Acetylcholineestarase-Inhibiting Alkaloids from Lycoris radiata Delay Paralysis of Amyloid Beta-Expressing Transgenic C. elegans CL4176
et al. (2013) Acetylcholineestarase-Inhibiting Alkaloids from Lycoris radiata Delay Paralysis of Amyloid Beta-
Expressing Transgenic C. elegans CL4176. PLoS ONE 8(5): e63874. doi:10.1371/journal.pone.0063874
Acetylcholineestarase-Inhibiting Alkaloids from Lycoris radiata Delay Paralysis of Amyloid Beta-Expressing Transgenic C. elegans CL4176
Lijuan Xin 0
Ritupriya Yamujala 0
Yuehu Wang 0
Huan Wang 0
Wen-Hsuan Wu 0
Michael A. Lawton 0
Chunlin Long 0
Rong Di 0
Hemachandra Reddy, Oregon Health & Science University, United States of America
0 1 College of Life and Environmental Sciences, Minzu University of China , Beijing , China , 2 New Brunswick Graduate School, Rutgers University , New Brunswick , New Jersey, United States of America, 3 Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, China, 4 Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University , New Brunswick, New Jersey , United States of America
The limited symptom relief and side effects of current Alzheimer's disease (AD) medications warrant urgent discovery and study of new anti-AD agents. The ''cholinergic hypothesis'' of AD prompts us to search for plant-derived acetylcholineesterase (AChE) inhibitors such as galanthamine that has been licensed in Europe for AD treatment. We used the unique amyloid b-expressing transgenic C. elegans CL4176, which exhibits paralysis when human Ab1-42 is induced, to study two natural benzylphenethylamine alkaloids isolated from Lycoris radiata (L' Her.) Herb, galanthamine and haemanthidine, and their synthetic derivatives 1,2-Di-O-acetyllycorine and 1-O-acetyllycorine for their anti-paralysis effects. Our data indicate that these Lycoris compounds effectively delay the paralysis of CL4176 worms upon temperature up-shift, and prolong the lives of these transgenic worms. Lycoris compounds were shown to significantly inhibit the gene expression of ace-1 and ace-2. Additionally, the Lycoris compounds may modulate inflammatory and stress-related gene expressions to combat the Ab-toxicity in C. elegans.
Funding: This work was partially funded by the National Natural Science Foundation of China (31161140345, 31070288, 20972166), and Ministry of Education of
China through its 111 and 985 programs (B08044, MUC985-9, MUC98506-01000101). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
With more than 20 million cases worldwide, Alzheimers disease
(AD) has been named the most common progressive
neurodegenerative disease . AD is characterized by cerebral degeneration,
neuronal cell death, and the hallmark accumulation of 4042
amino acid amyloid-b (Ab) in plaques  and tau tangles in the
affected brain nerve cells . According to the amyloid
hypothesis [3,4], Ab is produced by the cleavage of the
ubiquitous amyloid precursor protein (APP) and the imbalance
between the production and the clearance of Ab in central nervous
system, leads to neuronal degeneration. Up to now, there is no
cure for this debilitating disease. The widely accepted cholinergic
hypothesis of AD proposes that a serious loss of cholinergic
function in the central nervous system is associated with the
development of cognitive symptoms . A number of
acetylcholinesterase (AChE) inhibitor drugs, including tacrine, donepezil,
and galantamine, have been developed and are used to restore the
normal cholinergic function for synaptic transmission in the
central nervous system of AD patients . These drugs, which are
moderately effective in alleviating the symptoms of AD, have a
number of side effects, including gastrointestinal upsets [6,7].
Other drugs used to treat AD patients, such as memantine,
attempt to balance the cholinergic pathway by attenuating the
function of N-methyl-D-aspartate (NMDA) receptors. Like AChE
inhibitors, memantine offers only modest AD symptom relief along
with gastrointestinal side effects . As the world population
continues to live longer and AD patient numbers steadily rise, the
need for new, effective and safer pharmacological agents for AD
therapy becomes increasingly pressing.
In recent years, we have been working on the isolation of active
anti-AD compounds from traditional Chinese herbs. Lycoris Herb.
is a member of Amaryllidaceae and a genus endemic to East Asia.
Lycoris radiata (L Her.) Herb has been used in traditional Chinese
medicine to treat sore throat, rheumatoid arthritis and snake bites.
