GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism.
ABSTRACT Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet beta-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first of this new class of antihyperglycemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. We detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine-induced cell death, respectively. We found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Our findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Since the initial observation that a calorie-restricted (CR) diet can extend rodent lifespan, many genetic and pharmaceutical interventions that also extend lifespan in mammals have been discovered. The mechanism by which CR and these other interventions extend lifespan is the subject of significant debate and research. One proposed mechanism is that CR promotes longevity by increasing insulin sensitivity, but recent findings that dissociate longevity and insulin sensitivity cast doubt on this hypothesis. These findings can be reconciled if longevity is promoted not via increased insulin sensitivity, but instead via decreased PI3K/Akt/mTOR pathway signaling. This review presents a unifying hypothesis that explains the lifespan-extending effects of a variety of genetic mutations and pharmaceutical interventions and points towards new molecular pathways which may also be leveraged to promote healthy aging.SpringerPlus 01/2014; 3:735.
- [Show abstract] [Hide abstract]
ABSTRACT: Although the human brain is exceptional in size and information processing capabilities, it is similar to other mammals with regard to the factors that promote its optimal performance. Three such factors are the challenges of physical exercise, food deprivation/fasting, and social/intellectual engagement. Because it evolved, in part, for success in seeking and acquiring food, the brain functions best when the individual is hungry and physically active, as typified by the hungry lion stalking and chasing its prey. Indeed, studies of animal models and human subjects demonstrate robust beneficial effects of regular exercise and intermittent energy restriction/fasting on cognitive function and mood, particularly in the contexts of aging and associated neurodegenerative disorders. Unfortunately, the agricultural revolution and the invention of effort-sparing technologies have resulted in a dramatic reduction or elimination of vigorous exercise and fasting, leaving only intellectual challenges to bolster brain function. In addition to disengaging beneficial adaptive responses in the brain, sedentary overindulgent lifestyles promote obesity, diabetes and cardiovascular disease, all of which may increase the risk of cognitive impairment and Alzheimer's disease. It is therefore important to embrace the reality of the requirements for exercise, intermittent fasting and critical thinking for optimal brain health throughout life, and to recognize the dire consequences for our aging population of failing to implement such brain-healthy lifestyles.Ageing Research Reviews. 01/2015;
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ABSTRACT: Friedreich's ataxia is a neurodegenerative disorder associated with cardiomyopathy and diabetes. Effective therapies for Friedreich's ataxia are an urgent unmet need; there are currently no options to prevent or treat this orphan disease. Friedreich's ataxia is caused by reduced expression of the mitochondrial protein frataxin. We have previously demonstrated that pancreatic β-cell dysfunction and death cause diabetes in Friedreich's ataxia. This is secondary to mitochondrial dysfunction and apoptosis but the underlying molecular mechanisms are not known. Here we show that β-cell demise in frataxin deficiency is the consequence of oxidative stress-mediated activation of the intrinsic pathway of apoptosis. The pro-apoptotic Bcl-2 family members Bad, DP5 and Bim are the key mediators of frataxin deficiency-induced β-cell death. Importantly, the intrinsic pathway of apoptosis is also activated in Friedreich's ataxia patients-induced pluripotent stem cell-derived neurons. Interestingly, cAMP induction normalizes mitochondrial oxidative status and fully prevents activation of the intrinsic pathway of apoptosis in frataxin-deficient β-cells and neurons. This preclinical study suggests that incretin analogs hold potential to prevent/delay both diabetes and neurodegeneration in Friedreich's ataxia. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org.Human Molecular Genetics 12/2014; · 6.68 Impact Factor
GLP-1 receptor stimulation preserves primary cortical
and dopaminergic neurons in cellular and rodent
models of stroke and Parkinsonism
