Cyclic AMP and the Induction of Reactive Astrogliosis

Astrocytes play a major role in nerve regeneration of the central nervous system. In response to central nervous system (CNS) trauma or pathology such as neurodegeneration or ischemia, astrocytes react by exhibiting hypertrophy of their cellular processes and hyperplasia (proliferation), a process referred to as reactive astrocytosis. Reactive astrogliosis can be modeled in vitro using mechanical (scratch wound) or chemical stimuli to activate F98 rat glioblastoma cells from a quiescent state to a reactive-like state. Studies have shown monoclonal antibody (mAB) Jl-31 may recognize a phosphoepitope found on the nuclear intermediate filament, lamin, as well as the cytoplasmic intermediate filament characteristic to mammalian astrocytes, glial fibrillary acidic protein (GF AP), suggesting that mAB Jl-31 is an appropriate marker for astrocytes entering the reactive state. Reactive astrogliosis, as detected by increased labeling of cytoplasmic and nuclear epitopes by mAB Jl-31, can be modeled in vitro by treatment of F98 cells with forskolin, an activator of adenylyl cyclase. Here I tested the hypothesis that a cAMP-mediated intracellular pathway is involved in eliciting forskolin-induced reactivity in F98 cells, and I attempted to elucidate that pathway using inhibitors of known cAMP-dependent downstream signaling molecules and fluorescent Ca+2 indicators and characteristic nuclear labeling of F98 cells by mAB Jl-31. Forskolin was found to significantly increase labeling with mAB Jl-31 in as little as 7 minutes, reaching a maximum after 30 minutes. Pretreatment of F98 cells with the protein kinase A (PKA) inhibitor H89 abolished the effect of forskolin, while inhibitors of protein synthesis and L-type calcium channels had no effect. Forskolin was found to induce transient cytoplasmic increases in Ca+2 levels (as measured by Oregon Green conjugated BAPTA), and the relation of this to PKA is currently under investigation. On the basis of these results, it appears that induction of cAMP production in F98 cells results in a PKA-dependent increase in phosphorylation of cytoplasmic and nuclear intermediate filaments, which may indicate the first stages of F98 cell transition from normal to a reactive state.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Biology

Grantor

Texas State University-San Marcos

Year dc:date.issued

2010

Author dc:creator

• Patel, Mayuri P.

Advisor dc:contributor.advisor

• Koke, Joseph R.

Committee members dc:contributor.committeemember

• Garcia, Dana M.
• Raabe, Timothy
• Gross, Jeffrey M.

• cAMP pathway
• Forskolin
• F98 glioblastoma cells

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10877/4261

OAI identifier oai:identifier

oai:digital.library.txst.edu:10877/4261