 Lycoris plants are rich in benzylphenethylamine alkaloids such
as galanthamine, lycorine, lycoramine and lycorenine [10,11]. We
have previously reported the isolation of fifteen known
benzylphenethylamine alkaloids in 2011 from the bulbs and flowers of
Lycoris radiata . Some alkaloids were first reported from Lycoris
Herb. and some were first isolated from L. radiata, including
haemanthidine. Both galantamine and lycoramine from L. radiata
have been reported to have anti-AChE activities . Being orally
bio-available and stimulative to nicotinic acetylcholine receptors
(nAchR) , galanthamine is licensed in Europe for AD
treatment and is well tolerated by AD patients .
Besides the AChE inhibitory effects, galanthamine has been
found to modulate inflammation by attenuating TNF-a (tumor
necrosis factor-alpha) and NO (nitric oxide) release through the a7
nAChR  and p44/42 MAPK (mitogen-activated protein
kinase) pathway in murine microglia . Another AChE
inhibitor, donepezil, has also been shown to decrease cytokine
(oncostatin M, interleukin-1b and interleukin-6) levels in AD
patient lymphocytes . Tacrine, donepezil and huperzine, all
AChE inhibitors, have been demonstrated to prevent hydrogen
peroxide-induced cell death and Ab peptide-induced oxidative cell
death . Since inflammatory process and oxidative damage
have been implicated in neurodegenerative diseases, any AChE
inhibitory agent with the additive anti-inflammatory and/or
antioxidative effects would be expected to be superior for AD
Transgenic Caenorhabditis elegans (C. elegans) models have been
established for Alzheimers disease since 1995 . Nematode
disease models have been used to study the mechanisms of AD
toxicity  and to test the efficacies of drugs and
nutrisupplements. By using transgenic CL4176 worms, which express
the human Ab142 in muscle tissues under a temperature-inducible
system , it was reported that soy isoflavone glycitein could
protect worms from Ab-induced toxicity and this protection was
credited to the anti-oxidative activity of glycitein . Ginkgo biloba
extract EGb761 and ginkgolides were shown to suppress the
Abinduced pathological behaviors of several different Ab-transgenic
C. elegans, not by reducing oxidative stress but rather by
modulating Ab oligomeric species . Arya et al. used transgenic
C. elegans strains CL2006, which constitutively expresses Ab in the
body wall muscles, and CL2355, which has inducible neuronal Ab
expression, to show that reserpine (an FDA approved
antihypertensive drug) could ameliorate Ab toxicity .
In this study, we used transgenic C. elegans CL4176 to evaluate
the Ab toxicity- inhibitory effect of galanthamine and
haemanthidine purified from Lycoris radiata (L Her.) Herb. and their
derivatives 1,2-Di-O-acetyllycorine and 1-O-acetyllycorine. We
demonstrate that these L. radiata compounds strongly inhibit Ab
toxicity and prolong the lifespan of CL4176 worms. Attenuation of
Ab toxicity in this model system mostly results from the inhibition
of acetylcholineesterase gene expression. Modulation of
inflammation and stress-related genes may also contribute to the anti-Ab
toxicity of Lycoris compounds. Our study indicates that Ab
transgenic CL4176 nematodes can be efficiently used to screen
for AChE inhibitors.
Isolation and Synthesis of L. radiata Compounds
From the bulbs and flowers of L. radiata, we obtained fifteen
known alkaloids . Galanthamine and haemanthidine were
selected for further study. According to a previous report,
1-Oacetyllycorine possesses potent activity against electric eel
acetylcholinesterase (eeAChE, IC50 = 0.96 mM) . Therefore, we also
synthesized this compound and its analogue
1,2-di-O-acetyllycorine and used both in the following studies.
Natural and Synthetic L. radiata Compounds Delayed
Paralysis of Ab-Transgenic C. elegans CL4176 Nematodes
CL4176 worms were synchronously hatched and raised on
NGM plates containing OP50 bacteria and compounds at 16uC.
After two days, the temperature was shifted to the permissive 23uC
to induce expression of the Ab142 peptide. We consistently
observed that 26 hours after the temperature up-shift, non-treated
worms started to become paralyzed and die due to the expression
of human Ab142. In order to compare the efficacy of anti-Ab
activity of L. radiata compounds with memantine, the current
medication used to lessen symptoms in AD patients, we included
memantine as one of the experimental treatments in these studies.
While memantine has been shown to be effective in delaying the
paralysis of CL4176 upon temperature shift to 23uC, the
concentration used in that study was not reported . We tested
different concentrations of memantine in the paralysis assay with
CL4176 and found that it was effective only in the millimolar
range (data not shown). Consequently, we used 10 mM of
memantine as a reference throughout these studies. We showed
that galanthamine could reduce the mortality of CL4176
compared to the control (CK) worms which were not treated
with any compound (Fig. 1A). The control worms reached 100%
mortality 34 hr post temperature up-shift. In contrast, 8% of
worms exposed to 10 mM galanthamine were still alive at 36 hr.
