Diabetes Autoantibodies Mediate Neural- and Endothelial
Cell- Inhibitory Effects Via 5-Hydroxytryptamine- 2 Receptor
Coupled to Phospholipase C/Inositol Triphosphate/Ca2+
Mark B. Zimering *
Endocrinology, Veterans Affairs New Jersey Healthcare System, East Orange, New Jersey; Rutgers-Robert Wood Johnson Medical School, New
Brunswick, New Jersey.
Dr. Mark B. Zimering, Endocrinology, Veterans Affairs New Jersey Healthcare System, East Orange, New Jersey; Rutgers-
Robert Wood Johnson Medical School, New Brunswick, New Jersey; Fax: 908 604-5249;E-mail: @
Received: September 05, 2017; Accepted: September 19, 2017; Published: October 04, 2017
Mark B. Zimering (2017) Diabetes Autoantibodies Mediate Neural- and Endothelial Cell- Inhibitory Effects Via 5-Hydroxytryptamine- 2 Receptor Coupled to Phospholipase C/Inositol Triphosphate/Ca2+ Pathway. J Endocrinol Diab 4(4): 1-10. DIO: http://dx.doi.org/10.15226/2374-6890/4/4/00184
Aims: To identify the G-protein coupled receptor(s) on neuroblastoma and endothelial cells which mediate neural- and endothelial cellinhibitory effects in plasma autoantibodies from a subset of older type 2 diabetes with neurologic and vascular co-morbidity. To determine the
mechanism(s) of neurite retraction induced by diabetic pathologies’ auto antibodies.
Methods: Protein-A eluates from plasma of 11 diabetic patients having nephropathy, moderate-severe obesity and/or complications in which
increased inflammation plays a role (depression, Parkinson’s disease, atrial fibrillation, obstructive sleep apnea) were tested for neurite retraction
and decreased survival in N2A neuroblastoma cells, and decreased survival in pulmonary artery endothelial cells. Specific antagonists of G protein
coupled receptors belonging to the G alpha q subfamily of hetero trimetric G proteins or the phospholipase C/inositol triphosphate/Ca2+ pathway
were tested for modulatory effects on diabetic pathologies’ autoantibody-induced N2A neurite retraction, or cell survival.
Results: Co-incubation with specific antagonists of the 5-hydroxytryptamine- 2A receptor significantly prevented acute N2A neurite retraction
induced by 50-100 nM concentrations of diabetic pathologies’ autoantibodies. Protection against neurite retraction (M100907> spiperone>
ketanserin) closely paralleled the antagonists’ potency order at the 5-HT2-AR. Neuroblastoma or endothelial cell death (after 24 hours incubation)
with 50-100 nM autoantibodies was completely or nearly completely (91%) prevented by co-incubation with 200 nM M100907, a highly selective
5-HT2-AR antagonist. Alpha-1 adrenergic, angiotensin II, metabotropic glutamate 5, or endothelin A (100 nM-10μM) receptor antagonists did
not substantially inhibit autoantibody-induced cell death. The intracellular calcium chelator (BAPTA-AM, 50 μM) and inhibitors of the inositol
triphosphate (IP3) receptor (2-APB, 50μM), and phospholipase C-gamma (U73144, 1μM) each significantly protected against autoantibody-induced
acute N2A neurite retraction.
Conclusion: These data suggest that neural- and endothelial- inhibitory effects in autoantibodies from older adult diabetes with nephropathy
and obesity/inflammation-associated complications are mediated by agonist autoantibodies directed against the 5-hydroxytryptamine 2 receptor
positively coupled to the phospholipase C/inositol triphosphate/ cytosolic Ca2+ release pathway.
Keywords: 5-hydroxytryptamine-2 receptor; diabetes; autoantibodies; neurovascular diseases
Obesity and inflammation are risk factors for certain
diabetes-related neurovascular complications although the
underlying mechanism is unclear. Diabetic nephropathy is
characterized by increased micro vascular permeability  (e.g.
