v/v) mobile phase A; from 5.0 to 7.0 min, a linear
gradient from 30% (v/v) mobile phase A to 0% (v/v) mobile phase A; from 7.0 to 12.0 min,
an isocratic run at 0% (v/v) mobile phase A; from 12.0 to 15.0 min, a linear gradient from
0% (v/v) mobile phase A to 80% (v/v) mobile phase A.
2.3. Size Distribution and Surface Potential Measurements
Mean diameter, size distribution◦, polydispersibility index (PDI), and zeta potential of
the liposomes were determined at 20 C, by DLS (Zetasizer nano ZS, Malvern Instruments
Ltd., Malvern, UK). The instrument was equipped with a He–Ne laser operating at a
wavelength of 633 nm. Sizing measurements were made on the reconstituted liposomal
samples diluted 1/100 (0.1 mg/mL) with 10 mM NaCl. Transmission electron microscopy
(TEM) was performed, using an FEI Tecnai G2 20 Twin TEM (FEI, Hillsboro, OR, USA),
to observe the average particle size and distribution of the samples. Then 2 µL of the
liposomal suspension was placed on a formvar/carbon coated grid for 1 min, blotted off
with a #4 filter paper wedge, and stained for 1 min with 2% uranyl acetate, followed by
blotting with a #4 filter paper wedge. The grids were placed under a heat lamp for 5 min
and then into a grid box and stored in a desiccator prior to imaging.
2.4. Encapsulation Efficiency
The encapsulation efficiency of the liposomes was determined by ultraviolet spec-
troscopy, using Eppendorf UVette cuvettes (VWR; Radnor, PA, USA) at a 1 cm pathlength,
on an IMPLEN NanoPhotometer (Model NP80; Los Angeles, CA, USA). The liposomal
suspension (200
FSC231,
304 = 16,390 M−1
of free drug + amount of encapsulated drug)). Subsequently, 200
pension was pelleted in an ultracentrifuge (100,000 g), at room temperature, for 60 min.
The supernatant was discarded and the pellet was resuspended in 200 L of aqueous NaCl
µ
L) was mixed with 100% ethanol (200
cm−1) was determined (Abs liposome solution (amount
L of the liposomal sus-
µL), and the absorbance at 304 nm
(λ
max
ε
·
µ
×
µ

Pharmaceutics 2021, 13, 636
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(10 mM), and repelleted at 100,000
×
g for 60 min. The supernatant was discarded, the
pellet was resuspended in aqueous NaCl (10 mM, 200 µL) and diluted with 100% ethanol
(200 L), and the absorbance at 304 nm was once again determined (Abs pellet solution
µ
(amount of encapsulated drug)). The encapsulation efficiency was determined by using
the following equation:
abs pellet solution
(amount of encapsulated drug)
% encapsulation efficiency =
× 100
abs liposome solution
(amount of free drug + amount of encapsulated drug)
2.5. Animals
Adult (6–8 weeks old) male Sprague-Dawley rats (Charles River Labs, Wilmington,
MA, USA) arrived at our facility on week 5. Rats were housed in a temperature-controlled
facility with water and chow provided ad libitum. All animal protocols were approved by
the University of Montana Institutional Animal Care and Use Committee.
2.6. Preparation of Acute Rat Hippocampal Slices
Adult male (6–8 week) Sprague-Dawley rats were anesthetized with isoflurane and
rapidly decapitated. The brain was quickly removed and placed in an ice-cold cutting
media (75 mM sucrose, 80 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 24 mM NaHCO3,
25 mM glucose, 4 mM MgCl2, 1 mM L-Ascorbic acid, 3 mM Na pyruvate, 0.5 mM CaCl2,
pH 7.4). Both hippocampi were dissected, and coronal, 400-micron-thick slices were
made by using a Mcllwain tissue chopper (Vibratome, St. Louis, MO, USA). Slices were
equilibrated in oxygenated (95% O2, 5% CO2) aCSF (124 mM NaCl, 2.5 mM KCl, 1.25 mM
KH2PO4, 26 mM NaHCO3, 10 mM glucose, 1.5 mM MgCl2, 2.5 mM CaCl2, pH 7.4), at
37 ◦C, for 60 min, prior to OGD/R.
