Grant Proposal DSTCSRI Grant proposal 1 Unraveling the

Grant Proposal – DST/CSRI Grant proposal 1

Unraveling the molecular mechanisms involved in therapeutic role of Tylophorinidine on behavioral alterations in Animal models of Autism – Preclinical Approach Principal Investigator Dr. Shyamjith Manikkoth Associate Professor Department of Pharmacology Yenepoya Medical College Grant proposal 2

Contents • • Rationale Objectives Hypothesis Methodology Expected outcome Timeline and Budget References Grant Proposal 3

AUTISM Grant Proposal 4

Autism and Behavioral alterations • Neurodevelopmental disorder in which the central nervous system are impaired. • A stereotype pattern of behaviors characterized by severe irregularity in communication, social awareness and skillfulness are commonly observed[1] • Has two main symptoms: üsocietal communication deficit ürecurring or limited behaviors or interests. Grant proposal 5

Autism and Behavioral alterations • Individuals with autism regularly show various allied symptoms[2] such as – • Anxiety • Depression • Sleeplessness • Gastric disorders • Irritability and aggression • Hypo-sensory problems Grant proposal Self-injury Epilepsy Attention deficits Hyperactivity Mental retardation Hyper-sensory problems 6

Autism and Behavioral alterations • A variety of factors are implicated in the etiopathogenesis of autism or autism spectrum disorder (ASD): [3] 1. 2. 3. 4. 5. 6. 7. Weakened immune responses Neuro-inflammation Unusual neurotransmission Oxidative stress Mitochondrial dysfunction Environmental toxins Epigenetic modifications Grant proposal 7

Need for the Study • Despite advances in early diagnosis and intercession, no therapy has been yet proven to completely reverse the core symptoms of autism. In the modern day therapy, there is no effective curative treatment for autism. • This has, thus, driven the health care professionals to develop a new agent which can alleviate autism with less side effects. Grant proposal 8

• In this perspective, role of phytochemicals comes into picture. • Considerable importance given to the active plant metabolites with minimal adverse effects to treat autism and behavioral alterations. Grant proposal 9

Tylophora indica - Slender perennial climber Family- Asclepiadaceae Indian Ipecacuanha Traditional uses: bronchial asthma, rheumatism, allergies, diarrhoea Grant proposal 10

Tylophora indica Previous studies • Anti- asthmatic, anti- inflammatory, anti anxiety, antirheumatic, analgesic, anticonvulsant, hepatoprotective and antioxidant activity. Grant proposal 11

Tylophora indica From the HPLC-LCMS analysis it was found that Tylophorinidine is the active constituent present in the ethanolic extract of Tylophora indica and is responsible for many pharmacological properties. This phytoconstituent is an isoquinoline derivative. Grant proposal 12

Phytochemical Analysis Of TIEE using HPLC- LCMS Major Peaks with their Retention time in minutes 5. 79 Peak Area % Molecular Weight Observed Possible compound 39. 325 360. 0 Tylophorinidine Grant proposal 13

Objectives • To study the effect of Tylophorinidine – On behavioural abnormalities in Valproic acid induced autism in Wistar Albino rats. – On neuroinflammation seen in autism by estimating IL-6, TNF-α and BDNF levels. – On Neurotransmitters and Antioxidant levels • To study the protective role of tylophorinidine in autism by quantifying the JAK - STAT pathway. • To analyze whethere is any alteration in the methylation pattern in Promoter Cp. G Islands of GABRB 3. Grant proposal 14

Hypothesis Having shown promising neuroprotective activity in various studies, Tylophoridine can play a pivotal role in autism treatment. Grant proposal 15

Materials and methods VALPROIC ACID TYLOPHORINIDINE WISTAR ALBINO RATS Grant proposal TYLOPHORA INDICA 16