micro- and macro-albuminuria) and inflammation thought to
drive the progression to end-stage-renal disease (ESRD) . In
a prior study in 305 diabetic nephropathy patients, we reported
that albuminuria interacted significantly with endothelial cell
inhibitory autoantibodies in predicting the risk of substantial
decline in renal function, ESRD or death during three-five years’
randomized treatment in the VA NEPHRON D– a clinical trial
comparing angiotensin receptor blocker (ARB) alone vs. combined
ARB and angiotensin-converting enzyme inhibitor medications
[3,4]. Endothelial cell (EC) inhibitory autoantibodies were also
increased in subsets of diabetic patients suffering with specific
co-morbidities including: diabetes, schizophrenia and the comorbid
clusters: painful neuropathy/maculopathy/nephropathy
obstructive sleep apnea/atrial fibrillation/dementia and primary
open-angle glaucoma/dementia [5-9]. Mechanistically, potent
endothelial cell autoantibodies caused apoptosis through a
mechanism involving large, sustained increases in intracellular
Ca2+ leading to caspase-3 activation . Diabetic depression
autoantibodies caused neurite retraction, and prolonged N2A
neuroblastoma cell depolarization . A subset of dementia/
glaucoma autoantibodies caused contraction and process
retraction in cerebral cortical astrocytes . The pleiotropic
actions of the diabetic neurovascular autoantibodies led us to
test (in the present study) whether endothelial cell- and neuro
toxic diabetic autoantibodies may target a specific subclass
of G-protein coupled receptors (GPCR) linked to increases in
Participants & Methods
Informed consent for the Investigational Review Boardapproved,
local, Veterans Affairs New Jersey Healthcare System
proposal was obtained from all study patients prior to blood
drawing. Baseline clinical characteristics in the study participants
are shown in (Table 2). The cohort was comprised of older men,
having a mean age of 68 years, average BMI of 38 kg/m2, average
duration of type 2 diabetes of 13.8 years, and mean glycosylated
hemoglobin of 7.6%. In order to explore possible linkages
between autoantibodies, obesity, inflammation and diabetic
neurodegenerative or vascular complications, patients were
selected on the basis of a high concordance (in individual patients)
of these same risk factors and complications. Overall, the cohort
had a high prevalence of neurodegenerative disorder(s) (64%),
[i.e. major depression (n=5), Parkinson’s disease (n=2), glaucoma
(n=1)], nephropathy (82%), moderate-or severe-risk obesity
(82%), and one or more conditions linked to inflammation, e.g.
atrial fibrillation (64%), autoimmune thyroid disease (27%),
obstructive sleep apnea (45%). Nine of eleven moderate-severely
obese patients had an average of two or more complications
(atrial fibrillation, obstructive sleep apnea, painful neuropathy)
associated with inflammation. Our underlying hypothesis is that
diabetic nephropathy drives autoantibody production through
the release of endothelial cell auto antigens in the setting of
chronic inflammation. As a test of this hypothesis, all but two study
patients had diabetic nephropathy. Moderate-severely obese
patients were selected for this mechanistic study, since in prior
reports, obesity was significantly associated with pathologies
(e.g. atrial fibrillation, obstructive sleep apnea) in which neurotoxic
plasma autoantibodies were present at increased levels
compared to control diabetic patients .
Table 1: Autoantibodies in diabetes having co-morbid neurovascular complications: spectrum of autoantibody- mediated effects [5,6,7,8,9]
Autoantibodies Endothelial cell
Incr vs. control
Neurite retraction, depolarization
Incr vs. control
Incr vs. control
N2A & DG NPC retraction, depol
Incr vs. control
Neurite retraction, astrocytes effects*
Incr vs. control
Neurite rtrct/outgrowth 
Obstr sleep apnea
Inc vs. control
N2A neurite retraction, depolarization
Co-morbid with OSA
Incr vs. control
N2A neurite retraction, depolarization
*Autoantibodies in diabetic primary open-angle glaucoma and dementia caused contraction and process shortening in cerebral cortical astrocytes . Obstr (uctive);
Incr (eased) compared to Control diabetes populations lacking this complication; stim (ulate); NT- not tested; N2A mouse Neuroblastoma cell line; rtrct (retraction), cntr
(contraction); short (ening), depol (arization), DG dentate gyrus, NPC-neural progenitor cell, “ i.e. “NPC-neural progenitor cell, OSA- obstructive sleep apnea.
Diagnostic methods and subgroups
All patients having a diagnosis of major depression were
evaluated by psychiatry staff at the Veterans Affairs New Jersey
Health Care System as previously reported . Painful diabetic
neuropathy is defined according to previously reported criteria
. Diabetic nephropathy is defined as persistent micro-or
macro-albuminuria, the latter urinary albumin excretion >
300 mg/g creatinine or urinary protein excretion > 500 mg/g
Morbid obesity is defined as body mass index (BMI) > 40 kg/
m2. Moderate-severe obesity is defined as BMI > 35 kg/m2. Pt 1:
A 71 -year- old male with BMI 48 kg/m2, diabetic nephropathy,
atrial fibrillation, obstructive sleep apnea (OSA), glaucoma,
depression, and painful neuropathy.
Parkinson’s disease (PD)
PD 1: A 70- year- old male with diabetic nephropathy, atrial
fibrillation, obstructive sleep apnea, Parkinson’s disease and
PD 2: A 79 -year- old male with diabetic nephropathy,
autoimmune thyroid disease, and Parkinson’s disease.