2.7. OGD/R Procedure
Following equilibration, slices to be subjected to OGD/R were rinsed with glucose-
free aCSF (aCSF with 10 mM mannitol substituted for 10 mM glucose, pH 7.4) and then
transferred to deoxygenated glucose-free aCSF (0% O2, 95% N2, 5% CO2) and incubated for
40 min in a hypoxic glove box (Coy Laboratories, Grass Lake, MI, USA). Ten minutes prior
to the end of OGD, FSC231-containing liposomes (100
aCSF) or DDS (drug delivery system, empty liposomes) were added to the slices. The slices
were then transferred to oxygenated aCSF containing FSC231 (100 M in aCSF), DDS, or
µM, in deoxygenated, glucose-free
µ
no liposomal treatment for the time periods indicated. Normoxic controls were left in
oxygenated aCSF for the duration of the experiments and were time-matched to the last
reperfusion time-point of OGD-subjected slices.
2.8. Lysate Preparation
Slices were removed from aCSF at indicated reperfusion times and rinsed 3 times with
ice-cold phosphate-buffered saline (PBS). Slices were transferred into tubes containing lysis
buffer (250 mM sucrose, 20 mM HEPES, 2 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol,
1 mM 4-(aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 1% protease and
phosphatase inhibitor cocktail (Thermo, Rockland, IL, USA), 1% Triton X-100, 0.01% (w/v)
saponin, pH 7.4) and lysed immediately by sonication 3 times, for 5 s, at 25% power, with
a VirTis Ultrasonic Cell Disrupter 100 (Gardiner, NY, USA). Samples were centrifuged at
13,000
×
g, for 10 min, to remove cellular and nuclear debris. The supernatant was collected,
and a bicinchoninic acid assay (BCA; Thermo, Rockland, IL, USA) was performed to
determine protein concentration.
2.9. Western Blotting
◦
Samples were denatured in Laemmli buffer/
β
-mercaptoethanol and heat (100 C)
for 10 min and resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis

Pharmaceutics 2021, 13, 636
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(SDS-PAGE). Samples were transferred to a nitrocellulose membrane (Bio-Rad, Berkeley,
CA, USA) for subsequent detection, using immunoblotting. Blots were blocked for 1 h at
room temperature with 5% (w/v) nonfat dry milk in tris buffered saline (0.1% v/v Tween-
20, pH 7.5 (TBS-T)). Blots were then incubated with the following primary antibodies,
overnight, at 4 ◦C: anti-PICK1 (1:1000, rabbit IgG, Epitomics, Burlingame, CA, USA); anti-
GluA2 (1:1000, rabbit IgG, AbCam, Cambridge, MA, USA); anti GluA1 (1:1000, rabbit
IgG, Epitomics, Burlingame, CA, USA); anti-β-Actin (1:5000, mouse IgG, Calbiochem, San
Diego, CA, USA). Then they were washed 3 times with TBS-T and incubated with rabbit
or mouse horseradish peroxidase secondary antibody (1:2000, Jackson ImmunoResearch,
West Grove, PA, USA), for 2 h, at room temperature. Blots were then washed 3 times in
TBS-T and then incubated with chemiluminescence substrate (Thermo, Rockland, IL, USA).
Immunoreactive bands were visualized and captured with a Fuji Imaging System. Blots
were stripped and re-probed up to 2 times, using Restore Plus Western Blotting Stripping
Buffer (Thermo, Rockland, IL, USA).