The worm survival rates were further increased to 32% and 42%
by 30 mM and 50 mM of galanthamine 34 hr post temperature
upshift. Our data showed that the anti-paralysis effect of 50 mM
galanthamine was greater than that of 10 mM memantine. We
further demonstrated that 50 mM galanthamine could prolong the
lifespan of CL4176 up to 40 hr.
Our data also showed that haemanthidine, which we isolated
for the first time from L. radiata, could reduce the mortality of
CL4176, with 11%, 14% and 14% worms still alive 34 hr after the
temperature up-shift at concentrations of 10 mM, 30 mM and
50 mM (Fig. 1B). At these same concentrations, haemanthidine
appeared to be less effective at delaying the paralysis of CL4176
nematodes as compared to galanthamine.
The first synthetic alkaloid 1,2-Di-O-acetyllycorine at 50 mM
was shown to be comparable to10 mM memantine in preventing
paralysis of CL4176 worms expressing the Ab142 peptide (Fig. 1C).
At 30 mM, however, 1,2-Di-O-acetyllycorine increased the worm
survival rate to 48% 34 hr post temperature up-shift, which was
similar to the effect of 50 mM galanthamine (Fig. 1A).
The second synthetic alkaloid 1-O-acetyllycorine exhibited
similar anti-paralysis effects in CL4176 worms as 10 mM
memantine, and was comparable to 30 mM galanthamine at the
same concentration (Fig. 1D). However, 50 mM 1-O-acetyllycorine
increased the worm survival rate of CL4176 to the greatest level
(61% alive), compared to all the compounds tested.
Our data indicate that the new alkaloid haemanthidine from
L. radiata and our synthetic alkaloids 1,2-Di-O-acetyllycorine and
1-O-acetyllycorine could reduce Ab-toxicity with similar potency
to the known AChE inhibitor galanthamine. We have shown our
Lycoris compounds were effective at the micromolar concentration
range versus the millimolar concentration range required for the
current AD medication memantine. Therefore, 50 mM for all
Lycoris compounds and 10 mM for memantine were subsequently
used in the following experiments.
L. radiata Compounds Slightly Reduced the Levels of Ab
in Transgenic C. elegans CL4176
Since the paralysis and the subsequent death of CL4176 are
caused by the Ab toxicity, naturally we wanted to examine if our
Lycoris compounds would have any direct effect on the expression
of Ab Transgenic C. elegans CL4176 expresses Ab in the worm
muscle cells . It has been shown that CL4176 worms actually
do not form Ab plaques in the worm bodies. Rather, Ab is
expressed mostly in a soluble form [25,26]. We used the
fluorescent thioflavin T stain, which detects non-aggregated
proteins more specifically than thioflavin S , to quantify the
levels of Ab in CL4176 worms treated with L. radiata compounds.
Pure synthetic Ab obtained from Sigma (cat. # A9810) was
initially used to optimize the assay condition by preparing 0, 1, 5,
10 to 100 mg Abin 100 ml volume each with 20 mM thioflavin T. It
Figure 1. Time course of the paralysis assay of CL4176 C. elegans. Synchronized worms were hatched and fed on compound-containing NGM
medium for two days at 16uC. The temperature was then shifted to 23uC. The survival rate (% of live worms) was recorded and plotted against the
hours post temperature shift. The worms were treated with (A) galanthamine, (B) haemanthidine, (C) 1,2-Di-O-acetyllycorine and (D)
1-Oacetyllycorine at 10, 30 and 50 mM concentrations. In comparison, one group of worms were not treated (the non-treated control sample, CK) and the
other group ware treated with 10 mM memantine. Three independent experiments were carried out to give the average rates for worm survival and
to calculate standard deviations.
was determined that the Ab concentration effect could be assessed
by our Synergy HT Plate Reader using 440 nm excitation and
482 nm emission. At least 50 worms were collected from each
treatment and sonicated. Equal amount of total soluble protein
was used for each sample and assayed in triplicates and the
experiment was repeated three times. Our results demonstrated
that there was no reduction in the level of Ab by any of the Lycoris
compounds or by memantine 26 hr after the temperature shift to
23uC. As mentioned above, at 26 hr post temperature up-shift, the
worms became paralyzed and started to die. We then repeated the
treatments and collected worms 32 hr post temperature up-shift.