Bovine pulmonary artery (BPA) endothelial cells (Sigma,
St. Louis, MO) were maintained at 37 C in 5% CO2/95% air in
Medium 199 plus 10% fetal calf serum. BPA endothelial cells
were passaged continuously and used between passages 4-10.
Endothelial Cell Growth Assays
Endothelial cell number assays were carried out as previously
reported . After 48 hours’ incubation in the presence of
protein-A-eluate fractions, cell number, i.e. cell-associated acid
phosphatase activity, was determined as previously described
. Growth-promoting activity (determined in quadruplicate
for each test factor) is expressed as a percentage of the control
cell number (determined in quadruplicate) for cells grown in the
absence of protein-A- eluate fractions.
Protein A Affinity Chromatography
Protein A chromatography was carried out as previously
described . Eluate fractions were stored at 0-4 degrees C.
All fractions were sterile filtered (Millipore Corp., Bedford, MA;
0.22um) before assay for growth-promoting activity.
Mouse N2A cells
Mouse N2A neuroblastoma cells were cultured as previously
Neurite retraction assays
Mouse N2A cells were plated at low density in 35 mm dishes.
After one to two days incubation at 37 degrees C, protein A-eluate
fractions (7-10 μg/mL) from diabetic pathologies’ plasma
were added in the presence or absence of GPCR antagonists or
antagonists of downstream signaling molecules or an intracellular
calcium chelator. Thin neurite length was estimated (at baseline
and again after 25 minutes exposure to added fractions) as the
average length in all thin neurites (having diameter of ~ 0.5-1
microns) extending at least 2 cell diameters from the cell body at
baseline in each of 5-10 cells. Percent of antibody-induced neurite
retraction is expressed as a percent of acute neurite lengthshortening
(observed after 25 minutes) in cells exposed to test
factors plus autoantibodies compared to identical concentration
of same autoantibodies alone. Each point represents the mean of
two or more determinations which generally varied by < 15%.
N2A survival assays
N2a cells were plated at 103-104 cells/cm2 in 96 cell plates in
DMEM plus 10% fetal calf serum. After 2-3 days incubation, test
Table 2: Baseline clinical characteristics in study cohort
67.8 ± 7.0
Body mass index (kg/m2)
38.2 ± 5.8
Diabetes Duration (years)
13.8 ± 5.9
7.6 ± 1.4
Atrial fibrillation (yes/no)
Obstructive sleep apnea (yes/no)
Painful neuropathy (yes/no)
Autoimmune thyroid disease (yes/no)
Neurodegenerative disease* (yes/no)
*Depression (n=5), Parkinson’s disease (n=2).
fractions were added to cells in quadruplicate. Following (8 or 24
hours) incubation at 37 degrees C, cell survival was determined
using MTT assay. Twenty micro liters of a 5 mg/mL MTT solution
in PBS was added to each well; after 3.5 hours incubation at 37
degrees C, MTT solution was discarded and 200 micro liters
DMSO was added to each well. After 10 minutes, absorbance was
determined at 570 nm. Cell survival is expressed as a percentage
of the absorbance in control N2A cells to which no test factors
Adult neural progenitor cells
The stem cells were originally isolated from the dentate
gyrus of adult Fisher 344 rats and were obtained from Dr. Fred
Gage at the Salk Institute. The NPCs were cultured as previously
described . Test protein-A eluate fractions from diabetic
pathology plasma (5-7 μg/mL protein) was added to cells on DIV
3. Acute process shortening and cell contraction were determined
by continuous observation (at 5 minute intervals) using a Nikon
inverted microscope at 200X magnification for up to 75 minutes
after adding test factors.
M100907, ketanserin, spiperone, DOI, bosentan, losartan,
prazosin, U73211, BAPTA-AM, 2-APB were obtained from Sigma
Chemical Co. (St Louis, MO.) All other chemicals and reagents
were analytical grade.
Protein concentrations were determined by a bicinchoninic
acid protein assay kit (Pierce Chemical Co., Rockford, IL).
All data are the mean ± SD (Tables 1, 2, 4) or SEM (Figure 4-7)
as indicated. Comparisons were made by Student’s t-test for a
continuous variable with a significance level, p=0.05.
