Curcumin reverses the scopolamine-induced amnesia through the upregulation of synaptic plasticity proteins

doi:10.21203/rs.3.rs-5280585/v1

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Curcumin reverses the scopolamine-induced amnesia through the upregulation of synaptic plasticity proteins

https://doi.org/10.21203/rs.3.rs-5280585/v1

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Curcumin is widely used as an Ayurvedic medicine for inflammation-related disorders. Recent experiments have shown its anti-carcinogenic, chemoprophylactic, antioxidant, anti-angiogenic, nootropic and immunomodulatory properties. Biochemical studies have reported that Curcumin efficiently alleviates amnesia and reduces Aβ plaques in animal models of neurodegenerative disorders. However, there are no clear reports on the molecular mechanisms elicited by Curcumin against neurological disorders, particularly amnesia or memory loss. As memory is a result of the interaction of several synaptic plasticity proteins, we hypothesize that Curcumin modulates the specific synaptic plasticity proteins in different brain regions associated with memory processing. To prove this hypothesis, we have investigated the effect of curcumin treatment on the expression level of synaptic plasticity proteins (Arc and FMRP) in scopolamine-induced amnesic male mice. Amnesia was assessed through the Morris water maze test, and protein level was analyzed through Western blotting. We observed a significant downregulation of Arc and FMRP during scopolamine-induced amnesia, which gets reversed by the pre- and post-treatment with Curcumin in both the hippocampus and prefrontal cortex of these mice. Our study reveals a molecular pattern of how Curcumin ameliorates amnesia, but additional research on upstream signalling pathways would support the curcumins’ medicinal use in memory problems.

Cellular & Molecular Neuroscience

Scopolamine

Amnesia

Gene expression

Synaptic plasticity

Curcumin

Over the last few decades, the use of phytomedicines, along with their active ingredients, has gained momentum in alternative and complementary medicine systems. Out of the several known herbal drugs, turmeric (Curcuma longa) is well known to possess anti-bacterial, anti-inflammatory, immunomodulatory, anti-arthritic, anti-oxidative, metal chelator, and anti-cancerous properties (Maiti & Dunbar, 2018). Curcumin, the active constituent and principal curcuminoid of turmeric, is obtained from the root of this plant. Curcumin can evade the blood-brain barrier (BBB) and reduce complications due to different neurological disorders. It can potentially attenuate memory impairment and retrieve the lost synaptic connections better than other drugs like memantine and diclofenac (Ali & Arafa, 2011). Epidemiological studies have suggested that in comparison with Western nations, Alzheimer's disease and other kinds of dementia are less common in Asia and India. (Chandra et al., 2001) (Michelle Melgarejo da Rosa, 2022). It is believed that the consumption of a higher amount of turmeric in the curries of Indian foods has helped people keep the disease at bay. However, research studies exploring the molecular mechanisms that lead to such neuroprotective effects by Curcumin are scarce and often do not reach a consensus.

The development of learning and memory is believed to be molecularly based on synaptic plasticity. It is described as the synapses' capacity to alter their strength and adapt in response to knowledge and experience. Brain areas are linked to learning and memory formation rapidly, and the expression of synaptic plasticity genes is selectively upregulated after neuronal stimulation (Kennedy, 2016). Arc (activity-regulated cytoskeleton-associated protein) and FMRP (Fragile X mental retardation protein) are both synaptic plasticity proteins and have been shown to regulate learning and memory processes (Minatohara et al., 2016; Banerjee et al., 2010). Arc is also an immediate-early gene that is quickly and transitorily induced in response to neuronal activity. It is primarily localized at synapses and plays a vital role in synaptic plasticity and memory formation. FMRP plays an important role in various forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). It is involved in regulating synaptic strength, dendritic spine morphology, and the capability of neurons to adjust and change in response to experience.

In this study, we hypothesized that Curcumin attenuates amnesia by modulating the expression of synaptic plasticity proteins (Arc and FMRP). As a result, we investigated how curcumin administration affected Arc and FMRP levels in amnesic mice. Scopolamine hydrobromide, a powerful acetylcholine receptor antagonist, which suppresses central cholinergic neuronal activity and degrades memory, was used to create an amnesic mouse model (Shandilya et al., 2020; SoukhakLari et al., 2019). The pre-and post-treatment of Curcumin was done in these amnesic mice, followed by an analysis of their behavioural and molecular changes. We performed the Morris water maze to evaluate spatial memory and Western blotting to observe the protein expression changes in these mice's hippocampus and prefrontal cortex (PFC).