Yazhou Lia, TracyAnn Perrya, Mark S. Kindyb,c, Brandon K. Harveyd, David Tweediea, Harold W. Hollowaya,
Kathleen Powersd, Hui Shend, Josephine M. Egane, Kumar Sambamurtib, Arnold Brossif, Debomoy K. Lahirig,
Mark P. Mattsona, Barry J. Hofferh, Yun Wangd, and Nigel H. Greiga,1
aLaboratory of Neurosciences, Intramural Research Program, National Institute on Aging, Baltimore, MD 21224;bDepartment of Neuroscience, Medical
University of South Carolina, Charleston, SC 29425;cNeurological Testing Services, Mount Pleasant, SC 29466;dMolecular Neuropsychiatry Branch, Intramural
Research Program, National Institute on Drug Abuse, Baltimore, MD 21224;eLaboratory of Clinical Investigation, Intramural Research Program, National
Institute on Aging, Baltimore, MD 21224;fSchool of Pharmacy, University of North Carolina, Chapel Hill, NC 27599;gDepartment of Psychiatry, Indiana
University School of Medicine, Indianapolis, IN 46202; andhCellular Neurobiology Branch, Intramural Research Program, National Institute on Drug Abuse,
Baltimore, MD 21224
Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved December 5, 2008 (received for review
July 24, 2008)
Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic
intake. It enhances pancreatic islet ?-cell proliferation and glucose-
dependent insulin secretion, and lowers blood glucose and food
intake in patients with type 2 diabetes mellitus (T2DM). A long-
acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first
of this new class of antihyperglycemia drugs approved to treat
T2DM. GLP-1Rs are coupled to the cAMP second messenger path-
way and, along with pancreatic cells, are expressed within the
nervous system of rodents and humans, where receptor activation
elicits neurotrophic actions. We detected GLP-1R mRNA expression
in both cultured embryonic primary cerebral cortical and ventral
mesencephalic (dopaminergic) neurons. These cells are vulnerable
to hypoxia- and 6-hydroxydopamine–induced cell death, respec-
tively. We found that GLP-1 and Ex-4 conferred protection in these
cells, but not in cells from Glp1r knockout (-/-) mice. Administration
of Ex-4 reduced brain damage and improved functional outcome in
a transient middle cerebral artery occlusion stroke model. Ex-4
treatment also protected dopaminergic neurons against degener-
ation, preserved dopamine levels, and improved motor function in
the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse
model of Parkinson’s disease (PD). Our findings demonstrate that
Ex-4 can protect neurons against metabolic and oxidative insults,
and they provide preclinical support for the therapeutic potential
for Ex-4 in the treatment of stroke and PD.
diabetes ? exendin-4 ? neurodegeneration ? neuroprotection ? stroke
adult population now affected (1). Although T2DM now occurs
more often in the young, the incidence rises dramatically with
age, along with that of many of other conditions, including acute
and chronic neurologic disorders, exemplified by stroke (2),
Parkinson’s disease (PD) and Alzheimer’s disease (AD) (3,4),
which, like T2DM, were once considered relatively infrequent.
Indeed, the incidence of stroke, PD, AD, and several other
suggesting that shared mechanisms, such as insulin dysregula-
tion, may underlie these conditions (5). Although associated
with different cell types in divergent areas (e.g., cortical and
striatal neurons in stroke, substantia nigral and midbrain dopa-
minergic neurons in PD, pancreatic ?-cells in T2DM), parallel
biochemical cascades are triggered by specific environmental
and genetic signals and lead to the cellular dysfunction and death
characteristic of all of these disorders. Consequently, it is
possible that an effective treatment strategy for one such disor-
der may prove beneficial in others as well.
ype 2 diabetes mellitus (T2DM) is emerging as one of the
largest health issues worldwide; with some 6% of the world’s
The glucagon-like peptide-1 receptor (GLP-1R) agonist, ex-
endin-4 (Ex-4), is a long-acting analog of the endogenous
insulinotropic peptide GLP-1 (supporting information (SI) Fig.
S1). GLP-1 is derived from the posttranslational modification of
in response to food ingestion (6,7). GLP-1 and Ex-4 have potent
effects on glucose-dependent insulin secretion and insulin gene
expression through binding and activation of the G protein–
coupled GLP-1R on pancreatic ?-cells. Both peptides also have
trophic properties, inducing pancreatic ?-cell proliferation and
inhibiting apoptosis (7,8). Ex-4 has been approved for the
treatment of T2DM, in which it has been found to effectively
lower plasma glucose levels.
GLP-1R mRNA occurs widely throughout the brains of
rodents (9) and humans (6,7,10), and both GLP-1 and Ex-4 can
readily enter the brain (11) to modify feeding and satiety (12).