2.10. Immunoprecipitation
Rat hippocampal slices were prepared as described above. Rat hippocampal slices
were lysed in a buffer containing 50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 0.5% Triton X-100, and 1% protease and phosphatase
inhibitor cocktail. Protein concentrations were determined by using a BCA assay, and
lysates (500
µ
g/sample in 500
µ
L) were pre-cleared by using Protein-A/G 50/50 mix
◦
agarose beads (Bio-Rad, Redmond, WA, USA) for 1 h, at 4 C, followed by incubation
with a PICK1 antibody◦(15
µg per 500 µg protein, rabbit IgG, Epitomics, Burlingame, CA,
USA), overnight, at 4 C. The immunocomplex was then incubated for 4 h with 50 µL
Protein-A/G 50/50 mix agarose beads, at 4 ◦C, with rotation, and washed 3 times with
lysis buffer. Samples were eluted from the agarose beads by treatment with Laemmli
buffer/β
-mercaptoethanol and heat (100 ◦C) for 5 min and then subjected to SDS-PAGE.
After transfer to nitrocellulose membranes, blots were blocked and incubated as described
in Section 2.9, “Western Blotting”.
2.11. Statistical Analyses
The statistical analyses were performed by using GraphPad Prism 8 Software (Prism
8.4.0, 2020, GraphPad, San Diego, CA, USA). The p-values were calculated by using
One-Way Analysis of Variance (ANOVA) with post hoc Sidak. Data are expressed as
mean ± standard error of the mean (SEM). The results presented here are of at least three
independent experiments.
2.12. Data Availability
All data presented in this paper are contained within the manuscript or available upon
request of the corresponding author—D.A.J.
3. Results
3.1. Characterization of FSC231-Containing Liposomes
After initial rounds of experiments, FSC231 was found to possess poor solubility in
aCSF solutions. To improve the solubility characteristics of FSC231, we sought to utilize a
liposomal drug-delivery system.
Empty and FSC231-loaded liposomal suspensions were obtained by thin-film rehydra-
tion, using 10 mM aqueous NaCl (100 uM FSC231 final concentration), followed by 10x
freeze-dry/thaw cycles to yield liposomal solutions with a liposomal average mean diameter
of 173 nm (PDI 0.3) and 256 nm (PDI 0.3) (Table 1; Supplementary Figure S1), for the empty
and FSC231-loaded liposomes, respectively. The average zeta potential was determined to
be
−
2.89 and
−
7.01 for the empty liposomes and the FSC231-loaded liposomes, respectively
(Table 1; Supplementary Figure S1). Utilizing UV–Vis and LC/MS, we examined the stability

Pharmaceutics 2021, 13, 636
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of FSC231-loaded liposomes over a 1-month period. The FSC231 was chemically stable over
1 month when stored as a lyophilized powder at 4 ◦C (Supplementary Figure S2).
Table 1. Summary of size distribution results. Empty MLVs had an average particle size of 173 nm,
polydispersibility index (PDI) of 0.369 and zeta potential of
were found to have an average particle size of 256 nm, PDI of 0.364, and zeta potential of
−
2.89 mV. MLVs encapsulating FSC231
7.01 mV.
−
Article Particle Size
(nm)
Polydispersibility
Index (PDI)
Sample
Zeta Potential (mV)
Empty Multilamellar
Vesicles
FSC231-loaded
Multilamellar Vesicles
173.3
256.1
0.369
0.312
−2.89
−7.01
The encapsulation efficiency of the FSC231-loaded liposomes was obtained by uti-
lizing UV spectrophotometry (Supplementary Figure S3). The encapsulation efficiency
was determined to be 58% (Table 2). Utilizing TEM, the FSC231-loaded liposomes were
visualized to be fairly round in shape and size (~250 nm) (Figure 2), as confirmed by
dynamic light scattering (Table 1).