Table 3: Pharmacologic profile of endothelial cell-inhibitory activity in
% Neutralization of Diab Autoantibody-induced EC cell death
M100907 [2 μM]
M100907 [200 nM]
M100907 [100 nM]
Ketanserin [100 nM]
Bosentan [10 μM]
CTEP [10 μM]
A1-AR- alpha 1 adrenergic receptor; AT-1R- angiotensin II, type 1
receptor; ET-AR- endothelin A receptor, mGlu-5R- metabotropic
glutamate 5 receptor
Table 4: Effect of antagonists of the PLC/IP3R/Ca2+ pathway on
autoantibody- induced N2ANeurite retraction
% Diab Autoantibody-induced neurite retraction*
100 ±0 %
U73122 (PLC inhibitor)
12 ± 4 %
BAPTA-AM (Ca2+ chelator)
0 ± 0 %
2-APB (IP3R inhibitor)
*Neurite retraction was determined as percent of acute neurite
shortening (after 25 mins Exposure) in the presence of 50 nM diabetic
autoantibodies alone, or following a brief pre incubation (10 mins) with
indicated concentrations of specific antagonists of the PLC/IP3/Ca2+
path way or RhoA/ROCK signaling pathway. Results are mean ± SD in
two or more experiments.
Effect of diabetic autoantibodies in rat dentate gyrus neural
progenitor cells Rat dentate gyrus (DG) neural progenitor cells
(NPC) were sensitive to low concentrations of diabetic depression
autoantibodies (5-7 μg/mL) and were previously reported to
cause both NPC process retraction and decreased cell survival
. In the present study, diabetic autoantibodies (8 μg/mL =80
nM) from a patient suffering with nephropathy, Parkinson’s
disease, atrial fibrillation and painful diabetic neuropathy (i.e. PD
1) caused rapid NPC neurodegeneration in distal fine processes
after 15 minutes’ incubation. After 20-25 minutes exposure,
proximally-located processes extending from cell bodies had
undergone dendritic simplification and beading. After 65
minutes, several NPC cell bodies appeared highly refractile- a
characteristic of dying cells. Pre-incubation (for 5 minutes) with
M100907 at 2 μM concentrations completely prevented early
NPC neurodegeneration (during 30 minutes’ observation) in
identical concentrations (80 nM) of the PD, Pt 1 autoantibodies.
Ten-fold lower M100907 (200 nM) concentrations partially
protected against PD, Pt 1 autoantibody-induced early NPC
neuro degeneration in proximal dendrites. Twenty nanomolar
M100907 had no significant protective effect in either proximallyor
Effects of diabetic autoantibodies on N2a
neuroblastoma cells morphology and survival:
The effect of IgG autoantibodies on acute length- shortening
in N2A neuroblastoma thin dendrites was examined at five
-minute intervals using time-lapse microscopy (Figure 1).
Significant neurite retraction was evident as early as tenfifteen
minutes after application of 10 microgram per milliliter
concentration of diabetic autoantibodies from two different
patients (Figure 1).After 25 minutes, identical concentration of
Pt 1 (a morbidly obese diabetic patient (BMI 48 kg/m2) suffering
with nephropathy, depression, obstructive sleep apnea, painful
neuropathy, and atrial fibrillation) IgG caused mean N2A thin
neurite length reduction of 68% (average of ten cells each having
2-10 processes, not shown in Figure 1). Pre-incubation (for 5 minutes)
Figure 1: Inhibition of N2a neurite outgrowth by diabetic autoantibodies.
Ten microgram per milliliter concentrations of diabetic autoantibodies
from two different patients were incubated with N2A cells
and microscopic images (200 X) magnification were acquired after 15
(B) Or 10 minutes (D) compared to the respective baseline images (A,
C) in cells before autoantibody treatment. Blue arrows (A,C) and orange
lines (B D) indicate corresponding points along a particular neurite
segment at which acute length-shortening will have or already had occurred.
Neuroblastoma cell bodies appeared contracted and displayed
increased refractility (B, D) following autoantibody exposure. Loss of
dendritic branches, i.e. dendritic simplification was evident in a dendrite
segment after ten minutes exposure to diabetic painful neuropathy,
atrial fibrillation patient autoantibodies (uppermost three blue arrows
in C vs. uppermost three orange lines in D).
with 200 nM concentrations of M100907, a highly
selective 5-HT2A receptor antagonist, was ~ 94% protective
against IgG-induced process length reduction (assessed
after 25 minutes’ exposure time). M100907 at (5 -2000 nM)
concentrations caused a dose-dependent inhibition of process
length shortening in identical concentrations of Pt 1 IgG (Figure
2A). A log-log graph of the percent change in process length versus
M100907 concentration was linear, had a slope of unity (Figure
2B) and IC50 = 39 nM. Ketanserin, a moderate affinity, selective
5-HT2A receptor antagonist, and spiperone, a high affinity, less
selective 5-HT2AR antagonist, both caused dose-dependent
inhibition of Pt 1,IgG-induced neurite length shortening (Figure
3A-B). The IC50 calculated from log-log plots for ketanserin and
spiperone were 215nM and 50 nM, respectively, consistent with
the order of binding affinities of the three antagonists for the
5HT2A receptor:, i.e. M100907 > spiperone > ketanserin .
