National Institute of Pharmaceutical Education and Research,Guwahati -32, Assam (India)

INSULIN SIGNALLING: AN OPPORTUNISTIC TARGET TO MINIFY RISK OF ALZHEIMER’S DISEASE AND PERSPECTIVE

TREATMENT.

Final Presentation

PRESENTED BYRohit Mohan PardeshiM.S. (Pharm) Biotechnology NIPER, Guwahati

UNDER THE GUIDANCE OFDr. Ranadeep GogoiAssistant professor,

Dept. of BiotechnologyNIPER, Guwahati

CONTENTSINTRODUCTION

AIM & OBJECTIVES

HYPOTHESIS

MATERIALS & METHODS

RESULTS & DISCUSSION

CONCLUSION

ACKNOWLEDGEMENT

REFERENCE

3

INTRODUCTION

Alzheimer’s disease (AD) is progressive neurodegenerative disorder characterised by accumulation of senile plaques, neurofibrilatory tangles(NFT) and neurodegeneration.

Various studies have demonstrated that abnormalities such as diabetes, hypertension, sleep disorder, psychological stress increase the risk for Alzheimer’s disease .

Diabetes associated with insulin resistance and reduced insulin level mainly affects attention, learning memory, cognition and psychomotor efficiency.

Disturbance in down-regulation of insulin signalling leads to memory impairment through inhibition of serine phosphorylation of IRS-1, activation of GSK-3 β and decrease in level of Insulin Degrading Enzyme (IDE) which results in amyloid beta aggregation and formation of NFT.

Also the decrease in level of insulin or insulin resistance generate oxidative stress as well as reduce the central blood flow thus it marks over neurodegeneration.

DMInsulin

resistance / Insulin

OBJECTIVESTo develop and validate the diabetic cognitive and memory impairment induced by streptozotocin in SD rats.

To evaluate the effect of Edaravone and sEHI on the expression of β amyloid-42, Acetylcholine esterase,GSK-3β and IDE genes in diabetic cognitive and memory impairment.

To determine the effect of Edaravone and sEHI on the level of Acetylcholine esterase in diabetic cognitive and memory impairment rats.

To assess the biochemical and antioxidant parameters & level of cytokines involved in diabetic cognitive and memory impairment.

AIM :- To study the effect of Edaravone and sEHI on streptozotocin (STZ) induced diabetic cognitive and memory impairment in rats .

To evaluate the effect of Edaravone and sEHI on histological changes in diabetic cognitive and memory impairment.

To determine the combination effects of nutritional supplement (DHA) with sEHI ondiabetic cognitive and memory impairment.

IUPAC Name (2-deoxy-2-(3-methyl-3-nitrosourea)-1-D glucopyranose)

Molecular Formula C8H15N3O7

Molecular weight 265 g/mol

STREPTOZOTOCIN

Structure of STZ

Soluble epoxide hydrolase inhibitor(sEHI)

IUPAC Name= trans-4-[4-(3-adamantan- 1-ylureido)-

cyclohexyloxy]-benzoic acid (t-AUCB)Molecular weight = 403.24 g/mol

EDARAVONEIUPAC Name 5-methyl-2-phenyl-4H-pyrazol-3-one

Molecular Formula C10H10N2O

Molecular weight 174.19 g/mol

DOCOSAHEXAENOICACID (DHA)IUPAC Name all-cis-4,7,10,13,16,19-docosahexaenoic acid

Molecular Formula C 22H 32O 2

Molecular weight 328.48 g/mol

HypothesisDiabetes mellitus

Insulin OR -Insulin Resistance IDE

Aβ

Oxidative stressFOXO

IRS-1

PI3K

AKT

P

INSULIN

Memory impairment

TNFα

ALZHEIMER’S DISEASE

GSK3B

γ-secretase

Aβ

Formation of plaque

Tau Phosphorylation

NFT

Phosphatase

Neuronal Cell death

JNK Activation

EDV & sEHI

Materials and Methods

Species Rat

Strain and Number Sprague Dawley (6-animals per group)

Sex Male

Weight 200-250 g

Streptozotocin 50mg/kg i.p

Animals

Inducing Agents

Pharmacological InterventionsEdaravone (3mg/kg and 10mg/kg) i.psEHI ( 1mg/kg p.o.)DHA (100mg/kg p.o.)

