vitro and in vivo studies for novel therapeutic approaches. Roberto - - PowerPoint PPT Presentation

Neuronal oxidative injury in the development of the epileptic disease: in vitro and in vivo studies for novel therapeutic approaches.

Roberto Di Maio Ph.D.

Ri.MED Foundation, Italy Pittsburgh Institute for Neurodegeneratine Diseases

• Dept. of Neurology

University of Pittsburgh

Fifth annual Ri.MED Symposium

Priming the therapeutic pipeline: new strategies for drug discovery

Palermo, October 24th 2011

reduced Oxidized

Resting cell Stimulation of proliferation Mild oxidative insult release of reactive metals Severe oxidative insult mitochondrial DNA and DNA oxidative damage

Initiation of apoptosis

Apoptosis Extreme

• xidation

Oxidation of Caspases Apoptosis Necrosis Adaptative response Transcription factors Protective systems

O

X

Redox state

Redox state and biological events

Introduction

Epileptic disorders

• The most frequent neurodegenerative diseases after stroke
• Affect approximately 50 million of the world population
• 500,000 Italian have been diagnosed with epilepsy
• Contribute 0.5% of the global burden of disease
• Approximately 30% of epileptic patients are unresponsive to AEDs
• Temporal Lobe Epilepsies (TLEs) are the most common forms of

pharmaco-resistant epilepsy

Prevention of epilepsy is one of the main Epilepsy Research Benchmarks

Pilocarpine model of epilepsy Human Temporal Lobe Epilepsy NMDAR-mediated

• xidative damage and

exctitoxicity Apoptosis and Cell death Loss of neurons NMDAR subunits altered stoichiometry

The human Temporal Lobe Epilepsy Pilocarpine as model of Temporal Lobe Epilepsy

Glutamatergic paroxysm

?

Hippocampal damage

• Investigate the oxidative-related biochemical mechanisms

involved in altered NMDARs stoichiometry

• Develop a simple experimental model of hyper excitability

aimed to dissect the cellular mechanisms of epileptogenesis

Experimental design

Primary hippocampal neurons PILO 4µM

* * *

Horowitz, Milanese, Di Maio et al., Antiox. Redox Signal, 2011

Ratio SS/SH

Pilocarpine alters NMDA receptor expression and function in hippocampal neurons: NADPH oxidase and ERK1/2 mechanisms

Roberto Di Maio, Pier G. Mastroberardino, Xiaoping Hu, Laura Montero and J. Timothy Greenamyre Neurobiology of Disease, 2011

* * * *

Summary 1 PILO in primary hippocampal neurons recapitulates some features

• f epilepsy- associated hyper excitability

Primary hippocampal neurons PILO 4µM Time point: 24 hours PILO model of TLE in rat PILO 360 mg/Kg Time point: 24 hours

• How can thiol oxidation contribute to the development
• f chronic epilepsy?
• How can NR2B subunit lead to the development of the

epileptic disease?

Oxidative stress and NR2B containing NMDA autoreceptor up-regulation: relevance in epilepsy

Oxidative stress and NR2B containing NMDA autoreceptor up-regulation: relevance in epilepsy

N-Acetylcysteine (NAC) 300µM

Primary hippocampal neurons PILO 4µM Time point: 48 hours PILO model of TLE in rat PILO 360 mg/Kg; Ifenprodil 30 mg/Kg Time point: 48 hours

Summary 2

Apoptosis Cell death

pERK NMDAR2A upregulation

Oxidative stress and NR2B subunit’s function/expression could constitute selective targets for novel therapeutic approaches aimed to prevent the development of chronic epileptic diseases.

How can cannabinoids prevent chronic epilepsy?

The pharmacological stimulation of CB1 receptor during the epileptic

• nset could be effective to prevent the consequences of glutamatergic

paroxysm and the occurrence of the epileptic chronic brain damage

• CB1 receptor stimulation exerts antiepileptic effects in

animal models of acute epilepsy

• Cannabinoids are neuroprotective against oxidative

stress and neuronal inflamation

Experimental Design

Adult male Sprague-Daweley rats (initial weight: 250-280g) PILO 360 mg/Kg i.p. WIN 55,212-2 (CB1 receptor agonist; 2mg/kg s.c.)

Behavioral studies

Results

Behavioral studies

RACINE SCALE

(MODIFIED BY PINEL AND ROVNER)

0 = immobility 1 = facial automatisms 2 = head myoclonus 3 = forelimb myoclonus 4 = rearing 5 = falling 6 = more than three falls 7 = wild running 8 = tonic–clonic seizures

Results

Hippocampal sclerosis

Timm’s Staining

Results

Oxidative stress

Results

NR2A NR2B subunits expression

Results

GABA and CB1 receptor expression

Conclusions

CB1 receptor could represent a potential target for novel therapeutic approaches aimed to prevent the development of chronic TLE, the most common intractable form of epilepsy.

Future Directions

• Provide further insight into the efficacy of CB1 receptor agonists

in preventing the oxidative stress-mediated epileptogenesis in adult and in developmental brain

• Investigate the role of oxidative stress in the aberrant neuronal

proliferation of the epileptic hippocampus

• Test the potential efficacy of CB1 receptor agonists in the

prevention of the epileptic abnormal neurogenesis

Acknowledgements

• J. Timothy Greenamyre, MD, PhD

Professor & Vice-Chair of Neurology Director, Pittsburgh Institute for Neurodegenerative Diseases University of Pittsburgh

Xiaoping Hu

Cell cultures

Laura Maria Montero

Immunohistochemistry

Jason R. Cannon, PhD

University of Pittsburgh Neurochemistry