Saturating (1 μM) concentrations of 2,5-dimethoxy-4-
iodoamphetamine (DOI), a 5HT-2A receptor agonist, caused
Figure 2: Dose-dependent inhibition of diabetic autoantibody, N2a
neurite retraction by M100907, a highly selective 5-HT2AR antagonist.
Patient 1 autoantibodies (10 μg/mL) were incubated with N2A cells in
the presence or absence of (5-2000 nM) concentrations of M100907.
A)Percent of autoantibody-induced neurite retraction was determined
as described in Methods.
B)Log-log plot of (% of antibody retraction) vs. log M100907 concentration
is linear with a slope of unity indicative of a homogeneous population
of 5-HT-2A receptors.
Figure 3: Dose-dependent inhibition of diabetic autoantibody (Pt 1, 10
μg/mL), N2A neurite retraction by A) ketanserin, a selective 5-HT2AR
antagonist, or B) spiperone, a high affinity, less selective 5-HT2AR antagonist.
Percent of autoantibody-induced neurite retraction was determined
as described in Methods.
10-15% acute length-shortening in N2a neuroblastoma cell
processes after 15 minutes. After 25 minutes, process length
shortening began to reverse and after 45, and 60 minutes process
length had fully reverted to baseline levels, consistent with DOIinduced
desensitization . Contraction in N2a cell bodies
(which was evident after 15 mins) had also partially reversed
after 45 minutes continuous exposure to (1 μM) DOI. Taken
together, these results suggest involvement of 5-HT2A receptors
in the mechanism of diabetic autoantibody-induced N2a neurite
N2A cell survival
Co-incubation of N2a neuroblastoma cells with 200 nM
concentrations of the selective 5-HT2A receptor antagonist
M100907 completely protected against diabetic autoantibodiesinduced
accelerated N2a cell death (n= 4 different diabetic
patients; P < 0.001 Fig 4A). M100907 at identical (200 nM)
concentrations alone had no significant effect on N2a cell
survival (Figure 4A). Ketanserin (200 nM) or the selective
alpha-1 adrenergic antagonist prazosin (100 or 200 nM) did not
significantly protect against neuroblastoma cell death induced
by identical concentrations of diabetic autoantibodies (n=3
patients) (Figure 4B-C). Ketanserin (200 nM) or prazosin (100
or 200 nM) alone did not significantly affect N2A cell survival
(Figure 4B-C). Spiperone (200 nM) significantly protected (88%)
against diabetes autoantibodies-induced N2a cell death (n=2,
data not shown in Figure 4). These data suggest involvement of
5-HT2A receptors in diabetic autoantibody-induced accelerated
neuroblastoma cell death. Saturating concentrations (1 μM) of
the 5-HT2R agonist DOI (2,5-dimethoxy-4-iodoamphetamine)
alone caused dose-dependent modest decrease(s) in N2A cell
survival (Figure 5B). Co-incubation of DOI (1 μM) together with
10 nM concentrations of diabetic autoantibodies significantly
protected against autoantibody-induced death in a fraction of the
cells (Figure 5B) consistent with DOI-induced desensitization in a
fraction of cells. Dose-response curves indicated that fifty to onehundred
fold lower concentrations (10-20 nM) of diabetic IgG
autoantibodies caused an equivalent level of N2a cell death (after
24 hours) as saturating (1 μM) concentrations of DOI (Fig 5A),
indicative that diabetic autoantibodies caused enhanced longlasting
5-HT2AR activation via unknown mechanisms.
Mechanism of diabetic autoantibody-induced N2A
GPCRs belonging to the 5-HT2 family of serotonin receptors
typically couple to the Galpha q/11 subfamily of heterotrimeric
G-proteins . Gαq/11 activation causes phospholipase C
(PLC) activation resulting in increased intracellular Ca2+ via
inositol triphosphate (IP3)-mediated release from endoplasmic
reticulum stores . We used specific antagonists of the PLC/
IP3/Ca2+ pathway to test for possible involvement of a Gαq/11
-coupled mechanism underlying autoantibody-induced neurite
retraction. Autoantibody-induced neurite retraction was nearly
completed (85-90%) blocked by pre-incubation (15 minutes)
with (1 μM) concentrations of U73211, a specific phospholipase
C-gamma (PLC-γ) inhibitor (Table 4). It was completely
blocked by pre-incubations with (50 μM) concentrations of
the inositol 1,4,5-trisphosphate (IP3) receptor antagonist
2-aminoethoxydiphenyl borate (2-APB) or (10 μM) RhoA/
Rho kinase inhibitor Y27632 (Table 4). It was also completely
blocked by pre-incubation (15 mins) with (30 μM) Ca2+ chelator,
BAPTA-AM. Ten micromolar concentrations of Y27632 and fifty
micromolar 2-APB not only completely prevented autoantibody
neurite retraction, but they caused significant neurite lengthening
consistent with suppression of basal GPCR, Gαq/11 activation .