2.1. Reagents

Curcumin (catalogue # C13865G), Scopolamine hydrobromide (catalogue # PHR1470), and phosphatase inhibitor cocktail 3 (#P0044) were purchased from the Sigma Aldrich Co. USA. Di-methyl-sulphoxide (DMSO) (catalogue # MB058), HEPES solution (#TCL021) were procured from the HiMedia laboratories Pvt. Ltd. Mumbai, India. Phenylmethane Sulphonyl Flouride (PMSF) (#87606) was purchased from Sisco Research Laboratories Pvt. Ltd, Maharashtra, India. Anti-Arc antibody (sc-17839) and M-IgGκ BP-HRP secondary antibody (sc-516102) were obtained from Santa Cruz, USA. Anti-FMRP antibody (MAB-2160) and ECL Ultra Western HRP substrate (catalogue # WBULS0100) were purchased from the EMD Millipore Corp., Germany.

2.2. Animals and drug treatments

Male Swiss albino mice of 5 ± 1 weeks were purchased from the National Institute of Nutrition (NIN), Hyderabad, India. They were housed at the University of Hyderabad's animal home under 24 ± 2°C on a 12-hour light/12-hour dark cycle, with access to mice food and water at will. The animals were utilized in accordance with the institutional animal ethics committee's rules (Permission number UH/IAEC/AG/2021-22/11). There were four groups of mice (n = 5) - control, amnesic, amnesic pre-treated with Curcumin, and amnesic post-treated with Curcumin. The dosage treatment for mice started every day at 9 am. The control group was administered with 0.1% DMSO followed by 0.9% saline after 30 min. The amnesic group was administered with 0.1% DMSO followed by 3mg/kg body weight (BW) scopolamine after 30 min. The pre-treated group first received 50mg/kg BW of Curcumin and then 3mg/kg BW of scopolamine after 30 min. The post-treated mice group received 3mg/kg BW of scopolamine, followed by 50mg/kg BW of Curcumin after 30 min. Scopolamine hydrobromide was dissolved in 0.9% saline, whereas Curcumin was dissolved in 0.1% DMSO. All dosages were given in intraperitoneal (i.p.) injections, and the dosage treatment continued for five consecutive days (Table 1). All the doses and duration were standardized in the preliminary trials.

Table 1

The experimental procedure: Each mouse was administered with drugs/vehicle according to its group at around 9 am from day 1 till day 5 with an interval of 30 min between two i.p. injections. MWM was conducted every day after 3 h of the last drug treatment. Each mouse went through 3 trials every day. On the 5th day, mice brain was isolated after 30 min of MWM and processed further for Western blot.
	Control	Amnesic	Pre-treatment	Post-treatment
Day 1 to Day 5	0.1% DMSO (i.p)	0.1% DMSO (i.p)	Curcumin (i.p)	Scopolamine (i.p)
	30 min
	0.9% Saline (i.p)	Scopolamine (i.p)	Scopolamine (i.p)	Curcumin (i.p)
	3 h gap
	Morris Water Maze – 3 trials for each mice
Day 5	30 min gap
Day 5	Brain Isolation
	Western Blot for Arc and FMRP

2.3. Morris water maze test

The Morris water maze test was done per the procedure we stated earlier (Gautam et al., 2013). MWM was a 120 cm diameter, black, circular tank that was filled with water. North, South, East, and West were the four quadrants that divided the tank virtually. A platform with a height of 35cm was submerged 1cm underwater in the tank's northeast direction (target quadrant). There were enough cues placed around the four directions of the tank wall, and a video camera was set up on the roof to record the movement of the mice in the maze. MWM was conducted 3 h after the last drug treatment, from 12:30 to 16:00 every day for five days. Mice were gently placed in one of the quadrants of the tank and allowed to discover the hidden platform to escape from the water within 1 minute. Each mouse was given three trials from different quadrants of the tank, and the procedure was repeated for five days. Through the recorded video, the time required to touch the platform, i.e., the escape latency, total distance travelled, and speed, was noted and later analyzed using ANY-MAZE software (Stoelting USA, Version 6.05).