The study is approved by the Institutional Animal Ethical Committee(IAEC), GMCH Guwahati, with the approval no: MC/05/2015/73

10

Experimental Design

8 7 16 5 4 3 2

Checking Blood Glucose level weekly after STZ administration

Weeks

STZ 50mg/kg ip

Edavarone 3mg/kg & 10mg/kg i.psEHI 1mg/kg PODHA 100mg/kg PO

Neurobehavioral study. RNA extraction and Reverse transcriptase PCR for specific genes. Estimation of Biochemical and oxidative parameters . Histopathology of hippocampus .

Normal control Group

STZ (50mg/kg) IP single dose.

STZ (50mg/kg)IP + Edaravon (10mg/kg) IP for 2 weeks, twice a day

STZ (50mg/kg) IP + Edaravon(3mg/kg) IP for 2weeks,twice a day

Experimental animals have divided into six groups consisting of six animals in each group

STZ (50mg/kg)IP+ sEHI (1mg/kg) p.o.Group-5

Group-4

Group-3

Group-2

Group-1

Group-6 STZ (50mg/kg)IP+ sEHI (1mg/kg) p.o + DHA(100mg/kg)p.o

1. Effect of Edaravone & sEHI on Body weightRESULTS & OBSERVATIONS

2. Effect of Edaravone and sEHI on Blood Glucose Level

3. Effect of Edaravone and sEHI on neurobehavior

4. Effect of Edaravone and sEHI on neurobehavior

5. Effect of Edaravone and sEHI on the level of MDA

6. Effect of Edaravone and sEHI on nitrite level

7. Effect of Edaravone and sEHI on the level of GSH

8. Effect of Edaravone and sEHI on Serum Level of IL-1β

9. Effect of Edaravone and sEHI on Serum Level of IL-6

10. Effect of Edaravone and sEHI on the expression of APP

APPβ-actin

11. Effect of Edaravone and sEHI on the expression of AchE

Acetylcholinesteraseβ-actin

12. Effect of Edaravone and sEHI on the expression of GSK-3β

GSK-3 β

β-actin

13. Effect of Edaravone and sEHI on the expression of IDE

IDE

β-actin

14. Effect of Edaravone and sEHI on tissue level of AchE

HISTOLOGICAL CHANGES IN HIPPOCAMPUS TISSUE

1. Normal Control 2. Pathological control

histopathological changes in the hippocampus of STZ treated rats revealed by hematoxylin and eosin (HE) staining (400X).

Effects of Edaravone on histopathological changes in the hippocampus of STZ treated rats revealed by hematoxylin and eosin (HE) staining (400X).

3. Edaravone 3mg/kg 4. Edaravone 10mg/kg

HISTOLOGICAL CHANGES IN HIPPOCAMPUS TISSUE

5. sEHI 1mg/kg 6. sEHI+ DHA

Effects of sHEI and DHA on histopathological changes in the hippocampus of STZ treated rats revealed by hematoxylin and eosin (HE) staining (400X).

HISTOLOGICAL CHANGES IN HIPPOCAMPUS TISSUE

Diabetes associated with insulin resistance and reduce insulin level mainly affects psychomotor activity, attention, learning memory, cognition, mental flexibility in brain. [Bakris G.L 2009].

According to Garwood J et al., disturbance in insulin signalling leads to amyloid beta aggregation and neurofibrillatory tangles formation in brain which progressed into AD and memory impairment.

In DM the enhancing GSK-3β activity by inhibition of IRS-1 phosphorylation [Bedse G. et al.,2015] and insulin resistance leads to the reduced IDE expression which is associated with the significant risk factor for AD due to the decreasing metabolism of amyloid beta [David G.Cook et al.,2003].

