Introduction

1. Protection of rat primary hippocampal cultures from Aβ cytotoxicity by pro-inflammatory molecules is mediated by astrocytesNeurobiology of disease, Vol 19 (2005) 243-254presentation byAshim Malhotra, Brian Scharf, Sushil Pai & Ann-Marie Matei

2. Introduction • Central nervous system (CNS) consists of the brain and the spinal cord • In the brain there are two principle cell types, the neurons and the glia cells • Part of the glia are the astrocytes which are most commonly thought to provide a host of essential support functions for neurons

3. Protein misfolding disorders • Alzheimer’s disease • Huntington’s-Chorea • Parkinson’s disease

4. Introduction cont’d • Alzheimer’s disease (AD) - Multifactorial disease - The rate of synapse loss and neuronal cell death determines the onset and/or the progression of dementia - Impairment of mental function is preceded by the development of two lesions:  deposition of fibrillar β-amyloid peptide (Aβ) as insoluble extracellular aggregates forming the core of senile plaques  appearance of intracellular neurofibrillary tangles

5. Introduction cont’d • AD cont’d - β-amyloid plaques are characteristic hallmarks of AD - Aβ is derived from the cleavage of the amyloid precursor protein and varies in length from 39 to 42 amino acids - Aβ42 occurs more frequently and forms fibrillar aggregates more readily

6. Introduction cont’d • β-amyloid - Can activate inflammatory pathways by enhancing microglial secretion of inflammatory cytokines - It can trigger production of reactive oxygen species (ROS), nitrogen intermediates and TNF-α from microglia cells

7. Introduction cont’d • Microglial cells - Phagocytic cells - Major immunocompetent component of CNS - Serve as scavenger cells in the event of infection, inflammation, trauma and neurodegeneration - Produce several cytokines responsible for autocrine regulation and communication with neurons, astrocytes and leukocyte infiltrates

8. Introduction cont’d • Microglial cells cont’d - Gradual activation safeguards CNS homeostasis, tissue defense and immune reactivity - The AD brain is characterized by the presence of senile plaques surrounded by abundant activated microglia

9. Introduction cont’d • Astrocytes - The most abundant cell type in the brain - These cells fill the spaces between neurons - Contribute to brain homeostasis in several ways:  buffering of extracellular K+  regulating neurotransmitter release  forming the blood-brain barrier (BBB)  releasing growth factors  regulating the brain immune response - Play a role in a variety of diseases

10. Introduction cont’d • Astrocytes cont’d - Considered to be the structural and trophic support of the CNS - Antioxidant defense mechanism because they contain superoxide dismutase (SOD), glutathione peroxidase, glutathione - When stimulated by pro-inflammatory molecules they secrete IL-1β and nerve growth factor (NGF) potentially increasing the viability of damaged neurons

11. Introduction cont’d • Astrocytes cont’d - TGFβ1 mRNA increases in astrocyte cultures exposed to lipopolysaccaride from gram-negative bacteria (LPS), interferon gamma (IFN-γ) or tumor necrosis factor alpha (TNF-α) - TGF-β1 increases upon administration of IL-1β & elicits a synergism with NGF - There are however, contradictory reports regarding the protective role of astrocytes

12. Introduction cont’d • In this paper the effect of pro-inflammatory molecules LPS + IFN-γ was evaluated on the activation of glial cells and neurotoxicity induced by Aβ • LPS and IFN-γ have been widely used in different in vitro & in vivo experimental approaches for the study of Alzheimer’s & other neurodegenerative diseases

13. Hippocampus Anatomy CA2 CA1 Corpus Callosum Dentate gyrus

14. Materials & Methods Techniques • Cell culture • Hippocampal • Glial • Immunocytochemistry • Apoptosis assay: TUNEL • Cell Viability assay: MTT

15. Cell culture: Hippocampi 18 day SD rat hippocampi HBSS, pH 7.4 + trypsin 10% MEM stopped digestion After 24h 2uMAraC Anti- ( tubulin III & GFAP)

16. Principle of TUNEL assay 5’ 3’ DNA fragment w/ free 3’ -OH 5’ TdT BrDU 5’ Ab against BrDU

17. MTT Assay MOM mitochondrion SDH Formazan Courtesy: http://www.nikoderm.com/jyudakushiken/saibo_img/photo_001.jpg