2.4. Western blotting

All the mice were sacrificed 30 min after the MWM test on the fifth day. The hippocampus and PFC were isolated quickly on the ice to analyze protein expression. Both PFC and hippocampus are recognized to be major brain regions associated with memory and learning. The isolated brain regions were lysed with 10% RIPA buffer containing 1% PMSF and phosphatase inhibitor. The protein homogenate was centrifuged at 14000g at 4°C for 20 min. The resulting supernatant was collected and used to determine the concentration of proteins through the Bradford method (Bradford, 1976). 40 µg of protein from each mice group was mixed with 6X SDS sample buffer and boiled at 100°C for 5 min. The prepared protein was loaded on the 10% SDS-PAGE and ran at 120V. The separated proteins were transferred to the PVDF membrane with the help of a wet tank transfer system which was run at 60V for 7 h. The transferred proteins were blocked in 5% non-fat milk dissolved in TTBS (w/v) and incubated for 1 h. The blocked membrane was incubated overnight with primary antibodies: anti-Arc (1:500), anti-FMRP (1:500), and anti-GAPDH (1:10000) at 4°C. The membranes were washed thrice with TTBS for 15 min and then incubated with HRP-conjugated secondary antibody (1:5000) for 2 h. The membranes were again washed thrice with TTBS for 15 min. Finally, the chemiluminescent signal was detected by ECL reagents and captured with the help of BIORAD Image Lab Touch Software (version 2.4.0.03). The resultant images went through densitometrical analysis by ImageJ software (1.46r).

2.5. Statistical analysis

Data from different groups were compared and examined for the MWM test from the first to the fifth day. Each mouse completed three trials each day, and the data were statistically analysed using 2-way ANOVA followed by Bonferroni post-hoc test, taking p < 0.05 through IBM SPSS {28.0.1.0 (142)} for all parameters. All the molecular experiments were repeated thrice. The intensity of Western blot protein bands was visualized in BIORAD Image Lab Touch Software (version 2.4.0.03). The densitometry analysis of the proteins was done by ImageJ software (1.46r) after their normalization with GAPDH. The final results of protein expression levels were taken from three experiments and expressed as mean ± SEM. The significance between the groups was tested by individual t-tests taking p < 0.05.

3.1. Effect of curcumin treatment on the spatial memory of mice

The spatial learning and memory of mice were assessed via MWM for five days. Curcumin-treated amnesic groups were compared with the amnesic group for each day. The amnesic group was compared with the control group on each day, showing increased escape latency from day 4 onwards (day 4, F(3,200) = 14.967, amnesic p = 0.002; day 5, F(3,200) = 11.254, amnesic p < 0.001). On the other hand, the curcumin-treated groups showed decreased escape latency as compared to the amnesic group from 2nd day onward (day 2, F(3,200) = 9.697, pre-treatment p < 0.001, post-treatment p < 0.001; day 3, F(3,200) = 10.961, pre-treatment p = 0.001, post-treatment p = 0.003; day 5, F(3,200) = 11.254, pre-treatment p < 0.001, post-treatment p < 0.001) (Fig. 1).

To assess the learning rate for each group, the differences in escape latencies were examined day by day. Significant differences in escape latency were observed compared to the results from the previous day. Additionally, all the days were compared with day 1 to track the progress within each group. The control group showed a significant difference on day 5 when compared with day 4 (F(3,200) = 13.464, p < 0.001). No significant differences were observed upon comparison with day 1 except day 5 (p < 0.001). The curcumin-treated groups showed a significant decrease in escape latency on the second day compared to the first day (pre-treatment F(3,200) = 17.466, p = 0.002; post-treatment F(3,200) = 19.302, p < 0.001). This remained constant with no significant differences when compared with the previous day for the curcumin-treated groups. However, significant decrease in escape latencies was observed upon comparing days 2–5 with day 1 (pre-treatment day-2 p = 0.002, day-3 p < 0.001, day-4 p < 0.001, day-5 p < 0.001; post-treatment day-2 p < 0.001, day-3 p < 0.001, day-4 p < 0.001, day-5 < 0.001). On the other hand, the amnesic group did not have a significant difference when compared with the previous day. In comparison with day 1, the amnesic group showed a significant difference with only day 4 (amnesic F(3,200) = 9.302, p < 0.001) (Fig. 1).

Finally, the distance travelled and mean swimming speed of mice in each group were assessed to check for further differences in groups. Significant differences in distance travelled were observed from day 2 between groups. The amnesic group showed more distance travelled than the control group (day-2, F(3,200) = 7.084, p = 0.019). The curcumin-treated groups travelled less distance than the amnesic group (pre-treatment p < 0.001; post-treatment p < 0.001). No significant difference was observed on the other days for distance travelled. The mean swimming speed had no significant differences between groups.