We have previously reported that the activation of GLP-1R by
GLP-1 and Ex-4 is neurotrophic, inducing neurite outgrowth in
PC12 cells and protecting neurons against various insults (6,13–
15) through a cascade involving the second messenger, cAMP
(13). In light of these neurotrophic actions, the long-term
efficacy of Ex-4 in treating T2DM (7), and the elevated risk of
cerebrovascular disease and PD in T2DM (1–3,5), we evaluated
GLP-1R stimulation in well-characterized cellular and animal
GLP-1R Is Expressed and Functional in Cultured Embryonic Primary
Neurons. To establish the presence of GLP-1R in primary neurons,
cultured rat embryonic cerebral cortical (CC) and ventral mesen-
cephalic (VM) cells were probed for the presence of GLP-1R
mRNA by RT-PCR. Both neuron types were found to contain
GLP-1R mRNA (Fig. 1A). Incubation of cortical neurons with the
natural agonist GLP-1 (10 nM) led to a rapid, transient elevation
of intracellular cAMP level. This level peaked within 15 min and
then returned toward baseline by 30 min (Fig. 1B), demonstrating
Author contributions: Y.L., T.P., M.S.K., B.K.H., D.T., H.W.H., K.P., H.S., D.L., and Y.W.
data; T.P., J.E., K.S., M.P.M., B.H., Y.W., and N.H.G. designed research; K.S. and A.B.
contributed new reagents/analytic tools; and M.P.M., B.H., and N.H.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
January 27, 2009 ?
vol. 106 ?
no. 4 ?
protein levels, along with increased intracellular cAMP levels, in
response to Ex-4 (not shown).
GLP-1R Stimulation Decreased Hypoxia- and Dopaminergic Toxin–
are vulnerable to hypoxia, resulting in a loss of viability, as
assessed by a significant elevation in lactate dehydrogenase
(LDH) level (Fig. 1C) and a decline in (3-(4,5-dimethylthiazol-
tetrazolium, inner salt) (MTS) level (not shown) compared with
cells subjected to normoxia. Incubation with a GLP-1R agonist,
GLP-1 or Ex-4 (0.01–1 ?M), afforded significant protection
against hypoxia, lowering elevated LDH levels by as much as
76%. This effect was lost in the presence of the GLP-1R
antagonist Ex-9–39, indicating that the action was mediated
through GLP-1R. To confirm this, CC neurons from Glp1r?/?
mice were similarly cultured and exposed to hypoxia/normoxia
in the presence and absence of Ex-4 and the GLP-1R antagonist.
Glp1r?/?primary CC neurons were similarly vulnerable to
hypoxia but were not protected by Ex-4 (Fig. 1D).
The viability of mesencephalic cell cultures, known to be rich
in dopaminergic neurons, was determined by quantifying ty-
the dopaminergic toxin 6-hydroxydopamine (6-OHDA). As ex-
pected, 6-OHDA decreased TH(IR) significantly, by 30% (Fig.
2A). GLP-1 and Ex-4 (0.1 ?M) fully preserved TH(IR) from
6-OHDA toxicity, and, moreover, Ex-4 elevated TH(IR) in the
absence of 6-OHDA by an additional 60%. No significant
difference in the number of DAPI-positive nuclei was found
among the treatment groups (not shown). To elucidate the
universality of these protective effects, parallel studies were
performed in SH-SY5Y cells (Fig. 2B–D). Predictably, exposure
to 6-OHDA significantly reduced cell viability (Fig. 2B), with
elevations in caspase-3 activity and Bax and declines in Bcl-2
found by Western blot analysis (Fig. 2C and D). GLP-1 and Ex-4
resulted in elevated Bcl-2 and negligible caspase-3 and Bax
levels. To define the molecular pathways responsible for the
GLP-1R–mediated protection, specific inhibitors of PKA (H89;
10 ?M) and PI3K (LY294002; 10 ?M) were investigated; these
resulted in a loss of protection (Fig. 2D).