Edaravone substantially reduced the amyloid beta deposition, oxidative stress, tau phosphorylation, neurodegeneration in mice [Jiooss et al., 2015].

DISCUSSION

According to clinical trial reports DHA seen to be slower the progression of dementia, thus it can provide as nutritive supplement for AD treatment [Gerg M Cole et al., 2013].

Giorgio Poli et al., demonstrated that elevation of the level of epoxygenated fatty acid by inhibition of sEH may have the utility in treatment of neurodegenerative pathological conditions including prion and AD.

The present study also revealed that the decreased expression of GSK-3β and increased expression of IDE by Edaravone and sEHI improved the cognitive impairment in DM rats.

Reduced levels of cytokines such as IL-1β & IL-6 and results of antioxidant parameters reveals the protective roles of the drugs against neuroinflammation oxidative stress associated with AD.

Histopathological report also supports the activity of Edaravone and sEHI against neuronal loss showing marked improvement of morphological alteration.

DISCUSSION

CONCLUSION From the above results it has been suggested that Edaravone, sEHI and combination of sEHI and DHA improves the morphological and functional alterations associated with cognitive and memory impairment by the improvement of insulin signalling. Edaravone 10mg/kg dose shows more effective in cognitive and memory impairment than low dose (Edaravone 3mg/kg).

sEHI is more effective than Edaravone in diabetic cognitive and memory impairment.

The combination of sEHI with DHA shows more effect than the individual drugs, Edaravone and sEHI, in diabetic cognitive and memory impairment. So DHA can be a promising nutritive supplement for the treatment of AD.

But, the combination of DHA with sEHI elevated the blood glucose level even sEHI alone can reduce the blood glucose level. Hence, sEHI can be useful for the treatment of DM related AD rather than combination.

While managing diabetic patients, it is necessary to provide treatment for reduce the risk factors of AD as they are more prone to it.

REFERENCES• Peraza, L.R., et al., Regional functional synchronizations in dementia with Lewy

bodies and Alzheimer's disease. Int Psychogeriatr, 2016• Zhao, W.Q. and M. Townsend, Insulin resistance and amyloidogenesis as

common molecular foundation for type 2 diabetes and Alzheimer's disease. Biochim Biophys Acta, 2009.

• de la Monte, S.M. and J.R. Wands, Alzheimer's disease is type 3 diabetes-evidence reviewed. J Diabetes Sci Technol, 2008.

• Zhao, W.Q., et al., Insulin and the insulin receptor in experimental models of learning and memory. Eur J Pharmacol, 2004.

• Vekrellis, K., et al., Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme. J Neurosci, 2000.

• Yarchoan, M. and S.E. Arnold, Repurposing diabetes drugs for brain insulin resistance in Alzheimer disease. Diabetes, 2014.

• Reger, M.A., et al., Intranasal insulin improves cognition and modulates beta-amyloid in early AD. Neurology, 2008.

• Reger, M.A., et al., Intranasal insulin improves cognition and modulates beta-amyloid in early AD. Neurology, 2008. 70(6): p. 440-8.

ACKNOWLEDGEMENT I would, like to thank my guide Dr. Ranadeep Gogoi, Assistant Proffessor,NIPER-G;

Prof.A.K Adhikari, Director, NIPER-G;

Dr. B.K Bezbarauh,Project Director, NIPER-G;

Dr. Mangala Lahkar, Chief academic co-ordinator, NIPER-G;

Dr.J. Kotoky, Registrar, NIPER-G;

Dr.Pawan Kumar Samudrala, Assistant Professor, NIPER-G;

Dr. Utpal Mohan, Assistant Professor, NIPER-G;

Dr.Swapnil Sinha,DST women Scientist

Mrs. Neejara Sharma;

Mr. Ratan J. Lihite;

Special thanks to IBT hub NIPER-G ; HLA Lab, GMCH & Dept. of Pathology,

GMCH.

At last, I would like to thank all PhD scholars, My dear batch-mates and beloved

juniors

for your immense support throughout the NIPER-G life.