Download Reserpine ameliorates Aβ toxicity in the Alzheimer`s disease model

Experimental Gerontology 44 (2009) 462–466
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Experimental Gerontology
journal homepage: www.elsevier.com/locate/expgero
Short Report
Reserpine ameliorates Ab toxicity in the Alzheimer’s disease model
in Caenorhabditis elegans
Upasna Arya, Hemalata Dwivedi, Jamuna R. Subramaniam *
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India
a r t i c l e
i n f o
Article history:
Received 1 December 2008
Received in revised form 17 February 2009
Accepted 20 February 2009
Available online 3 March 2009
Keywords:
Ab
Reserpine
Delay in paralysis
Lifespan extension
Movement
a b s t r a c t
Earlier we have reported that reserpine, an antihypertensive drug, known to downregulate biogenic
amines through inhibition of the vesicular monoamine transporter (VMAT), increases longevity of Caenorhabditis elegans with a high quality of life, namely, enhanced and prolonged mobility (Srivastava et al.,
2008). As neurodegenerative diseases are of adult onset, we addressed the protective ability of reserpine
against neurodegenerative diseases, especially Alzheimer’s disease (AD). In the well established AD
model in C. elegans, Amyloid b (Ab) is expressed in the muscles and Fb toxicity is manifested as paralysis
(Link, 1995). In this model, reserpine significantly delayed paralysis and increased the longevity. In addition, reserpine provided thermotolerance, but interestingly the Fb transcript and expression levels
remains grossly unchanged.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
Neurodegenerative diseases lead to progressive and gradual
loss of neurons that severely compromise the function of nervous
system. Alzheimer’s disease, AD, is one of the most common form
of neurodegenerative disease condition and leading cause of progressive dementia followed by other complications in the older
people worldwide. AD is highly prevalent and is predicted to become a huge burden on societies. It is characterized by the deposition of toxic Ab deposits and intraneuronal tau tangles in the brain
(Goedert and Spillantini, 2006; Taylor et al., 2002). To date, there is
no cure for this devastating disease, with limited protection being
provided by acetylcholinesterase inhibitors and memantine (Klafki
et al., 2006). Hence, there exists, the dire need of pharmacological
agents that can serve as a potential therapy for AD. To this effect,
the available in vivo experimental model system for the Alzheimer
disease in mice (Zheng et al., 1995), Drosophila (Chan and Bonini,
2000) and Caenorhabditis elegans (Link, 1995; Wu et al., 2006; reviewed by Link, 2001, 2006) serve both as a drug discovery tool
(Wu and Luo, 2005) and for deciphering the incompletely understood disease mechanism.
The established model of AD in C. elegans that expresses the human
toxic Ab species namely Amyloid beta 1–42 in the muscles (Link,
1995) have greatly aided in understanding the possible mechanism
of AD pathology. This model has helped to identify the nexus between
lifespan extension pathways and proteotoxicity in neurodegenerative
* Corresponding author. Tel.: +91 512 2594042; fax: +91 512 2594010.
E-mail address: [email protected] (J.R. Subramaniam).
0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.exger.2009.02.010
diseases (Cohen et al., 2006; Steinkraus et al., 2008) and could be used
as a drug evaluation model as shown for Ginkgo biloba extract (Wu
et al., 2006). In addition, neuronal expression of Ab in C. elegans causes
hypersensitivity to exogenous serotonin. This hypersensitivity could
be reversed by Ginkgo biloba extract (Wu et al., 2006).
Reserpine is an FDA approved antihypertensive drug. Reserpine’s action is attributed to its ability to inhibit the vesicular
monoamine transporter, VMAT, thereby reduce the level of the biogenic amines (serotonin, dopamine, octopamine/norepinephrine,
tyramine and histamine) neurotransmitters in the synaptic vesicles. We have recently reported that reserpine is able to positively
modulate several characteristics associated with aging in C. elegans
viz. movement, lifespan, pharyngeal pumping and stress tolerance
(Srivastava et al., 2008). Moreover, several reports have implicated
that crucial lifespan extension pathways can delay adult onset neurodegenerative diseases in C. elegans (Cohen et al., 2006; Steinkraus
et al., 2008). This led us to investigate the pharmacological efficacy
of reserpine in the most common and severe of the neurodegenerative disease condition, Alzheimer disease.