Ex-4 Treatment Reduces Infarction Size and Improves Functional
Outcome in Stroke.Todefinethetranslationalpotentialofourcell
culture studies, the protective effect of Ex-4 was evaluated in a
cell cultures. The expected RT-PCR product size is 190 bp. GAPDH was used as
an external control and showed equal expression across lanes. Lane 1, nega-
tive control; lane 2, positive control: RNA from CHO-GLP-1R cells (CHO cells
stably transfected with rat GLP-1R); lanes 3 and 4, RNA from primary CC and
VM neurons, respectively. (B) GLP-1–stimulated release of cAMP from CC
neurons. Time-dependent cAMP levels were assayed after incubation with 10
nM GLP-1 (n ? 3). (C) Pretreatment with GLP-1/Ex-4 protects CC neurons from
hypoxia-induced loss of cell viability, as indicated by elevated levels of se-
creted LDH. Compared with normoxia (21% O2, 5% CO2), a 3-h exposure to
hypoxia (1% O2, 5% CO2) induced a significant elevation in LDH (P ? .05),
defined as a 100% response. GLP-1 and Ex-4 (0.01–1.0 ?M) protected cells,
ameliorating the hypoxia-induced elevation in LDH by up to 76%. This effect
was abolished by the GLP-1R antagonist Ex-9–39. n ? 5 for each treatment,
(D) CC neurons from Glp1?/?mice are vulnerable to hypoxia, as assessed by
.05 vs. hypoxia; 1-way ANOVA plus posthoc Dunnett’s test, n ? 5).
(A) One-step RT-PCR shows rat GLP-1R mRNA expression in neuronal
neurons from 6-OHDA treatment and likewise protects SH-SY5Y cells from
6-OHDA–induced cell death. (A) TH(IR) of primary VM cells pretreated with
TH(IR) was significantly different versus PBS-treated controls (P ? .05; Dun-
with vehicle (Veh), GLP-1, or Ex-4 (0.1 u?M) for 2 h and then subjected to
6-OHDA (30 ?M) for 24 h. Subsequently, cell survival was quantified by MTS
assay. Whereas 6-OHDA reduced cell survival to 83% (*P ? .05 vs. control),
GLP-1 and Ex-4 protected against this 6-OHDA loss of cell viability (*P ? .05 vs.
markers of apoptosis were elevated by 6-OHDA (30 ?M) and lowered by Ex-4
inhibitors of PKA (H89; 10 ?M) or PI3K (LY294002; 10 ?M) and was retained
with insulin (0.01 ?M; positive control) (*P ? .05, Dunnett’s t-test, n ? 5 vs.
vehicle plus 6-OHDA).
Pretreatment with GLP-1 or Ex-4 protects TH(IR) of VM primary
www.pnas.org?cgi?doi?10.1073?pnas.0806720106Li et al.
well-characterized rodent model of stroke, middle cerebral
artery occlusion (MCAo), which mimics the most common type
of human stroke. A 1-h transient occlusion produced a well-
demarked area of infarction that, as assessed by triphenyltetra-
zolium chloride (TTC) staining at 48 h, spanned the right
frontal, parietal, and occipital cerebral cortices (Fig. 3A). The
infarct volume, assessed by measuring the number of 2-mm-thick
brain slices affected and the infarct area, was reduced by ? 50%
in Ex-4–pretreated rats compared with controls. Ex-4 signifi-
cantly reduced each measured parameter of infarction size (Fig.
3B–D) and was accompanied by improved functional outcome,
as assessed by locomotor activity measures at 2 days (Fig. 3E).
Because changes in body temperature, blood pressure, and
arterial blood gases may affect the outcome of stroke, these
parameters were measured in Ex-4–treated and control rats both
before and after treatment (Table S1); no significant changes
were found. Likewise, cerebral blood flow remained unchanged
before, during, and after Ex-4 administration (Fig. S2). Ex-4’s
lack of effects on these parameters suggests that its beneficial
effects in stroke are due primarily to its central actions. To
confirm that these actions are mediated through GLP-1R,
parallel studies were performed in wild-type (WT) and Glp1r?/?
mice. Ex-4 was found to afford protection in the WT mice, but
not in the Glp1r?/?mice (Figs. 3F and S3).