Taken together, these data suggest involvement of the PLC-γ/
IP3R/Ca2+ pathway (which positively couples to Gαq/11 and
5-HT-2A receptor) in the mechanism of diabetes autoantibodiesdependent
Figure 4: Effect of GPCR antagonists on diabetic autoantibody-induced
accelerated N2A cell loss. N2A neuroblastoma cell autoantibody- induced
accelerated loss was A) completely prevented by co-incubation
with 200 nM concentrations of M100907, B-C) lack of significant protection
by co-incubation with 200 nM ketanserin(B) or 100 nM prazosin
(C). N2A cells were incubated in the presence of absence of specific
GPCR antagonists for 24 hours at 37 degrees C; cell number was determined
as described in Methods. Results are the mean ± SEM. NS- not
N2A accelerated cell death (after 8 hours incubation) with
autoantibodies from three different diabetic patients was
significantly prevented by co-incubation with (non-toxic) 1 μM
concentrations of the IP3R inhibitor 2-APB (Figure 5C) suggesting
involvement of sustained Ca2+ release in the mechanism of
neuroblastoma cell death.
Figure 5: Effect of 5-HT-2AR agonist (DOI) on N2A survival in the absence
(A-B) or (B) presence of diabetic autoantibodies; C) effect of
2-APB (inositol triphosphate 3 receptor antagonist) on N2A survival in
the presence of diabetic autoantibodies. N2A cells were incubated for
24 hours (A-B) or for 8 hours (C) at 37 degrees C; cell number was determined
as described in Methods. Results are the mean ± SEM.
Effect of diabetic autoantibodies on endothelial cell
M100907 at (200-2000 nM) concentrations completely
protected endothelial cells from cell death induced by diabetic
Parkinson’s disease (10 μg/mL) (Figure 6A) or diabetic
depression autoantibodies (not shown in Figure 6A). Lower
M100907 concentrations (50-100 nM) caused dose-dependent
significant (16-27%) protection (Figure 6B) against diabetic
autoantibodies – induced EC death (n= three different patients).
The autoantibodies (10 μg/mL) in a Parkinson’s disease-sufferer
(PD2), who also had autoimmune thyroid disease and diabetic
nephropathy promoted endothelial cell proliferation (Figure 6C);
and the modest EC proliferation was completely neutralized by
200 nM concentrations of M100907 (Figure 6C). After brief storage
(7 days at 4 degrees C), re-assay of an identical concentration of
the PD2 autoantibodies revealed EC inhibitory activity which was
sensitive to neutralization (22%) by low (25 nM) concentrations
of M100907 (Figure 7A). Substantially higher concentrations of
ketanserin (100 nM) or prazosin (1μM) alone had no significant
effect on the EC inhibitory activity in PD2 autoantibodies (Figure 7B,C).
Figure 6: Effect of highly selective 5-HT-2AR antagonist M100907 on
endothelial cell survival induced by diabetic autoantibodies. M100907
dose-dependently protected against (A,B) endothelial cell survival inhibitory
effect in Parkinson’s disease, pt 1 autoantibodies (10 μg/mL).
C) M100907 (200 nM) completely neutralized EC stimulation from Parkinson’s
disease, pt 2 autoantibodies (10 μg/mL). Bovine pulmonary
artery endothelial cells were incubated in the presence or absence of
Parkinson’s disease autoantibodies for 48 hours at 37 degrees C; cell
number was determined as described in Methods. Results are the mean
Figure 7: Effect of M100907 (A) or other GPCR antagonists (B,C) on
Parkinson’s disease Pt 2 autoantibody-induced inhibition of endothelial
cell survival. Bovine pulmonary artery endothelial cells were incubated
in the presence or absence of Parkinson’s disease, Pt 2 autoantibodies
(10μg/mL) for 48 hours at 37 degrees C; cell number was determined
as described in Methods. Results are the mean ± SEM. NS- not statistically
These data suggest involvement of a 5HT-2 receptor in the
inhibitory effects of diabetic neurovascular autoantibodies on EC
Vascular cells express endothelin A receptor (ET-AR),
angiotensin II, type 1 receptor (AT-1R) and metabotropic
glutamate 5 receptor (mGlu-5R). Saturating concentrations (10
μM) of each of the specific GPCR antagonists: bosentan (ET-AR),
losartan (AT-1R) or CTEP (mGlu-5R) had a modest inhibitory
effect on autoantibody-mediated EC survival (Table 3) consistent
with GPCR cross-talk, but lack of direct involvement of these
receptors in autoantibody-mediated EC survival inhibition.