In this report, we address whether reserpine, which had positive effects on lifespan extension and quality of life in wild type C. elegans
could provide any protection/delay in the neurodegenerative disease,
AD, using the AD model in C. elegans. Our results show that: (i) reserpine is able to alleviate the AD pathogenesis in C. elegans by delaying
toxic Ab expression-mediated paralysis. (ii) But reserpine did not significantly alter the Ab expression of deposits in vivo. (iii) In addition,
reserpine increased the stress tolerance and extended the lifespan in
AD model worms. (iv) Reserpine action seems to be independent of
Ab induced serotonin hypersensitivity in the neurons in C. elegans.
U. Arya et al. / Experimental Gerontology 44 (2009) 462–466
2. Materials and methods
463
all the worms got paralysed approx. within 30 min. The assay
was repeated minimum three times.
2.1. Strains
2.7. Statistical analysis
The transgenic C. elegans line that constitutively expresses
Ab1–42 in the body wall muscles (CL2006) (AD model), CL2355
strain which has inducible neuronal Ab expression and it’s control
strain CL2122 is a kind gift from C.D. Link. The Bristol strain N2 was
used as the wildtype strain. Both the CL2006 strain and N2 worms
were maintained on NGM plates with OP50 Escherichia coli as food
at 20 °C following standard protocols (Brenner, 1974). The CL2355
and it’s control strain was maintained at 16 °C and was given a
temperature upshift at 23 °C for inducing Ab expression as described (Wu et al., 2006).
2.2. Reserpine treatment assays
We followed the protocol that had been described recently (Srivastava et al., 2008; Duerr et al., 1999). Briefly, 5 ll of reserpine
(50 mM reserpine prepared in acetic acid) was added to 198 ll of
M9 buffer and spread on the bacterial lawn on NGM plates
(35 mm) and allowed to dry. The final concentration of reserpine
is 60 lM/150 lg in the plates. The spread plates were used within
a week. Unless otherwise specified all assays were carried out at
20 °C. Around 30–40 synchronous worms (CL2006) at L4 stage,
grown on OP50 E. coli lawn on NNGM agar were shifted to the control and reserpine plates and followed for all the assays described
below. The reserpine treatment was chronically given throughout
the lifespan of the transgenic worms from L4 stage onwards.
Regarding the age of the worms, L4 stage was considered as day
0. Each assay was repeated at least three to four times. The worms
which were missing or died due to internal hatching were not included in the assays.
2.3. Paralysis assay
For the paralysis assay, the worms were monitored everyday
and were considered paralysed when they did not move even after
repeated prodding. The paralysis was followed till day 12 of adulthood as described (Cohen et al., 2006).
2.4. Movement assay
The CL2006 has a roller marker coinjected. Because of this, these
worms roll rather than move around sinusoidally. Hence, movement was measured as number of rolls made by the animals per
20 s (Huang et al., 2004; Srivastava et al., 2008). The worms were
considered not moving when they did not move in response to tapping of the plate or prodding.
2.5. Thermotolerance
For monitoring endpoint thermotolerance, the synchronous
four day old adult CL2006 worms with and without reserpine
treatment were shifted to 35 °C, and the worms were scored as
dead or alive every 1 h till they died (Srivastava et al., 2008).
2.6. Exogenous serotonin sensitivity assay
Synchronised neuronal Ab expressing CL2355 worms and the
control strain CL2122 with and without reserpine treatment (from
L4 onwards) were collected after 36 h of temperature upshift from
16 to 23 °C. They were placed in 5 mg/mL serotonin in M9 buffer
(Dempsey et al., 2005) and scored for paralysis every 5 min till
Mean, standard deviation and P values were calculated using
SigmaPlot, and Sigmastat. Survival graph was plotted using the
logRank survival plot in SigmaStat.