Ex-4 Treatment Preserves Dopaminergic Cells in a MPTP-Induced PD
Model. The neuroprotective actions of Ex-4 were quantified in a
well-characterized model of PD. Exposure to 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) induces a PD-like syndrome
in humans, monkeys, and mice. In the brain, MPTP is converted
to MPP?, which is selectively transported into dopaminergic
neuron axon terminals, causing oxidative stress, mitochondrial
dysfunction, and cell death (16). Analyses of dopaminergic
markers in mice given MPTP demonstrated a cell loss that
culminated in motor function impairment. Ex-4 afforded com-
plete protection against dopaminergic neuron damage and mo-
tor impairment. Specifically, compared with controls, MPTP
significantly reduced the number of TH-immunopositive (?)
neurons within the substantia nigra (SN) by 63%, as assessed by
immunohistochemistry (Fig. 4A and B), and depleted TH(?)
intensity by 71%, as assessed by immunoblotting (Fig. 4C). In
parallel, levels of dopamine (DA) and metabolites [dihydroxy-
reduced dramatically, and the ratio of metabolites to DA con-
centration was elevated, consequent to MPTP (Fig. 4D). In
contrast, mice given Ex-4 before MPTP showed no differences
from controls in terms of the number and intensity of SN TH(?)
At 48 h after ischemia/reperfusion, rats were killed, the brain was sliced into
2-mm sections, and stained with TTC. Marked infarction (white areas) within
the right cerebral cortex was found. The size of infarction was significantly
decreased in animals treated with Ex-4, compared to vehicle (n ? 10/group),
with regard to (B) the volume of infarction ? [sum of the infarction area in all
brain slices (mm2)] ? [slice thickness, 2 mm], (C) the area of the largest
48 h after ischemia/reperfusion in an activity chamber. Vertical activity
(VACTV) and vertical movement time (VTIME) were determined from the
number of beam interruptions that occurred in vertical sensors and the time
(s) spent in vertical movement during a 30-min test, respectively. (F) Likewise,
Ex-4 (1 ?M ? 5 ?L left lateral ventricle) decreased infarct volume in the WT
mice but was ineffective in the Glp1r?/?mice (WT control, n ? 6; WT Ex4, n ?
and Student’s t-test.
were protected from MPTP-induced damage of the dopaminergic system,
quantitatively assessed by TH immunohistochemical analysis of the SN and TH
immunoblot analyses of the striatum at 7 days. (A) Representative SN sections
from control, and MPTP-treated mice with and without Ex-4. (B) Compared
with controls, TH(?) cells in SN were reduced by MPTP (*P ? .05). Those from
mice given Ex-4 and MPTP were no different from controls (P ? .05). (C)
Similarly, as assessed by immunoblotting in striatum, TH levels were signifi-
cantly reduced by MPTP (*P ? .05 vs. controls) and no different from controls
given Ex-4 were protected from MPTP-induced depletion of brain DA and
metabolites (DOPAC and HVA). DA, DOPAC, and HVA from striatum were
quantified by HPLC at 7 days in mice treated with PBS, MPTP, and MPTP plus
Ex-4. Levels of each were reduced by MPTP (P ? .05 vs. PBS) and preserved by
Ex-4 (P ? .05 vs. PBS; P ? .05 vs. MPTP) compared with controls (Dunnett’s
t-test, n ? 10). The ratios of DOPAC:DA and HVA:DA were 0.08 and 0.065 in
Mice given Ex-4 (20 nM, 0.25 ?L/h in the lateral ventricle over 7 days)
Li et al.
January 27, 2009 ?
vol. 106 ?
no. 4 ?
neurons, as assessed by immunoblotting, and DA and metabolite
levels and ratios. Motor function was quantified by several
paradigms over multiple days, including mean score of behavior,
rotarod, pole test (Fig. 5), beam walk, and open-field activity
(Fig. S4); performance in all animals was significantly impaired
by MPTP. In contrast, motor function was fully preserved after
Ex-4 treatment and for all paradigms was similar to that of
controls not treated with MPTP.
The risk of both stroke and PD is elevated in persons with T2DM
(17,18), even in newly treated patients, in whom the short-term
risk of stroke is doubled (17). Clearly, an effective neuropro-
tective strategy would be valuable for this vulnerable patient
group, as well as for the general population, given the lack of
effective treatments for stroke and PD. Increasing evidence
suggests that cortical and dopaminergic neurons die through
apoptosis after a stroke and through a related form of pro-
grammed cell death during PD (19). Evidence for classic apo-
ptosis in both conditions includes elevated levels of the apoptotic
ptotic genes and proteins (19,21,22), as was evident in our cell
culture studies. Analogous elevations in markers of apoptosis
have been described in pancreatic ?-cells during T2DM (7,23),
one of many commonalities shared by these degenerative con-
ditions. The ability to initiate a degenerative process in different
cell types by widely varying insults suggests the existence of a
common cell death network that can be entered from different
points but, once activated, follows similar interrelated biochem-
ical pathways, with little dependence on the site of entry (22). In
such a system, a strategy that effectively halts the death network
process in one disease, such as T2DM, may hold promise for
another, particularly when the molecular machineries underpin-
ning this action share commonalities.