Table 2. Determination of encapsulation efficiency (%). The encapsulation efficiency of the liposomes
was determined with UV–Vis by measuring the absorbance at 304 nm (bolded in table) and utilizing
amount of encapsulated drug
the equation
× 100.
amount of free drug + amount of encapsulated drug
Wavelength (nm)
Encapsulation Efficiency (%)
302
303
304
305
306
58
58
58
58
57
Figure 2. Representative transmission electron micrographs (TEM) of the liposomes. TEMs of the liposomes stained with
uranyl acetate. ( ) Representative TEM (lower magnification) demonstrating round shape and size with scale of 500 nm.
) Representative TEM (higher magnification) demonstrating round shape and size with scale of 200 nm. The liposomes
are roughly 250 nm in size, confirmed by dynamic light scattering.
A
(
B
A concentration of 100
µM FSC231 was chosen as a drug-saturating concentration
which is approximately 10 times the Ki (10.1
µM) of FSC231 for the PDZ binding domain
of PICK1. This concentration was subsequently used in all the following experiments. The
bin/amphiphysin/rvs (BAR) domain on PICK1 has been suggested to bind to liposomes of

Pharmaceutics 2021, 13, 636
7 of 14
specific size [25]. Though the exact radius of liposomes capable of binding with BAR have
not been elucidated, we performed all subsequent experiments in the presence of empty
liposomes to determine if there were any BAR associated effects, as shown in the following
figures. We found no changes between untreated controls and our empty liposome drug-
delivery system with regards to the PICK1–GluA2 interaction. This suggests that the empty
liposomes alone have no effect on the PICK1–GluA2 interaction.
3.2. FSC231 Treatment Antagonizes NMDA-Induced PICK1–GluA2 Interaction
Previous studies have observed that NMDAR activation leads to increased interaction
between PICK1 and GluA2 [18]. Treatment with FSC231 blocked the NMDA-induced
increase in association between PICK1 and GluA2 (Figure 3). As a positive control, we
have shown that FSC231 is able to inhibit NMDA-mediated PICK1–GluA2 association
(Figure 3).
Figure 3. FSC231 prevents NMDA-induced association of GluA2 and PICK1. Fifteen minutes after
hippocampal slices underwent 10 min NMDA (20
association with PICK1. FSC231 treatment prevents this increase in association, whereas empty
liposome drug-delivery system (DDS) does not, in hippocampal slices. ( ) Immunoprecipitation of
PICK1, and representative Western blot for GluA2. ( ) Quantification of Western blots normalized to
µM) stimulation, there was an increase in GluA2
A
B
the unstimulated, untreated samples. N = 3, one-way analysis of variance (ANOVA) with post hoc
Sidak; ** denotes p < 0.01; data are expressed as mean ± standard error of the mean (SEM).
3.3. Optimal PICK1–GluA2 Association
Previous studies under OGD conditions suggests that the GluA2 AMPAR subunit com-
position switch is dependent on PICK1 trafficking [26] and that this trafficking contributes
to delayed neuronal death in OGD-induced hippocampal slices [19]. These conditions have
not been examined under reperfusion conditions. To determine the time point at which
to carry out our OGD/R experiments, we first conducted a time-response experiment in
which we used 40 min OGD and increasing reperfusion times (0, 5, 15, 30, or 60 min). In
previous studies [27], 40 min of OGD was determined to be optimal in hippocampal slices.
PICK1 was immunoprecipitated from all samples and then immunoblotted for GluA2.
We found an increase in PICK1–GluA2 association with reperfusion when compared to
the normoxic control, with maximal association between 30 and 60 min of reperfusion
(Figure 4). A reperfusion time of 30 and 60 min gave statistically significant results. In
subsequent experiments, the 30-min reperfusion time point was used.

Pharmaceutics 2021, 13, 636
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Figure 4. OGD/R-induced PICK1–GluA2 interaction increases with reperfusion time. Post-OGD/R
immunoprecipitation of PICK1 and subsequent immunoblotting for GluA2 show an increase in
PICK1–GluA2 interaction in hippocampal slices at 40 min of OGD and 5, 15, 30, and 60 min of
reperfusion. (A) Representative Western blot of GluA2 following immunoprecipitation of PICK1 at
indicated OGD/R time-points. (B) Quantification of Western blots normalized to normoxic control.