2.8. RT-PCR
The standard PCR, RT-PCR and agarose gel analysis were performed as described (Sambrook, 2001). Total RNA isolation for
RT-PCR analysis was performed using Trizol Reagent (Sigma). The
cDNA was synthesized using random hexamers and the MMLV Reverse Transcriptase (Fermentas). The Ab and actin were PCR amplified from the cDNA using already reported Ab primers (Link, 1995)
and C. elegans actin primers as an endogenous gene control at a linear range of 25 cycles for both the reserpine treated and untreated
worms.
2.9. Immunohistochemistry
The whole-worm immunohistochemistry for determining Ab
expression pattern in both the reserpine treated and untreated
CL2006 worms was done according to the available protocol after
paraformaldehyde fixation (Koushika et al., 2004). Bristol strain
N2 was used as staining control. The primary antibody staining
was done using the monoclonal antibody 6E10 (Covance) at
1:100 dilution. Anti-mouse FITC secondary antibody was used at
1:50 dilution. Ab immuno-reactive deposits were viewed using
10, 40 and 63 objective in the Zeiss Axioscope. The photomicrographs were taken using the CCD camera and the Axiovision
version 4.1 software.
3. Results
3.1. Reserpine alleviates the paralysis phenotype of Ab1–42 expressing
transgenic C. elegans
In the well established transgenic AD model in C. elegans (Link,
1995; Cohen et al., 2006; Wu et al., 2006), the toxic human Ab(1–
42) peptide is expressed in the body wall muscles under the control of unc-54 promoter (CL2006) ( Link, 1995). This human Ab
expression leads to manifestation of several pathological features
including progressive paralysis. The progressive paralysis phenotype is quite significant in this constitutively Ab expressing transgenic strain, CL2006. Therefore, we investigated the effect of
reserpine treatment on the paralysis phenotype in these transgenic
worms. For this, reserpine treatment was chronically given starting
from L4 stage onwards or from day 2 of adulthood. Interestingly,
reserpine treatment remarkably delayed the progressive paralysis
phenotype of the transgenic Ab worms both when given from
either L4 stage onwards (protection – 50%) or day 2 of adulthood
(protection – 43%) (Fig. 1A.a and b).
Moreover, as mentioned before, the Ab expressing worms manifest Ab toxicity as progressive paralysis. In this, at younger age, the
worms movement is normal, but as they age they cannot move and
become paralysed. Hence, we also scored for the movement along
with paralysis. Basically, the Ab expressing worms move by rolling,
hence the number of rolls made by the worms can be easily scored.
We noticed that the reserpine treated Ab1–42 expressing worms
were hyperactive and kept rolling for longer time in comparison
to the untreated controls (Fig. 1B). Thus we observed both overall
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U. Arya et al. / Experimental Gerontology 44 (2009) 462–466
Fig. 1. Protective effect of reserpine in Ab expressing transgenic C. elegans. (A.a and A.b) Reserpine suppresses progressive paralysis phenotype of Ab expressing CL2006
worms. (A.a) Reserpine delays the progressive paralysis phenotype of CL2006 worms when chronic reserpine treatment is given from L4 stage. Control (black) n = 105,
reserpine (blue) n = 103. (A.b) Reserpine delays the progressive paralysis phenotype of CL2006 worms when chronic reserpine treatment is given from day 2 of adulthood.