The incretin, GLP-1, and long-acting Ex-4 induce numerous
biological actions in the pancreas, including stimulation of
glucose-dependent insulin secretion, elevated insulin synthesis,
decreased glucagon levels, and, notably, ?-cell proliferation and
inhibition of ?-cell apoptosis (7,8). These and other actions are
mediated through the G protein-coupled GLP-1R, and Ex-4 has
demonstrated therapeutic value in T2DM (7). GLP-1R is a
member of the class B family of 7-transmembrane-spanning,
heterotrimeric G protein-coupled receptors. In humans and
rodents, a single structurally identical GLP-1R has been iden-
tified that is expressed in a wide range of tissues, including the
brain. GLP-1–immunoreactive fibers and GLP-1Rs are widely
expressed throughout the brain (6,9,10). Ligand activation of the
G? subunit of GLP-1R on pancreatic ?-cells leads to activation
of adenylate cyclase activity and production of cAMP, the
primary mediator of GLP-1R activation (7).
GLP-1Rs are present in rodent cultured CC and VM primary
cells. Adding GLP-1 to primary neurons induced a time-dependent
elevation in cAMP, indicative of a functional receptor. cAMP-
mediated pathways are central to the antiapoptotic actions of
GLP-1 in ?-cells (6–8), and the neuroprotective effects of cAMP-
elevating agents are seen in many neuronal cells, including sensory
(24), dopaminergic (25), septal cholinergic (26), cerebellar granule
(27,28), and spinal cord motor neurons (29).
Our previous studies have established that a 50% GLP-1R
occupancy in primary neurons is achieved by 14 nM GLP-1 (14), a
value similar to that for ?-cells. Here we show that administration
of GLP-1 and Ex-4 to primary CC and VM neurons or SH-SY5Y
cells proved to be neuroprotective against insults that modeled
stroke and PD. Specifically, these cells were vulnerable to hypoxia
and a dopaminergic toxin, as assessed by classic markers of cell
viability and the presence of cell death markers. GLP-1 and Ex-4
concentrations as low as 10 nM conferred protection against
hypoxia. This effect was lost in the presence of the GLP-1R
antagonist Ex-9–39 and was absent in Glp1r?/?neurons, indicating
that it is GLP-1R–mediated. Interestingly, not only were VM
neurons fully protected from 6-OHDA–induced toxicity by GLP-1
and Ex-4 (100 nM), but also Ex-4 substantially elevated TH(IR)
beyond that of untreated controls (Fig. 2A), indicating both neu-
rotrophic and neuroprotective activity. TH also is expressed in
catecholamine neurons in the area postrema, and Ex-4 has been
shown to significantly elevate TH levels in these neurons by
contains a cAMP-responsive element (31), representing a further
modulatory mechanism that may account in part for the Ex-4–
induced rise in TH(IR).
These neurotrophic/protective actions are in accordance with
previous findings establishing that GLP-1R stimulation protects
hippocampal neurons from amyloid-? peptide–, Fe2?-, and
glutamate-induced toxicity (15,32,33). The pathways that under-
pin the antiapoptotic actions of many endogenous neuroprotec-
tive agents commonly converge on activation of the transcription
factor cAMP response element–binding protein by phosphory-
lation. Those mediating GLP-1’s antiapoptotic actions in neu-
rons remain to be fully elucidated. Previous work has demon-
strated a clear involvement of PKA; neuroprotection by GLP-1
was abolished by Rp-cAMP, which blocks cAMP activation of
PKA (13). PI3K and MAPK are other important signaling
pathways involved in GLP-1–mediated events. A selective inhib-
itor of the former (LY294002), but not of the latter (PD98059),
has been reported to inhibit GLP-1–mediated protective effects
in neuronal cells (13). In the present study, each of these
pathways appeared to contribute to the protection afforded by
Ex-4 and GLP-1 to SH-SY5Y cells (Fig. 2D). Potential GLP-1
actions mediated through MAPK-independent signaling and
growth factor–dependent Ser/Thr kinase AktPKB have been
reviewed recently (31–33).