3.2. Effect of curcumin treatment on the protein level

Arc expression: In the hippocampus, we observed a 23% decrease in Arc protein in the amnesic group compared to the control group (p = 0.03, t score = 5.08, df = 2). On the contrary, there was an insignificant increase of 48% and 31% Arc protein levels in the pre-and post-treated groups, respectively, as compared to the amnesic group (p > 0.05 for both) (Fig. 2a). In the prefrontal cortex, we observed a decrease of 45% Arc protein level in the amnesic group as compared to the control group (p = 0.007, t score = 11.76, df = 2). On the other hand, the pre-and post-treatment with Curcumin significantly increased the Arc protein level by 89% and 77% as compared to the amnesic group, respectively (p = 0.03, t score=-4.95, df = 2, and p = 0.03, t score=-5.08, df = 2 respectively) (Fig. 2b).

FMRP expression: We observed a decrease of 39% in the hippocampal FMRP protein level of the amnesic group compared to that of the control group (p = 0.02, t score = 6.60, df = 2). There was a significant increase of 98% and 55% hippocampal FMRP protein levels in the pre-and post-treated groups, respectively, as compared to the amnesic group (p = 0.003, t score = 15.78, df = 2 and p = 0.02, t score = 3.92, df = 2 respectively) (Fig. 3a). On the other hand, the prefrontal cortex showed a significant decrease of 40% FMRP protein level in the amnesic group as compared to the control group (p = 0.03, t score = 5.06, df = 2). The pre-and post-treatment with Curcumin in this amnesic mouse group significantly increased the FMRP protein level by 73% and 37% as compared to the amnesic group, respectively (p = 0.04, t score = 3.40, df = 2, and p = 0.03, t score = 3.03, df = 2 respectively) (Fig. 3b).

Curcumin has been in focus for treating all kinds of disorders, i.e. cardiovascular disorders, autoimmune diseases, neurological disorders, diabetes, etc. (Pari et al., 2008). In the current study, we have established a scopolamine-induced amnesic mice model and showed that Curcumin diminishes the scopolamine-induced amnesia through the upregulation of Arc and FMRP and hence possesses the nootropic effect. Arc is largely localized in neuronal dendrites and plays a critical part in memory consolidation. Studies have revealed the relation of Arc with LTP as well as LTD (Korb & Finkbeiner, 2011). The Fmr1 gene encodes FMRP, which plays a significant role in developing connections between neurons. It regulates the mRNA translation of various synaptic plasticity genes, including Arc, to maintain LTD (Sidorov et al., 2013).

Interestingly, pre-treatment with Curcumin in amnesic mice showed more pronounced upregulation of synaptic plasticity proteins compared to the post-treatment in both brain regions. PFC and hippocampus have been linked with spatial and episodic memory (Churchwell & Kesner, 2011). Behavioural studies have shown the integrated contribution of the PFC and the hippocampus in performing tasks (Avigan et al., 2020). Our results have shown that alleviating scopolamine effects on Arc by Curcumin is significant in PFC rather than the hippocampus. On the other hand, in the case of FMRP, Curcumin was effective in alleviating amnesic effects in both PFC and hippocampus. Therefore, this suggests that Curcumin has more pronounced effects in PFC than the hippocampus. Apart from our results, an earlier study by SoukhakLari et al., 2018 has also reported alleviating the scopolamine effect in p-Akt and p-GSK-3β after treatment with Curcumin. Another study by Hussain et al., 2022 reports the positive effects of curcumin analogues treatment on AChE and BuChE after scopolamine induction. Curcumin also alters the expression levels of other synaptic plasticity genes, such as BDNF, which is reported to be an upstream gene for IEG (Franco-Robles et al., 2014; Zhang et al., 2015). With this said, studies have also shown the ability of curcumin to impair fear memories in the amygdala by downregulating the upregulated IEGs- Arc and Egr-1 after fear conditioning (Melissa S Monsey, 2015). This suggests that curcumin undergoes various mechanisms in different brain areas.

Scopolamine has been proven as a non-selective muscarinic antagonist, which blocks acetylcholine's effects and disturbs synaptic plasticity homeostasis. Depending upon the dose, it can block NMDA receptors (Falsafi et al., 2012). The diminution of IEGs by scopolamine has been reported earlier (Lu et al., 2018; Gautam et al., 2013). The downstream process of FMRP is associated with the cholinergic system (Antar et al., 2004). There is not a lot of information connecting the cholinergic system to the FMRP upstream pathway. As our results demonstrate the decreased expression of both Arc and FMRP in the amnesic groups, we assume that the cholinergic system is also a part of the upstream process of FMRP. The Arc and FMRP protein levels demonstrate the recovery of scopolamine-induced amnesic effects of the curcumin treatment. However, these assumptions require further testing.