Control (black) n = 110, reserpine (blue) n = 115. The paralysis assay has been done three times. (B) Reserpine improves movement of Ab expressing CL2006 worms. Prolonged
and improved locomotion till the age of day 17 in reserpine treated worms in comparison to day 12 for control untreated worms. Control (black) (N = 100) and reserpine
treated (blue) (N = 100). The movement assay has been done four times and the improvement in movement was found statistically significant. P < 0.001. (C) Reserpine
extends Ab expressing transgenic CL2006 worms lifespan. Chronic reserpine treatment from L4 stage onwards extends lifespan of reserpine treated worms. Control (N = 161)
(black) and reserpine treated (N = 163) (blue). The difference is statistically significant with P < 0.001.The lifespan assay has been done four times. (D) Reserpine confers
thermotolerance to CL2006 worms. Reserpine treated worms (N = 102) (blue) were more tolerant to heat stress (at 35 °C) compared to the control worms (N = 100) (black
line). The difference was statistically significant with P < 0.001. The thermotolerance assay has been done three times. (E) Reserpine action is independent of Ab induced
exogenous serotonin hypersensitivity. The serotonin sensitivity assay was performed on CL2355 (neuronal Ab) and CL2122 control strain. The CL2355 strain (n = 85) (black
triangle) is hypersensitive to exogenous serotonin in comparison to control CL2122 (n = 114) (black circles). Reserpine treatment does not improve the serotonin sensitivity of
CL2355 (n = 78) (open triangles) and CL2122 (n = 88) (open circles) in comparison to untreated controls. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this paper.)
improvement in movement of the worms accompanied with
delayed paralysis for reserpine treated worms (Fig. 1A and B).
3.2. Increased survival rate and enhanced stress (thermo) tolerance of
Ab expressing worms treated with reserpine
(Johnson et al., 2001). Hence, we also investigated the possibility
of stress (thermo)tolerance in the reserpine treated worms. It
was repeatedly observed in different trials that reserpine treated
Ab expressing animals survived for a longer time under thermal
stress (Fig. 1D).
3.2.1. Reserpine enhances lifespan
Caenorhabditis elegans serves as an ideal model system to study
longevity and age-related mechanism. In order to explore whether
the beneficial effects of reserpine can also be extended to increase
in longevity of the transgenic Ab worms, we carried out survival assays on the constitutive human Ab worms, CL2006. For this, we
subjected the Ab worms to reserpine treatment from the L4 stage
onwards. Quite interestingly, reserpine treatment robustly extended the lifespan of human Ab expressing transgenic animals
in comparison to the untreated controls (Fig. 1C). The normal mean
lifespan of Ab expressing CL2006 worms was 12–13 days and the
reserpine treated worms lived up to 19 days.
3.3. Reserpine does not alter serotonin hypersensitivity of the neuronal
Ab worms
3.2.2. Reserpine increases stress tolerance
Moreover, as reported before, most of the known longevity mutants are also known to provide stress tolerance to the worms
In order to compare the Ab transcript and protein levels in the
CL2006 reserpine treated and untreated worms, we carried out
the RT-PCR analysis and performed whole-worm immunohisto-
Caenorhabditis elegans responds to exogenously provided serotonin and gets paralysed. This assay has greatly aided in identifying
the molecular players in the movement pathway. Neuronal expression of Ab causes serotonin hypersensitivity in C. elegans (Wu et al.,
2006). Hence, we evaluated whether reserpine treatment could reverse this. We observed that reserpine does not reverse the serotonin hypersensitivity in the neuronal Ab worms (Fig. 1E).
3.4. Reserpine treatment does not change the overall Ab
U. Arya et al. / Experimental Gerontology 44 (2009) 462–466
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Fig. 2. Ab transcript and expression levels remain unaltered upon reserpine treatment of CL2006 worms: (A) RT-PCR performed on the cDNA of reserpine treated and control
CL2006 worms using Ab primers. The endogenous C. elegans gene actin is used as an internal control. The RT-PCR analysis has been done two to three times. C, control; t,
treated (reserpine). (B) Whole-mount immunohistochemistry of reserpine treated and control CL2006 worms. (a) The Bristol strain N2 was used as a staining control. (b)
CL2006 control (c) CL2006 reserpine treated. Almost similar pattern of expression of Ab as detected by monoclonal antibody 6E10 was observed in both the control and
treated worms. Arrows indicates Ab immuno-reactive deposits. Scale bar = 10 lm. The immunohistochemistry staining has been done three times.
chemistry for observing immuno-reactive deposits in vivo. RT-PCR
performed on the cDNA using Ab primers showed similar level of
Ab amplification in both the reserpine treated and untreated animals (Fig. 2A). Similarly, the expression pattern of Amyloid beta
reactive immuno deposits remained largely unchanged upon
reserpine treatment (Fig. 2B).