N = 3, one-way ANOVA with post hoc Sidak, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes
p < 0.0001, data are expressed as mean ± standard error of the mean (SEM).
3.4. FSC231-Containing Liposomes Attenuate OGD/R-Induced PICK1–GluA2 Interaction
No previous studies have evaluated PICK1 expression under OGD/R conditions.
Increased PICK–GluA2 association could be attributed to an increased expression of PICK1.
We therefore performed Western blot analysis on total lysate treated with or without FSC231
to investigate if total PICK1 expression was altered during 40/30 OGD/R. There was no
significant change in PICK1 protein levels as a result of OGD/R treatment (Figure 5A).
To investigate the ability of FSC231 to inhibit the PICK1–GluA2 interaction, we next
performed immunoprecipitation experiments on OGD/R exposed hippocampal slices in
the presence or absence of FSC231 and empty liposomes. We found that FSC231 treatment
significantly reduced the PICK1–GluA2 interaction (Figure 5C). Furthermore, slices that
were treated with empty liposomes exhibited no significant change in PICK1–GluA2
association compared to untreated controls. These results suggest that FSC231 may be
blocking the PDZ binding domain of PICK1, preventing the interaction with GluA2.
3.5. FSC231 Treatment Prevents OGD/R-Induced Degradation of GluA2 AMPAR Subunit
Since GluA2 AMPAR subunit trafficking is, at least in part, dependent on PICK1, we
next sought to examine whether PICK1 is involved in the OGD/R-induced degradation of
GluA2. Total GluA1 protein levels were unaffected by OGD/R, and FSC231 treatment also
had no effect in GluA1 protein levels (Figure 6A). However, OGD/R-induced degradation
of GluA2 was prevented in slices treated with FSC231 (Figure 6C).

Pharmaceutics 2021, 13, 636
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Figure 5. FSC231 prevents OGD/R-induced interaction of PICK1 and GluA2. (
indicating that PICK1 protein levels are unchanged with OGD/R in hippocampal slices. (
A
) Representative Western blot of PICK1
) Quantification of Western blots
) Representative Western blot demonstrating that 40 min of OGD and 30 min of reperfusion increases
the association between PICK1 and GluA2. FSC231 treatment prevents the OGD/R-induced increase in PICK1–GluA2
interaction. ( ) Quantification of post-OGD/R immunoprecipitation of PICK1 and subsequent immunoblotting for GluA2
B
represented in ( ). (
A
C
D
normalized the normoxic untreated control. N = 12, one-way ANOVA with post hoc Sidak; * denotes p < 0.05; ns denotes
no significance.
Figure 6. FSC231 treatment prevents the OGD/R-induced degradation of GluA2. (
GluA1 indicating that 40 min of OGD and 30 min of reperfusion does not alter GluA1 protein levels in hippocampal slices.
) Quantification of Western blots from ( ). ( ) Representative Western blot for GluA2 demonstrating that 40 min of OGD
and 30 min of reperfusion decreases the amount of GluA2 protein in hippocampal slices. This effect is ameliorated with
treatment of FSC231. ( ) Quantification of immunoprecipitation/ immunoblotting experiments from ( ). N = 12, one-way
A) Representative Western blot for
(B
A
C
D
C
ANOVA with post hoc Sidak; ** denotes p < 0.01, ns denotes no significance, and data are expressed as mean
error of the mean (SEM).
±
standard

Pharmaceutics 2021, 13, 636
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4. Discussion
FSC231 has been utilized in studies examining pulmonary hypertension [28
,
29], acute
liver injury [30], hyperalgesia [31,32], willed-movement in focal cerebral ischemia [33], and
cocaine-seeking behavior [34]. This study is the first to analyze the therapeutic potential of
this small molecule in ischemia/reperfusion injury. FSC231 binds to the PDZ domain of
PICK1, hindering its ability to associate with the GluA2 AMPAR subunit.