Administration of Ex-4 (10 ?g s.c) achieved plasma levels of
200 pg/mL (48 nM) in humans (34), which compare favorably to
the doses studied here. To evaluate the translational relevance
of the aformentioned cellular effects, the actions of centrally
administered Ex-4 were assessed in classical rodent models of
stroke and PD. Whereas Ex-4 and GLP-1 readily enter the brain
has behavioral consequences. (A) Rotarod: The ability of mice to remain on a
rotating rod at 7 days was reduced (67%; P ? .05 vs. PBS) by MPTP and
preserved by Ex-4 (P ? .05 vs. PBS; P ? .05 vs. MPTP). (B) Pole test: Assessed on
2 consecutive days, initially 3 h after MPTP. The time taken for mice to turn
around (T-Turn) and descend a pole (T-Total) was slower in the MPTP-treated
mice (P ? .05 vs. PBS and Ex-4 plus MPTP) and no different from PBS controls
(P ? .05) in the MPTP plus Ex-4 mice. (C) Mean score of behavior: A composite
of tests were rated daily. Whereas the MPTP plus Ex-4 mice were no different
than the PBS controls, the MPTP mice could be differentiated on and after 7
days (*P ? .05 vs. PBS, Dunnett’s t-test, n ? 10/group).
Ex-4 protection of MPTP-induced toxicity of dopaminergic neurons
www.pnas.org?cgi?doi?10.1073?pnas.0806720106 Li et al.
after systemic administration (11), and Ex-4 given by this route
has proven effective in alleviating peripheral neuropathy in
rodents (35), direct administration into the brain allowed dif-
ferentiation of centrally mediated GLP-1R actions from numer-
ous systemic ones. Ischemic brain injury activates apoptotic
cascades within the ischemic core and penumbra that peak on
day 2 after MCAo. p53 mRNA and protein are up-regulated
shortly after stroke, leading to p53-dependent programmed cell
death in penumbra (36). Administration of Ex-4 substantially
decreased infarct size, as assessed by 3 related measures of TTC
staining at 48 h in rats (Fig. 3A and B). The reduced stroke
volume (?50%) was similar to that achieved by inhibition of
p53-dependent apoptosis (37), suggesting protection from apo-
ptotic rather than necrotic cell death and translating to signifi-
cant improvements in measures of motor activity. Blood flow, as
well as a wide number of physiological parameters (Table S1)
that can influence ischemic damage, remained unchanged by
Ex-4 administration, supporting a direct central GLP-1R–
mediated effect. Parallel studies in WT mice confirm that the
neuroprotective actions of Ex-4 in MCAo translate across spe-
cies, and the loss of this action in Glp1r?/?mice reaffirm that
neuroprotection is mediated through GLP-1Rs.
Administration of MPTP in mice induces a consistent dopami-
nergic cell loss that parallels many aspects of PD (16,20). In the
present study, the mice receiving MPTP demonstrated classic
reductions in both the number of TH-immunoreactive cells, a
marker of dopaminergic cells in the SN (63% loss), and of TH
intensity in immunoblot analyses of striatum (71% loss). These
animals demonstrated motor function deficits. General behavioral
assessment, combining multiple paradigms, detected differences
between the MPTP and control animals starting at 7 days after
MPTP administration and increasing with time. Specific tests of
motor function (i.e., pole test, beam traverse, open-field activity,
and rotarod) confirmed MPTP-induced impairment. To correlate
reductions in dopaminergic cells with motor function losses, con-
a 75% drop in HVA level, was evident, in line with the results of
previous MPTP studies (20). These declines resulted in a 2- to
4D). Ex-4 provided complete protection, as assessed by quantifi-
cation of TH(?) cell number, TH immunoblot analysis results, DA
and metabolite levels and ratios, and all behavioral paradigms
studied. Overall, the MPTP mice treated with Ex-4 were indistin-
guishable from controls.