The present evidence is the first to suggest that autoantibodies
in subsets of diabetes suffering with nephropathy, atrial
fibrillation, morbid obesity, depression, and/or Parkinson’s
disease mediate neural- and endothelial cell- inhibitory effects
via 5-hydroxytryptamine-2 receptors. The 5-hydroxytryptamine
receptor-2A (5-HT2AR) is expressed in neuroblastoma cells
; and 5HT-2AR is the predominant subtype which is highly
concentrated in brain regions involved in perception, mood,
learning and memory, pain, cognition, appetite, and sleep/
wakefulness . The 5HT-2A receptor is a target of atypical
antipsychotic drugs , and anti-depressant medications, such
as amitriptyline , the latter is also useful in the treatment of
painful diabetic neuropathy.
We previously reported highly potent neural- and ECinhibitory
autoantibodies enriched in subsets of diabetes having
co-morbid depression, schizophrenia and/or painful neuropathy
compared to age-matched control diabetic populations lacking
these specific complications (Table 1). The present finding that
three different 5HT-2AR antagonists prevented autoantibodyinduced
acute N2A neurite retraction and displayed an order of
potency (M100907> spiperone> ketanserin) which mirrored their
relative affinities for the 5-HT-2AR  suggests involvement
of 5-HT-2A receptors in diabetic autoantibody-induced neural
The 5-HT-2A receptor couples to a Gα q/11 subclass of
heterotrimeric G proteins in neurons and other cell types .
Following receptor activation, GTP-bound Gαq/11 activates
phospholipase C (PLC-γ) which in turn hydrolyzes PIP2 to yield
inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes
release of Ca2+ from intracellular stores, the latter plays a role
in the activation of protein kinase C. In the current study, specific
inhibitors of phospholipase C (PLC-γ), IP3R and an intracellular
calcium chelator each nearly or completely prevented acute
autoantibody-induced neurite retraction. The selective RhoA/
ROCK inhibitor Y27632 (10 μM) also completed prevented
acute neurite retraction consistent with results in previous
studies [5,6,7,8,9]. The precise mechanism linking PLC/IP3R/
Ca2+ activation with RhoA/ROCK-signaling in N2A cells is not
clear, however, evidence from other laboratories indicates that
ROCK activation can occur through the coordinated action(s) of
activated Gαq/11 and beta (β) - arrestin-1 .
Large increases in intracellular Ca2+ are known to be toxic
to neurons . Substantial protection against accelerated N2A
cell death by nontoxic concentration of 2-APB (1 μM) suggests an
important role for release of Ca2+ from sequestered intracellular
stores in the mechanism of accelerated neuroblastoma cell loss.
A striking feature of autoantibody-induced neurite retraction
was the absence of its spontaneous cessation, e.g. as occurred in
N2A cells exposed to high concentrations of the hallucinogenic
5-HT2A agonist 2,5-dimethoxy-4-iodoamphetamine, (DOI). Beta
arrestin -1 and -2 knock-out-bearing mouse embryonic fibroblasts
displayed ‘enhanced agonism’ at both the angiotensin II receptor
and beta adrenergic receptors  consistent with a general role
for β-arrestins in GPCR- desensitization . The mechanism of
diabetic autoantibodies ‘long-lasting agonism’ at the 5-HT-2AR
is unknown. One possibility (which requires more study) is that
it may interfere with β -arrestin binding to regulatory domains
located in the third intracellular loop of GPCR . Dimeric or
oligomeric forms of IgG autoantibodies might preferentially
target 5-HT-2A receptor heterodimers in the membrane which
(in some cases) are comprised by highly stable non-covalent
interactions between oppositely-charged amino acid residues in
the intracellular domains of physically neighboring GPCRs .
Stable heteromers involving GPCRs positively coupled to Gq/11
heterotrimer G proteins may contribute to sustained activation of
PLC/IP3R/Ca2+ pathway causing long-lasting Ca2+ release (>10
minutes) similar to that which was observed in different subsets
of diabetic autoantibodies .
The 5-hydroxytryptamine-2A receptor can activate multiple,
different signaling pathways depending (in part) on differences in
the chemical structure(s) among particular agonist ligands .