Delayed neuronal death following OGD/R has been attributed to decreased surface
expression of GluA2-containing AMPARs and increased expression of surface GluA2-
lacking Ca2+-permeable AMPARs [10
–15]. The present findings demonstrate that OGD/R-
induced AMPAR trafficking is mediated by the PICK1 interaction with GluA2. Previous
studies have shown that disrupting the PICK1–GluA2 interaction with a c-terminal GluA2
peptide (EVKI) effectively attenuates OGD-induced GluA2 internalization [19]. The present
work aimed to observe this interaction with the use of the recently described PICK1 PDZ
inhibitor, FSC231 [20], with the added implementation of reperfusion following OGD.
While c-terminal peptide inhibitors possess higher selectivity due to mimicking natural
binding partner sequences, they possess poorer pharmacokinetic profiles when compared
to small molecules [35]. Compared to endogenous peptides, FSC231 was found to have a
similar binding affinity to PICK1.
Consistent with previous findings using c-terminal peptide inhibitors of the PICK1
PDZ domain, we found a significant decrease in PICK1–GluA2 interaction with FSC231.
It is unclear whether FSC231 completely blocks the internalization of GluA2-containing
AMPARs or inhibits the slow recycling of internalized GluA2 back to the surface. Future
studies are needed to evaluate the balance between endocytosis and exocytosis of AMPARs
with OGD/R, and the effect of FSC231 on this tightly regulated process. PICK1 expressed
in HEK293 cells was found to regulate the trafficking and recycling rate of its PDZ binding
partners through a Rab11-dependent recycling pathway [36]. PICK1 knockout (KO) mice
were shown to have no considerable difference in NMDA-induced internalization of
GluA2 compared to wild-type neurons, but exhibited increased acceleration rates of GluA2
recycling to the neuronal plasma membrane [18].
The phosphorylation of GluA2 at the serine 880 residue has been shown to be impor-
tant for the surface removal of GluA2. Serine 880 phosphorylation of GluA2 diminishes its
affinity for glutamate receptor-interacting protein 1 (GRIP1), an AMPA binding protein
(ABP) scaffold protein that localizes AMPARs to the synaptic membrane [37]. When GluA2
is phosphorylated at the serine 880 residue, it increases its affinity for binding to the PICK1
PDZ binding domain [37
for GluA2 phosphorylation at the serine 880 residue [39
gested to be facilitated by PICK1, acting as a scaffold, bringing PKC
,
38]. There are reports implicating PKC
40]. This phosphorylation is sug-
to GluA2 by direct
α as the kinase responsible
,
α
binding of PICK1 to ABP [39], but it is unclear if this mechanism holds true under OGD/R
conditions. Recently, we provided evidence that the increase in serine 880 phosphorylation
resulted in the dissociation of GluA2 from GRIP1/ABP, thus, enabling the association of
GluA2 with PICK1 [41]. This study also reported that there is an increase in the association
of activated PKCα with PICK1 following OGD/R [41].
Further studies using specific pharmacological inhibitors and/or knockdown of PKC
are necessary to determine the exact role of PKC in OGD/R. It is possible that inhibiting
the interaction between PICK1 and PKC with FSC231 may decrease phosphorylation of
α
α
α
GluA2, resulting in less internalization, and subsequent degradation, of GluA2-containing
AMPARs following OGD/R (Figure 7). However, this is speculative and further investiga-
tion is required.