Sl. No. Work Plan Duration required 6 months 1. Isolation of tylophorinidine from Tylophora indica 2. Studies on therapeutic role of tylophorinidine on behavioral abnormalities in Valproic acid induced autism in Wistar albino rats Estimation of IL-6 , cytokines and BDNF levels to study the protective role of tylophorinidine in autism 8 months 4. Quantification of neurotransmitter release in the brains of autistic Wistar rats 1 month 5. Evaluating the protective role of tylophorinidine on oxidative stress induced in autistic animals 1 month 6. To assess the role of tylophorinidine in molecular alterations associated with JAK-STAT pathway 3 months 7. To analyze whethere is any alteration in the methylation pattern in Promoter Cp. G Islands of GABRB 3 8 months 3. Grant proposal 3 months 17

Methodology 1. Behavioral Abnormalities - Rota – rod test, touch response and pain response - Actophotometer test and Open field test - Elevated plus maze and light dark arena tests - Morris water maze and runway apparatus test - Forced swimming test and tail suspension test 2. Biochemical Estimations - ELISA kits - Immunofluorescence - Western Blotting technique Grant proposal 18

Methodology 3. Neurotransmitter and Antioxidant Investigations - Spectrophotometric and Spectrofluorimetric techniques - Flow cytometric assays 4. JAK – STAT pathway detection - ELISA kits 5. DNA alteration - q. RT – PCR Grant proposal 19

Expected outcome and Applicability The study aims at understanding and unraveling the molecular mechanisms associated with therapeutic potential of tylophorinidine in autism using experimental models. Results of the study will pave an insight into the possibility of developing tylophorinidine as a novel sole therapeutic agent for mitigating behavioral abnormalities in autism. Grant proposal 20

Activities 1 -6 Time schedule (Months) 13 -18 19 -24 7 -12 25 -30 31 -36 Literature survey Isolation of tylophorinidine from Tylophora indica Evaluation of therapeutic potential of tylophorinidine on autism model Estimation of IL-6 , cytokines and BDNF levels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Quantification of neurotransmitter release 18 Protective role of tylophorinidine on oxidative stress in autism 19 Assess the role of tylophorinidine in molecular alterations associated with JAK-STAT pathway Analysis of alteration in DNA methylation 20 21 22 23 Data compilation, publications Grant proposal 24 25 26 27 28 29 30 31 32 33 34 35 36 21

Budget ITEMS A. B. 1 st Year BUDGET 2 nd Year 3 rd Year Salaries/wages 1, 92, 000 5, 76, 000 Consumables 10, 000 9, 000 8, 000 27, 000 Travel 50, 000 1, 50, 000 Other costs 20, 000 60, 000 Equipment Grand total (A+B) Total FEC* 10, 000 Total (INR) Recurring 10, 000 44, 86, 000 Grant proposal 22

References 1. Christina M. Corsello. Early Intervention in Autism. Infants & Young Children, 2005; 18(2): 74 -85 2. Emily JH. Jonesa, Teodora Gligaa, Rachael Bedfordc, Tony Charmanb, Mark H. Johnsona. Developmental pathways to autism: A review of prospective studies of infants at risk. Neuroscience and Biobehavioral Reviews, 2014; 39: 1– 33 3. Necmi Namala, Hayriye Ertem Vehitb, Selcuk Koksalc. Do autistic children have higher levels of caries? A cross-sectional study in Turkish children. J Indian Soc Pedod Prev Dent, 2007; 97 -100 4. Muthu MS, Prathibha KM. Management of a child with autism and severe bruxism: A case report. J Indian Soc Pedod Prevent Dent, 2008: 82 -84 5. Daniel A. Rossignol. Novel and emerging treatments for autism spectrum disorders: A systematic review. Annals of Clinical Psychiatry 2009; 21(4): 213 -236. 6. Alexis Tchaconasa and Andrew Adesman. Autism spectrum disorders: a pediatric overview and update. Curr Opin Pediatr 2013, 25: 130– 143 7. Helen V. Ratajczak. Theoretical aspects of autism: Causes—A review. Journal of Immunotoxicology, 2011; 8(1): 68– 79 8. D. Ebrahimi-Fakhari, M. Sahin, Autism and the synapse: Emerging mechanisms and mechanism-based therapies. Curr. Opin. Neurol. 28, 91– 102 (2015) 23 Grant proposal

THANK YOU Grant proposal 24