The thioflavin T assay results showed that the Ab levels in the
galanthamine, haemanthidine, 1,2-Di-O-acetyllycorine,
1-O-acetyllycorine and memantine treated worms were 91.5% 64.86
(p = 0.019), 91.37% 67.72 (p = 0.062), 91.23% 69.26 (p = 0.088),
93.61% 61.41 (p = 0.001) and 98.27% 61.83 (p = 0.085),
compared to the non-treated worms. These data indicate that
galanthamine significantly reduced the Ab level by approximately
8.5% and the synthetic 1-O-acetyllycorine significantly reduced the
Ab level in worms by approximately 6.4% compared to the
nontreated worms. In comparison, the data also indicate that
memantine did not seem to reduce the Ab level much.
Quantitative real-time RT-PCR (qRT-PCR) was carried out to
assess the capability of Lycoris compounds to inhibit the Ab gene at
the transcript level. By using the primers designed for the Ab
transgene and the F23B2.13 gene encoding an RNA polymerase
small subunit as a non-variable endogenous control  (Table 1),
the relative gene expression of Ab was assessed by the 22DDCt
method. Our qRT-PCR results from three independent
experiments showed that galanthamine significantly reduced the Ab
transgene expression by an average of 2.72-fold (p,0.01)
compared to the non-treated sample. The results also showed
that 1,2-Di-O-acetyllycorine reduced the Ab gene expression by
1.32-fold (p,0.05) and 1-O-acetyllycorine by 1.3-fold (p,0.01).
Since it is generally accepted that a 2-fold change in gene
expression is considered significant, we can assume the effect of
1,2-Di-O-acetyllycorine and 1-O-acetyllycorine on Ab gene
expression was negligible. Haemanthidine and memantine,
however, did not seem to have any inhibitory effect on the Ab
transgene expression at the transcript level.
L. radiata Compounds Inhibited Acetylcholine Esterase
Gene Expression in Ab-Transgenic C. elegans CL4176
To delineate the anti-paralysis mechanisms of natural and
synthetic L. radiata compounds in Ab-transgenic C. elegans CL4176
nematodes, we investigated the ability of the natural and synthetic
L. radiata compounds to inhibit the AChE gene expression in
Abtransgenic C. elegans CL4176 nematodes. Total worm RNA was
isolated from CL4176 nematodes 26 hr after the temperature
upshift. At this point, the worms had fed for a total of 48 hr on
medium containing both the compounds and OP50 bacteria and
just started to become paralyzed. Unlike vertebrates with only one
AChE gene , C. elegans and other nematodes have multiple ace
genes. C. elegans ace-1 gene is expressed in all body-wall and vulval
muscle cells  while ace-2 is expressed almost exclusively in
neurons . ace-3 is expressed in pharyngeal muscle cells and
neurons . ACE-1 and ACE-2 account for 95% of the total
enzymatic activity, ACE-3 for the remainder 5% and ACE-49s
activity is normally undetectable. Consequently, we focused on the
effects of L. radiata compounds on gene expression of ace-1 and
To study the steady state expression levels of the ace-1 and ace-2
genes, we performed qRT-PCR assays using specific forward and
reverse primers for these genes (Table 1) with total nematode RNA
isolated from Ab-transgenic C. elegans CL4176. The relative levels
of ace-1 and ace-2 gene expression were assessed by the 22DDCt
method using the F23B2.13 gene as a non-variable endogenous
control, averaged from three independent experiments.
Galanthamine reduced the gene expression of ace-1 by 7.61-fold, the
synthetic 1,2-Di-O-acetyllycorine decreased ace-1 expression by
4.62-fold and haemanthidine decreased ace-1 expression by
2.73fold (Fig. 2A). Our results also demonstrated that the synthetic
1O-acetyllycorine reduced the expression of ace-1 in CL4176 worms
by only 1.6-fold and that memantine had no inhibitory effect on
ace-1. Additionally, we demonstrated that the synthetic
1,2-Di-Oacetyllycorine reduced ace-2 expression by 2.97-fold (p,0.05),
galanthamine reduced ace-2 expression by 2.94-fold (p,0.05),
synthetic 1-O-acetyllycorine decreased ace-2 expression by
2.67fold (p,0.01) and haemanthidine decreased ace-2 expression by
2.04-fold (p,0.01). Memantine had a slightly inhibitory effect,
reducing ace-2 levels by 1.39-fold which was negligible.
