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Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a <em>C. elegans</em> Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

The City University of New York (CUNY)

In stroke and several neurodegenerative diseases, malfunction of glutamate (Glu) transporters causes Glu accumulation and triggers excitotoxicity. Many details on the cascade of events in the neurodegenerative process remain unclear. As molecular components of glutamatergic synapses are assembled in Caenorhabditis elegans and as many fundamental cellular processes are conserved from nematodes to humans, we studied Glu-induced necrosis in C. elegans and probed its genetic requirements. We combined deltaglt-3, a Glu transporter-null mutation, with expression of a constitutively active form of the alpha subunit of the G protein Gs. While neither deltaglt-3 nor expression of the constitutively active form of the alpha subunit of the G protein Gs is severely toxic to C. elegans head interneurons, their combination induces extensive neurodegeneration. deltaglt-3-dependent neurodegeneration acts through Ca2+-permeable Glu receptors of the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, requires calreticulin function, and is modulated by calcineurin and type-9 adenylyl cyclase (AC9). We further show that mammalian AC9 hyperactivates mammalian AMPA-receptors (AMPA-Rs) in a Xenopus oocyte expression system, supporting that the relationship between AMPA-Rs hyperactivation and AC9 might be conserved between nematodes and mammals. AMPA-Rs-AC9 synergism is thus critical for nematode excitotoxicity and could potentially be involved in some forms of mammalian neurodegeneration.

Caenorhabditis elegans glutamate transporter deletion induces AMPA-receptor/adenylyl cyclase 9-dependent excitotoxicity.

Excitotoxicity (the toxic overstimulation of neurons by the excitatory transmitter Glutamate) is a central process in widespread neurodegenerative conditions such as brain ischemia and chronic neurological diseases. Many mechanisms have been suggested to mediate excitotoxicity, but their significance across diverse excitotoxic scenarios remains unclear. Death Associated Protein Kinase (DAPK), a critical molecular switch that controls a range of key signaling and cell death pathways, has been suggested to have an important role in excitotoxicity. However, the molecular mechanism by which DAPK exerts its effect is controversial. A few distinct mechanisms have been suggested by single (sometimes contradicting) studies, and a larger array of potential mechanisms is implicated by the extensive interactome of DAPK.

Itzhak Mano

Cellular,
Molecular,
and Biomedical Science,
CUNY School of Medicine,
PhD Program in Biology,
The CUNY Graduate Center,
Cellular, Molecular, and Biomedical Science, CUNY School of Medicine,
PhD Program in Biology, The CUNY Graduate Center

The City University of New York (CUNY)

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model