Our findings indicate that the neuroprotective actions of
GLP-1R agonists appear to effectively translate across a number
of classic cellular and animal models, including stroke and PD,
as well as cholinergic ablation (14), kainic acid–induced CA3
hippocampal loss (38), and peripheral neuropathy (35). In
contrast, the Glp1r?/?mice demonstrated impaired learning, as
well as increased brain injury and associated behaviors after a
lesion (32). Together, the findings of these studies suggest a loss
of function in -/- mice and a physiological role for GLP-1R
activation in the normal brain, as in the pancreas, that can be
augmented by pharmacologic concentrations of agonists and
inhibited by antagonists. Recent studies have demonstrated that
Ex-4 can induce neurogenesis of neural stem cells both in culture
(38,39) and promote differentiation toward a neuronal pheno-
type (38), as has been reported for PC12 cells (13). Ex-4’s ability
to improve dopaminergic markers and function when adminis-
tered a week or more after 6-OHDA– or cytokine-induced
apoptosis, rather than at the time of insult as in our study, is
indicative of neuroregenerative action (38,39).
In synopsis, the role of GLP-1R stimulation in balancing cell
survival versus death in pancreatic cells is well established (7)
and, together with the insulinotropic actions of Ex-4, supports its
clinical utility in T2DM. Likewise, the parallel GLP-1R–
mediated trophic and protective actions of Ex-4 in neurons may
be of clinical utility in acute and chronic neurologic disorders,
epitomized by stroke and PD. Not only are persons with T2DM
at increased risk for stroke and PD, but also several studies have
reported a high prevalence of insulin resistance in PD, vascular
dementia, and other neurodegenerative conditions (2,4,17), with
impaired glucose tolerance seen in 50%–80% of subjects (4,40).
Dopaminergic neurons and insulin receptors are both densely
localized within the SN, and dopaminergic agents used in PD
(e.g, L-DOPA) have been reported to induce hyperglycemia
(40). Together, these findings suggest that GLP-1R agonists may
exert various useful actions in persons at high risk for stroke or
with PD, a hypothesis that is amenable to clinical testing.
Materials and Methods
cells were probed for GLP-1R mRNA and stimulated with GLP-1 (10 nM) to
assess the presence and functionality of GLP-1R. CC cultures were challenged
with transient hypoxia (1% O2, 5% CO2, 37 °C, 3 h) followed by normoxia
(21%O2, 5% CO2, 37 °C, 48 h). VM cultures were exposed to 6-OHDA (30 ?M,
90 min), in the presence and absence of GLP-1 and Ex-4 (10 nM–1.0 ?M), and
studies by MTS (Promega) or LDH (Sigma) assays and in 6-OHDA studies by TH
Some studies used primary neurons from Glp1r?/?mice, and others used
assessed by Western blot analysis as described previously (20,41,42).
Animal Studies. Stroke (MCAo) Model. At 15 min after left lateral ventricle
administration of Ex-4 (1 ?M ? 20 ?L; 83 ng) or vehicle (PBS), adult male
monitored before, during, and after MCAo. Motor function, assessed in a loco-
at 48 h (SI Materials and Methods). Likewise, transient (90 min) MCAo was
performed in adult male WT and Glp1r?/?ICR mice 15 min after left lateral
size determined at 48 h (SI Materials and Methods).
PD (MPTP) Model. At 2 h after left lateral ventricle administration of Ex-4 (20
given the dopaminergic toxin MPTP (20 mg/kg in 0.1 mL of PBS i.p. at 2-h
throughout the SN were processed for immunostaining using TH antibody
(T-1299; Sigma), and TH(?) cells were quantified by image analysis. Levels of
DA, DOPAC, and HVA were measured by HPLC from striatum, and TH immu-
noblotting was performed using a TH (phospho S40) antibody (AbCam) (20).
behaviors, rotarod, pole test, beam walk, and open-field activity as described
previously (44,45) (SI Materials and Methods).
Statistics Dunnett’s t-test and 1-way ANOVA with Student-Newman-Keul
(SNK) posthoc analysis were used for statistical comparison, with P ? .05
considered statistically significant. Data are presented as mean ? SEM.
ACKNOWLEDGMENTS. This work was supported in part by the Intramural
Research Programs of the National Institute on Aging and the National
Institute on Drug Abuse. Animal studies were performed in accordance with
approved protocols, in compliance with the National Institutes of Health’s
Guidelines for Animal Experimentation. Daniel J. Drucker, MD, University of
Toronto, kindly provided the Glp1r?/?mice.
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