L. radiata Compounds Inhibited Inflammation and
StressAssociated Gene Expression Which May Contribute to
the Anti-Ab Paralysis Effect in C. elegans CL4176
As inflammation is implicated in Ab-toxicity and AChE
inhibitors have been shown to modulate inflammation to improve
the cholinergic health [7,15,28], we decided to evaluate the ability
of L. radiata compounds to reduce the expression of inflammation
and stress-associated genes in CL4176 C. elegans. Pro-inflammatory
genes and cytokines such as TNFa, IL-1 and IL-6 are associated
with human neurodegenerative diseases. While C. elegans contains
homologues of TNFA1P1 (TNFa-induced protein 1), this gene has
not been associated with Ab toxicity in human . However, it
has been reported that the treatment of TNFa in human cell
TCCTCCTCCAACTTTTCCAAA TCTTCCTTGAACCGCTTCTTTC TGGAGCCTCAATTTGGAGTTTTC GGGCGTCGTACACCATCA
cultures exposed to Ab can protect hippocampal neurons from Ab
toxicity . Link et al. have shown that the gene expression levels
of two C. elegans homologs of human TNFA1P1, F22E5.6 and
ZC239.12, were up-regulated in transgenic CL4176 upon
temperature up-shift . They also showed that TNFA1P1
expression was increased in AD patient brain tissues .
Additionally, Link et al. reported that the expression of human
aB-crystallin (CRYAB), a well-studied stress-inducible chaperone
protein, was enhanced in AD patient brain tissues. Equivalently,
the C. elegans CRYAB-homologous HSP16 (heat shock protein
16)2 (Y46H3A.D) and HSP16-4 (Y46H3A.E) gene expressions were
up-regulated in transgenic CL4176 worms following the
temperature up-shift .
We used the same primers in Link et al.  (Table 1) to
perform qRT-PCR on total RNA isolated from CL4176 26 hr
after the temperature shift. We found that galanthamine,
haemanthidine and 1,2-Di-O-acetyllycorine at 50 mM significantly
reduced the gene expression of TNFA1P-homolog F22E5.6
(p,0.01,0.05), with haemanthidine being the most effective
compound with an average of 4.8-fold reduction compared to the
non-treated sample (Table 2). 1,2-Di-O-acetyllycorine at 50 mM
was also shown to affect another TNFA1P1-homolog, ZC239.12,
whose gene expression was reduced by 5.5-fold (a significance level
of p = 0.006). Additionally, our data indicate that 50 mM
haemanthidine reduced the gene expression of two stress-related
HSP-16 genes by 1.99- and 2.07-fold, corresponding to a statistical
significance values of p = 0.007, and 0.025, respectively. These
results show us that galanthamine, haemanthidine and
1,2-Di-Oacetyllycorine may be involved in limiting inflammation and
stress-related cellular damage caused by Ab toxicity in transgenic
CL4176 worms. Interestingly, we found that 10 mM memantine
could reduce the expression of these four genes related to
inflammation and stress in CL4176 C. elegans (Table 2).
Antioxidant Activity did not Contribute to the Anti-Ab
Paralysis of CL4176 Nematodes by L. radiata Compounds
Besides Ab toxicity, oxidative stress has been widely postulated
to contribute to the etiology of Alzheimers disease [7,26].
Abtransgenic C. elegans CL4176 have been shown to be under
increased oxidative stress upon the temperature shift to 23uC .
We used the dye DCF-DA to measure intracellular levels of
H2O2related reactive oxidative species (ROS) in C. elegans CL4176
following exposure to compounds and temperature shift for 26 hr.
Our data showed that exposure to the Lycoris compounds had no
effect on ROS levels in treated worms compared to non-treated
worms. This result indicates that antioxidant activity was not one
of the factors contributing to Lycoris compounds anti-paralysis
activity in CL4176 worms.
Cytotoxicity of L. radiata Compounds in Mammalian Cells
Ultimately, we would like to screen for natural and synthetic
AChE inhibitors that have potent anti-AD effects but do not
induce undesirable side effects for patients. The crinine and
lycorine alkaloids have been reported to have noteworthy
cytotoxicity [9,34,35]. We tested the cytotoxicity of our Lycoris
compounds at the highest concentration of 50 mM that was used in
the C. elegans in FHs 74 Int cells (human fetal small intestine
normal cells, ATCC # CCL-241). With the MTS proliferation
test, we demonstrated that the natural haemanthidine, a crinine
type of alkaloid, was the most toxic and exhibited an average (from
two independent experiments) of 28% 62.62 inhibition rate of cell
proliferation compared to non-treated cells which coincided with
the previous report. Our results showed that the natural
galanthamine and the synthetic 1,2-Di-O-acetyllycorine were not
toxic at all to FHs 74 Int cells at 50 mM concentration. The
synthetic 1-O-acetyllycorine was also shown to inhibit the
proliferation of FHs 74 Int cells by 12.8% 63.98.