Arc expression is PKA-dependent, which is activated by NMDA receptors (Bloomer et al., 2008). Also, the activation of NMDA receptors enables the dendritic transference of Arc mRNA (Bramham, 2008). Thus, transportation and activation are disrupted in amnesic mice as scopolamine precludes NMDARs, leading to the ineffective activation of the gene and protein. Curcumin is shown to be effective in reversing these effects and resuming the activation of Arc. BDNF is a known upstream factor required for the activation of Arc. Curcumin also increases BDNF levels in rodent models (Franco-Robles et al., 2014). Curcumin might be involved in the activation of NMDA receptors, acetylcholine receptors and BDNF to increase the expression levels of Arc. Regulation of Fmr1 and FMRP is processed by the activation of metabotropic glutamate receptors (mGluR), which couples with phospholipase C (PLC) and activates protein kinase C (PKC) that leads to the transcription of the Fmr1 gene (Antar et al., 2004). Previous studies report that mGluR controls cholinergic synapses, but there is no evidence that activation of mGluR is associated with acetylcholine receptors.

Our study demonstrates the decline in the expression of FMRP after induction of scopolamine. Evidence suggests that the activation of mGluR translates to FMRP; thus, there is a possibility that the cholinergic system is involved in the indirect activation of FMRP by regulating the activation of mGluR or may be directly involved in the activation of the gene. Most synaptic plasticity proteins are localized in dendritic spines by NMDA receptors. However, the localization of FMRP is facilitated by mGluR itself. FMRP is also involved in translating proteins activated by mGluR and in mGluR-LTD by inhibiting the translation of proteins like Arc. FMRP knockout mice studies have reported elevated Arc and other LTD protein levels (Sidorov et al., 2013). Based on our results and previous studies, we hypothesize that the curcumin may reactivate the cholinergic receptors and the mGluR and restart the FMRP synthesis, leading to the translation of Arc and other LTD proteins (Fig. 4).

AChE

Acetylcholinesterase

ANOVA

Analysis of Variance

Arc

Activity Regulated Cytoskeleton–Associated Protein

BBB

Blood Brian Barrier

BDNF

Brain–derived neurotropic Factor

BuChE

Butyrylcholinesterase

Body Weight

degree of freedom

DMSO

Di–methyl–sulphoxide

DNA

Deoxyribonucleic Acid

ECL

Enhanced Chemiluminescence

Fmr1

Fragile X Messenger Ribonucleoprotein 1

FMRP

Fragile X Mental Retardation Protein

GAPDH

Glyceraldehyde − 3 Phosphate Dehydrogenase

HEPES

4–(2–hydroxyethyl)–1–piperazine ethane sulphonic acid

HRP

Horseradish Peroxidase

i.p.

intraperitoneal

IEG

Immediate Early Gene

LSD

Least Significant Difference

LTD

Long–Term Depression

LTP

Long–Term Potentiation

mGluR

Metabotropic Glutamate Receptor

IgGκ BP–Mouse IgGκ light chain Binding Protein

mRNA

messenger Ribonucleic Acid

MWM

Morris Water Maze

NIN

National Institute of Nutrition

NMDA

N–methyl–D–aspartate

NMDAR

N–methyl–D–aspartate Receptor

Akt–Protein Kinase B

PFC

Pre–Frontal Cortex

GSK–3β–Glycogen Synthase Kinase 3 beta

PKA

Protein Kinase A

PKC

protein kinase C

PLC

phospholipase C

PMSF

Phenylmethane Sulphonyl Flouride

PVDF

Polyvinylidene Fluoride

RIPA

Radioimmunoprecipitation assay buffer

SDS

Sodium Dodecyl Sulphate

SDS

PAGE–Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

SEM

Standard Error of Mean

TTBS

Tween–20 Tris–Buffered Saline

Conflict of interest:

6. Authors declare no conflict of interest.

Acknowledgements:

5. This work was supported financially by grants from the International Society for Neurochemistry Committee for Aid and Education in Neurochemistry (ISN-CAEN 1B) and principally by the University of Hyderabad Institution of Eminence (UoH-IoE), India.

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The authors declare no competing interests.

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Curcumin reverses the scopolamine-induced amnesia through the upregulation of synaptic plasticity proteins

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Abstract

Figures

1. Introduction

2. Methodology

2.1. Reagents

2.2. Animals and drug treatments

2.3. Morris water maze test

2.4. Western blotting

2.5. Statistical analysis

3. Results

3.1. Effect of curcumin treatment on the spatial memory of mice

3.2. Effect of curcumin treatment on the protein level

4. Discussion

Abbreviations

Declarations

Conflict of interest:

Acknowledgements:

References

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