Pharmaceutics 2021, 13, 636
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Figure 7. Potential mechanism of FSC231 treatment following OGD/R. Under normal physiologic conditions, AMPARs
in the hippocampus are GluA2-containing Ca2+-impermeable receptors. GRIP1 is a scaffold protein that aids in tethering
GluA2-containing AMPARs to the cell membrane. Under pathologic OGD/R conditions, there is a massive release of
neurotransmitters, including glutamate, which activates AMPARs and subsequently activates NMDARs and L-type voltage-
gated calcium channels. An increase in intracellular Ca2+ activates PKC
plasma membrane. PICK1 binds to GluA2, GluA2 is phosphorylated by PKC
α
binding to PICK1, which translocates to the
, and is subsequently endocytosed and
α
degraded. There is a subsequent increase in GluA2-lacking Ca2+-permeable AMPARs. This leads to intracellular calcium
overload, resulting in excitotoxicity and cell death. FSC231 blocks the interaction of GluA2–PICK1, prevents the GluA2
AMPAR subunit from being degraded, and acts in a neuroprotective manner.
Treatment with FSC231 antagonized the OGD/R-induced degradation of the GluA2
AMPAR subunit. It is unclear whether this is due to decreased endocytosis of the GluA2
AMPAR subunits following OGD/R or if this is a result of increased endosomal sorting
to the slow recycling endosome. Either of these actions would result in GluA2 not being
targeted towards a degradative pathway with OGD/R. Further studies are needed to
confirm PICK1’s role in the endocytic trafficking and degradation of the GluA2 subunit
with OGD/R.
PICK1 has been recently suggested to be a valuable drug target for the treatment of
neurological disorders, including excitotoxicity [42], cancer [43], and neuropathic pain [44],
through its PDZ binding domain. PICK1 has also been an ongoing subject of interest in re-
search involving synaptic plasticity [45,46]. It will be important to understand the multiple
roles this protein plays in the trafficking of its binding partners. FSC231 has the potential
to be a small-molecule therapeutic for the prevention of ischemia/reperfusion-induced
delayed neuronal death in vivo, by preventing the degradation of GluA2-containing AM-
PARs. Many studies have utilized a liposomal drug-delivery system to target the brain
following ischemia/reperfusion in vivo; see the recent review from Fukuta et al. [47]. It
has been previously demonstrated that PEGylated liposomes under 200 nm successfully
targeted the brain in vivo and delivered Fasudil, a Rho-kinase inhibitor, to the ischemic
region [48]. With these parameters in mind, it is possible that FSC231 may also translate to
an in vivo therapy for ischemia/reperfusion injury, utilizing a liposomal system to deliver
this therapeutic small molecule to the brain.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/pharmaceutics13050636/s1, Figure S1: Size distribution and zeta potential of FSC231-loaded
MLVs, Figure S2: FSC231 is stable when encapsulated in MLVs as a lyophilized powder for up to at
least 30 days, Figure S3: Representative UV scans for determination of encapsulation efficiency.
Author Contributions: Conceptualization, D.A.J.; methodology, F.A.-D., D.A.J., N.R.N., T.T.D.;
software, L.M.A.; validation, D.A.J., L.M.A.; formal analysis, L.M.A.; investigation, F.A.-D., P.H.B.,
N.R.N., T.T.D.; data curation, P.H.B., L.M.A., T.T.D.; writing—L.M.A.; writing—review and editing,
D.A.J., L.M.A., F.A.-D., T.T.D.; supervision, D.A.J. All authors have read and agreed to the published
version of the manuscript.

Pharmaceutics 2021, 13, 636
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Funding: This research was funded by NIH grant R15NS074418 and start-up funding from Washing-
ton State University, Department of Pharmaceutical Sciences.
Institutional Review Board Statement: All animal protocols were approved by the University of
Montana Institutional Animal Care and Use Committee; PHS Assurance Number: D16-00210.
Informed Consent Statement: Not Applicable.
Data Availability Statement: All data are contained in the manuscript or available upon request to
the corresponding author: D.A.J.; Darrell.jackson@wsu.edu.
Conflicts of Interest: The authors declare no conflict of interest.
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