As the world population is growing older, the number of
patients with Alzheimer disease (AD) is steadily increasing
worldwide. With the limitations of improving the AD symptoms
and the unfavorable side effects of current AD medications, it is
imperative that we actively search and screen for new
ADcontrolling agents. We describe here the utilization of
Abtransgenic C. elegans to study the inhibitory effects of two natural
and two synthetic compounds from Lycoris radiata on paralysis of
the worms caused by the Ab toxicity. Our results showed that the
natural galanthamine and haemanthidine and the synthetic
1,2Di-O-acetyllycorine and 1-O-acetyllycorine could effectively
attenuate the toxicity of Ab expressed in transgenic CL4176 worms
after the temperature shift from 16uC to 23uC (Fig. 1). Although it
has been reported that the popular AD medication memantine
could delay the paralysis of CL4176 nematodes , its effective
concentration was not reported. We showed in this study that the
Ab toxicity attenuation by the Lycoris compounds was achieved at
1050 mM concentrations. In comparison, 10 mM memantine
had to be used to achieve similar anti-paralysis effect in CL4176
worms. Memantine, a known attenuator of N-methyl-D-aspartate
(NMDA) receptors, is used to keep the degeneration of cholinergic
cells in check . One of our Lycoris compounds, galanthamine, is
a known acetylcholine esterase (AChE) inhibitor that has been
licensed in Europe for AD treatment. Both galanthamine and
haemanthidine isolated naturally from Lycoris are alkaloids. Both
the NMDA attenuator and AChE inhibitors are presumed to
function by maintaining the healthy cholinergic status of cells.
Therefore, the nearly 1000-fold lower concentration needed for
Lycoris compounds to produce similar anti-paralysis effects in
CL4176 indicates that they are much more effective than
memantine in this system.
In order to evaluate the effects of Lycoris compounds on AChE
genes, real-time quantitative RT-PCR analysis was conducted
with total RNA isolated from worms that had been treated and
incubated at higher temperature (23uC) for 26 hr. This time point
was chosen since this is when the first symptoms of paralysis were
observed and it allowed us to examine early changes in gene
expression associated with the results of the treatment itself, rather
than the downstream consequence of paralysis . Our data
demonstrated that the natural compound galanthamine was the
most inhibitory to the gene expression of ace-1 followed by the
synthetic 1,2-Di-O-acetyllycorine and the natural compound
haemanthidine (Fig. 2A). We have also shown that all of our
Lycoris compounds could inhibit the gene expression of ace-2 by
around 3-fold. Interestingly, we did not find memantine inhibitory
to the expression of ace-1 and ace-2. To our knowledge, there has
been no previous report of using Ab-transgenic C. elegans CL4176
nematodes to study the anti-AChE activity of any AChE
inhibitors. It is understood that CL4176 expresses Ab in muscle
cells of worms upon temperature up-shift and the paralysis
phenotype is a result of the Ab toxicity. However, as the creators
of CL4176 pointed out that 67 genes were up-regulated and 240
genes were down-regulated in CL4176 upon temperature up-shift
. Since there are many mechanisms proposed for Ab toxicity,
so far there is no strong indication of which gene changes are
responsible for the Ab toxicity in CL4176 worms . It is likely
that the AChE inhibiting activities of our Lycoris compounds
provide some protection to CL4176 worms against Ab toxicity,
just as memantine, an attenuator of N-methyl-D-aspartate
(NMDA) receptors for cholinergic health, was found to be able
to delay the paralysis of CL4176 . At least, our data suggested
that CL4176 C. elegans can be used effectively to screen compounds
that are inhibitory to AChE gene expression.