4. Discussion
Reserpine is of natural origin. It is a plant alkaloid purified
from the roots of the plant Rauwolfia serpentina. Ancient Indians
(1000–2000 BC) had known, documented, and used it to treat
snakebites and insanity (Lopez-Munoz et al., 2004). In recent
20th century, in the 1940s and ‘‘50s, reserpine was one of the
first psychopharmacological drug to treat the psychiatric diseases and also as an antihypertensive (Vakil, 1949; Bleuler and
Stoll, 1955). Reserpine is still used for hypertension at the dosage of 0.1–0.25 mg. Reserpine is widely known for its function
of downregulation of biogenic amines through inhibition of the
vesicular monoamine transporter (VMAT). As reserpine was able
to enhance longevity, provide stress tolerance and improve
movement in wildtype C. elegans (Srivastava et al., 2008), we
speculated that we can evaluate reserpine as a therapeutic in
late-onset neurodegenerative disease conditions. Here, our results show that reserpine indeed can provide protection against
the Alzheimer disease pathogenesis in C. elegans, wherein reserpine is able to delay paralysis, improve movement, provide
stress tolerance and enhance longevity of Ab expressing worms
(Fig. 1A–D). Thus, reserpine can be regarded as a general modulator of lifespan and movement in both the proteotoxic models
of cellular toxicity in C. elegans and otherwise.
In order to understand the possible mechanism of reserpine
action, we evaluated the effect of reserpine on Ab expression
and deposits. The Ab mRNA and in vivo protein deposits remains
unaffected upon reserpine treatment (Fig. 2A and B). Along similar lines, recently, Steinkraus et al. showed that though bacterial
deprivation provided suppression of proteotoxicity in polyglutamine disease model in C. elegans, the overall amount of
Q35YFP protein was unchanged and the protection was medi-
ated by the master heat shock factor, hsf-1 (Steinkraus et al.,
2008). This suggests that distinct pathways of protection can
also be operative other than reduction of toxic protein levels
per se in such pathological conditions. In addition to this, reserpine inducing stress tolerance makes hsf-1 as one possible mechanism for reserpine action. Nevertheless, the possibility of
modulation of distinct Ab species in this scenario cannot be ruled out and further investigation is needed in this regard to
identify whether reserpine can alter the toxic Ab species to mitigate the disease phenotype in the transgenic Amyloid beta
expressing worms.
Another neuronal model of AD in C. elegans, which does not
show any Ab deposits, manifests higher sensitivity to exogenous
serotonin (Wu et al., 2006). As reserpine is a biogenic amines
downregulator, and earlier we found serotonin to be partly required for reserpine mediated lifespan extension (Srivastava
et al., 2008), we evaluated the reserpine treated neuronal Ab
worm’s exogenous serotonin sensitivity and found that reserpine
could not alter this (Fig. 1E). Since, in the exogenous serotonin assays, serotonin is provided in high doses, probably reserpine’s effect was masked. Further studies on the serotonin deficient
mutant, tph-1 could help to determine whether reserpine acts
through serotonin modulation.
The dramatic protective effects of reserpine on the Ab expression induced paralysis and lifespan in C. elegans was brought about
at the reserpine dosage of around 60 or 150 lg. This dosage, which
is in line with that given to hypertension patients, 100–250 lg,
makes it a potential drug to be screened in higher in vivo experimental models of Alzheimer disease such as mice and evaluated
for AD therapeutics.
Acknowledgements
The authors would like to thank Dr. C.D. Link, University of Colorado, Boulder, USA for the AD model worms and Dr. K. Subramaniam, BSBE, IIT-Kanpur, India, for the generous help with the worm
expertise. This work was financially supported by Indian Institute
of Technology, Kanpur. U.A. thanks University Grants Commission,
Government of India for the scholarship.
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U. Arya et al. / Experimental Gerontology 44 (2009) 462–466
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