The ability of a given ligand to activate a specific intracellular
signaling pathway downstream of 5-HT-2AR binding has been
referred to as ‘biased agonism’ or ‘functional selectivity .’ In
a prior report in three hundred and five diabetic nephropathy
patients from the VA NEPHRON D randomized clinical trial,
patients harboring less- inhibitory or mildly-stimulatory EC
plasma autoantibodies were at substantially higher risk of
experiencing a composite chronic kidney disease (CKD) endpoint,
(i.e. significant decline in renal function, end-stage renal disease
or death), compared to patients harboring strongly- inhibitory
EC plasma autoantibodies . Here, the protein-A eluate in a
diabetic CKD, Parkinson’s disease patient (i.e. PD2) contained
components which evoked either mild EC stimulation or potent
EC inhibition and unexpectedly, both kinds of responses were
blocked by M100907 suggesting 5-HT-2AR-mediated functional
selectivity. Although the precise make-up of the autoantibody
ligand(s) is unknown, evidence from prior studies suggests that
EC stimulation may have resulted from an immune complex
ligand. Autoantibodies in four additional diabetic nephropathy/
CKD patients evoked initial EC stimulation and upon re-assay
following brief storage, EC inhibition (M. Zimering, unpublished
data). These data suggest that immune complexes in diabetic CKD
protein-A eluates were less stable (in dilute solution) compared
to component IgG. More study is needed to determine whether
diabetic CKD autoantibodies may also induce proliferation in
mesangial cells, via 5-HT-2A receptors coupled to ERK signaling
The pharmacologic profile of antagonism of diabetic
autoantibody-induced accelerated endothelial cell death suggests
possible involvement of additional receptor subtypes besides
5-HT-2A. For example, 100 nM ketanserin did not protect against
autoantibody-induced endothelial cell death despite having low
nanomolar affinity at the 5HT-2A receptor. Since 5-HT-2BR is the
predominant 5HT receptor subtype expressed on pulmonary
artery endothelial cells  and ketanserin has low micro-molar
affinity at the 5-HT-2B receptor , these data suggest possible
involvement of the 5-HT-2B receptor subtype in autoantibody
mediated accelerated endothelial cell death.
Adult neurogenesis in the dentate gyrus has been shown
to correlate with the efficacy of clinical response(s) to various
anti-depressant treatments . The present data showing that
diabetic autoantibodies inhibitory effects on DG NPC cells could
be completely prevented by the selective 5-HT-2AR antagonist
M100907 is of interest. Adult DG neural progenitor cells express
multiple different 5-HT-1 and 5-HT-2 receptor subtypes .
The inhibitory effects of long-lasting 5-HT-2R agonist diabetic
depression autoantibodies are consistent with a report that
chronic (7 days) administration of a 5-HT-2R agonist, α-methyl-
5-HT, suppressed newborn DG neuron survival in adult female
C57Bl/6 mice .
The ontogeny of agonist 5-hydroxytryptamine-2A receptor
autoantibodies is unknown. One possibility is endothelial injury
which releases sequestered auto-antigens, e.g. basic fibroblast
growth factor (bFGF) , whose increased concentration in
plasma correlated with the spontaneous appearance of agonist
FGF-like autoantibodies in various pathologies including cancer
subsets [11, 30] and obese type 2 diabetes . Vascular
injury also causes platelets to release serotonin together with
polyphosphates, the latter of which, like heparin, is a highly
anionic substance capable of binding and protecting (locallyreleased)
bFGF from inactivation . Since platelet activation
increases in obesity and inflammation , it is possible that
repeated vascular injury through the release of sequestered
antigens promotes immune response(s) (in susceptible
individuals) leading to the formation of agonist 5-HT-2A receptor
autoantibodies. Kidney was reported to have among the highest
5-HT-2R mRNA expression levels among peripheral organs .
Thus it is possible that 5-HT-2R released through ongoing renal
endothelial damage (e.g. in diabetic nephropathy populations)
may contribute (in part) to the development of agonist 5-HT-2
In summary, diabetic autoantibodies from patients suffering
with nephropathy, and/or depression, atrial fibrillation, painful
neuropathy or another neurodegenerative disease caused
N2A neurite retraction and accelerated neuron loss through a
mechanism involving 5-HT-2A receptors positively coupled to
the PLC/IP3/Ca2+ pathway. These findings have relevance for
understanding possible role(s) for agonist 5-hydroxytryptamine-
2A receptor autoantibodies in pathophysiology in diabetesrelated
complications affecting brain regions and peripheral
organs in which the 5-HT-2 receptor is highly concentrated.
Dr. Janet Alder, Dr. Smita Thakker-Varia, and Shavonne Teng
(Department of Neuroscience & Cell Biology, Rutgers-Robert
Wood Johnson Medical School, Piscataway, New Jersey) for
providing the mouse N2A neuroblastoma and rat dentate gyrus
neural progenitor cells used in these experiments. Veterans
Biomedical Research Institute (East Orange, New Jersey) for
grant support to MBZ.
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