Since inflammation and oxidative-induced stress are associated
with Ab-toxicity and some AChE inhibitors including
galanthamine have been shown to modulate inflammation process [13,14],
we evaluated our Lycoris compounds for their inhibition of two C.
elegans human homologs of TNFA1P1 genes, F22E5.6 and
ZC239.12 and two homologs of human aB-crystallin (CRYAB),
HSP16-2 (Y46H3A.D) and HSP16-4 (Y46H3A.E). These four
genes have been shown to be up-regulated in CL4176 worms
following the temperature up-shift . Our results indicated that
galanthamine and haemanthidine significantly reduced the gene
expression of F22E5.6 and 1,2-Di-O-acetylcorine highly
significantly reduced the expression of ZC239.12. Haemanthidine was
also found to be noticeably inhibitory to expression of the two
HSP16 genes (Table 2). These data suggest that Lycoris compounds
can both suppress AChE gene expression and are able to modulate
expression of inflammation-related and stress-related genes in
transgenic C. elegans CL4176. Since C. elegans really do not have the
inflammation system per se, the benefits of Lycoris compounds
antiinflammation activity will need to be further evaluated in animal
Our data suggested that only galanthamine could reduce the
transgene Ab expression by 2.7-fold in CL4176 worms and the
inhibitory effect on Ab transcript level from haemanthidine,
1-Oacetyllycorine and 1,2-Di-O-acetyllycorine was negligible.
However, all four Lycoris compounds could slightly reduce the Ab peptide
level in CL4176 worms by 6.4% to 8.5%. Since all four Lycoris
compounds were shown to inhibit paralysis of CL4176 after
temperature up-shift (Fig. 1), the slight reduction in Ab transgene
expression at both the transcript and peptide levels by these
compounds may play a small role in extending the lifespan of
CL4176 worms. Whether this slight Ab reduction results directly
from the AChE inhibition/cholinergic health-promoting activity
of our Lycoris compounds needs to be further elucidated.
Additionally, we did not find anti-oxidant activity contribute to
the anti-paralysis effect of the four Lycoris compounds. Therefore,
our findings in this study strongly indicate that the anti-paralysis
effects of our Lycoris compounds mainly result from their AChE
gene inhibition and inflammation/stress-related gene modulation.
However, it will be interesting to find out the inter-relationship
amongst the Ab toxicity and the cholinergic health and the
lifespan extension promoted by the Lycoris compounds in CL4176
worms in future studies.
The present study demonstrates that human Ab-expressing
transgenic C. elegans CL4176 can be used effectively to screen for
compounds that are inhibitory to AChE gene expression.
Although we have shown that human fetal intestinal epithelial
cells provide an easy system to test the cytotoxicity of AChE
inhibitors, further efficacy studies need to be conducted in mouse
and monkey models of Alzheimers disease before any new
compound can be studied in human clinical trials.
Materials and Methods
Isolation and Synthesis of L. radiata Compounds
The isolation of galanthamine and haemanthidine from L.
radiata has been reported in our previously published paper .
1O-Acetyllycorine  and 1,2-di-O-acetyllycorine  were
prepared using established procedures.
Paralysis Assay of Ab-Transgenic C. elegans CL4176
Ab-transgenic C. elegans CL4176 nematodes [genotype:
smg1(cc546ts); dvIs27] were obtained from CGC (Caenorhabditis
Genetics Center) and maintained on NGM (Nematode Growth
Medium) plates (60 mm petri dishes) at 16uC. Paralysis assays
were performed according to the method of Dr. C. Link . In
brief, one week before initiating the paralysis assay, gravid adult
worms were picked onto NGM plate spread with OP50 bacteria to
lay eggs for 24 hr at 16uC. The gravid adults were picked off the
plates and the progeny were allowed to grow for 7 days into
second day gravid adults. One day before the initiation of the
paralysis assay, NGM plates were prepared with 50 ml of fresh
OP50 bacterium mixed with Lycoris compounds at different
concentrations or 10 mM memantine and incubated at 37uC
overnight. At day 1 of the initiation of the paralysis assay, the
second day gravid adults were allowed to lay eggs on NGM plates
containing OP50 and compounds. Approximately 50 progeny
were maintained on each plate for each treatment and incubated
at 16uC for two days. The worms were then incubated at 23uC
and the survival rates were recorded. Worms without movement
after prodding were recorded as dead.
