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

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 NEURONS CONDUCT ELECTRICAL IMPULSES AND ARE THE PRINCIPLE MEDIATORS OF MEMORY FUNCTION

Protein misfolding disorders Alzheimer’s disease Huntington’s-Chorea Parkinson’s disease

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  The most affected brain structures are the hippocampal formation and cerebral cortex

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

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  Induce cell damage

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

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

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  MS, epilepsy, age-related disease such as AD or Parkinsons, neurodegenerative disease

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 Interleukins are a group of cytokines that are expressed by white blood cells (leukocytes, hence the -leukin) as a means of communication (inter-). The function of the immune system depends in a large part on interleukins, and rare deficiencies of a number of them have been described, all featuring autoimmune diseases or immune deficiency. NGF-Nerve growth factor is a naturally occurring protein that is neurotrophic

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 TGFb is secreted by platelets, macrophages and lymphocytes. It has many functions which include increasing IL-1 production by activated macrophages, inducing a class switch to IgA by proliferating B cells, acting as a chemo-attractant for monocytes and macrophages, and inhibiting the proliferation of cells required for the inflammatory process. This means that TGFb actually aids in wound healing because it limits the inflammation caused by injury. TGFb is a very powerful inhibitor of lymphocyte function but stimulates other cells

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 Toll-like receptors (TLRs) are type I transmembrane proteins and their function is the recognition of pathogens and the activation of immune cell responses directed against those pathogens. The eukaryotic nuclear factor kB (NF-kB) plays an important role in inflammation, autoimmune response, cell proliferation, and apoptosis by regulating the expression of genes involved in these processes.

Hippocampus Anatomy CA2 CA1 Corpus Callosum Dentate gyrus

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

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

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

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