Quantitative Staining of Ab with Thioflavin-T
Twenty-six or 32 hours post temperature shift from 16uC to
23uC, all the CL1476 worms for each treatment plate were
collected by washing the plate with 1 ml 16 PBS (diluted from
106 PBS, Fisher) and transferred into a microfuge tube. The
worms were pelleted by centrifugation at14k rpm for 2 min and
sonicated with Branson Sonifier 150 at setting 2 for 15 sec 64
times. The sonicated worms were centrifuged at 14 krpm for
2 min and the supernatant was transferred into a new tube. The
concentration of total soluble protein in each sample was
quantified by the Bradford method (BioRad). Equal amount of
total protein from every sample was used in each independent
experiment, which was divided into 3 replicates. Each replicate
was mixed with 10 ml 106 PBS (Fisher) and 2 ml 1 mM
thioflavinT (Sigma) (final concentration of 20 mM) in a final volume of
100 ml. Fluorescence resulting from Ab stained by thioflavin-T was
measured by the Synergy HT Plate Reader using excitation at
440 nm and emission at 482 nm and averaged from at least three
Measurement of H2O2 Levels in CL4176 C. elegans
Twenty-six hours after the temperature up-shift, exactly 50
CL4176 worms from each treatment plate were collected by a
platinum worm-picker into 100 ml 16PBS (diluted from 106PBS,
Fisher) containing 1% Tween-20 in a microfuge tube and
sonicated with Branson Sonifier 150 at setting 2 for 15 sec 64
times. The intracellular levels of H2O2-related reactive oxidative
species (ROS) in CL4176 worms were measured as described
using 2,7-dichlorofluorescein diacetate (DCF-DA, Sigma Aldrich)
. Briefly, sonicated worms were pipetted into the wells of a
96well plate containing 100 ml of 16 PBS plus 100 mM DCF-DA.
The final concentration of DCF-DA in the assay is 50 mM. The
fluorescence was recorded by Synergy HT Plate Reader at
485 nm excitation and 545 nm emission and averaged from three
Quantitative Real-Time RT-PCR (qRT-PCR) Analysis
Twenty-six hours post temperature shift from 16uC to 23uC,
treated CL4176 worms were collected by washing the plate with
1 ml 16PBS and transferred to microfuge tubes. The worms were
pelleted by centrifugation at 14 krpm for 2 min. The total
nematode RNA was isolated by the freeze-cracking method as
described in the WormBook (http: //www.wormbook.org). Briefly,
500 ml Trizol reagent (Life Technologies) was added to each
sample. The freeze-thaw was repeated three times by freezing in
dry ice/ethanol bath and 5 min 37uC -incubation. After 100 ml
chloroform was added, the worm suspension was then vortexed
vigorously and centrifuged at 14 krpm for 3 min. Total nematode
RNA in the supernatant was precipitated by ethanol, sodium
acetate and centrifugation. Precipitated RNA was further purified
by in gel-DNase digestion following the suggestion of Qiagen using
the RNeasy column. RNA concentration and integrity was
measured by a Nanodrop spectrophotometer.
cDNA was produced from total nematode RNA by reverse
transcription using the High Capacity cDNA Synthesis Kit and
random primers (Applied Biosystems/Life Technologies).
Quantitative, real-time PCR (qPCR) was performed by Applied
Biosystems SDS 7300 instrument using gene-specific forward
and reverse primers (Table 1) with the following parameters: 1
cycle of 50uC for 2 min, 1 cycle of 95uC 10 min and 40 cycles of
95uC for 15 sec followed by 60uC for 1 min. The levels of relative
gene expression were assayed by the 22DDCt method using the
F23B2.13 gene encoding an RNA polymerase small subunit as a
non-variable endogenous control.
Cytotoxicity Assay of Lycoris Compounds
Human fetal intestinal epithelial (FHs 74 Int) cells were
purchased from ATCC and grown in complete Hybri-care
medium (ATCC) supplemented with 30 ng/ml epidermal growth
factor, 10% fetal bovine serum, 20 units/ml penicillin and 20 mg/
ml streptomycin at 37uC with 5% CO2. 16104 FHs 74 Int cells in
100 ml medium were seeded in each well of the 96-well tissue
culture plate. After overnight growth, cells were washed with 16
PBS and incubated with 100 ml complete medium containing the
Lycoris compounds at the final concentration of 50 mM. After 24 hr
incubation, wells were washed with 16 PBS, 100 ml complete
medium was added to each well. MTS (Promega) reagent (20 ml)
was added into each well and the plate was incubated at 37uC with
5% CO2 for 2 hr. The cell viability was measured at 490 nm using
the Synergy HT Plate Reader. The cytotoxicity of Lycoris
compounds was assessed as the percentage reduction of cell
viability compared to the cells-only controls. All samples were
tested in triplicate and the assays were repeated twice.
The authors are greatly thankful to the critical review of the manuscript by
Dr. Monica Driscoll (Rutgers University, New Jersey, USA).
Conceived and designed the experiments: RD MAL CL. Performed the
experiments: LX RY HW YW WHW RD. Analyzed the data: LX RY
WHW RD. Contributed reagents/materials/analysis tools: RD MAL CL.
Wrote the paper: RD.
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