Inhibition of GFAP synthesis by antisense RNA in astrocytes

Abstract

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with Lipofectin (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H-antisense GFAP RNA alone and reached an equilibrium of 7-8.8 eta g per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 eta g per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose- and time-dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)-treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30-40% decrease in GFAP content 2 days after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuritic Outgrowth Associated with Astroglial Phenotypic Changes Induced by Antisense Glial Fibrillary Acidic Protein (GFAP) mRNA in Injured Neuron–Astrocyte Cocultures

“…The medium was changed 19 hr after antisense addition. Antisense GFAP mRNA was produced according to Yu et al (1991). A 1.25 kb HindIII restriction fragment was obtained from the 5Ј coding end of a 2.5 kb cDNA clone encoding GFAP (generous gift from Dr N. J. Cowan, New York University) and subcloned into PGEM 3z, a SP6/T7 riboprobe vector system (Promega, Madison, WI).…”

“…Using antisense GFAP mRNA (Yu et al, 1991), we indeed have observed that phenotypic changes, consecutive to the decrease in GFAP synthesis, induced a functional shift to neurite-promoting elements.…”

Inactivation of the Glial Fibrillary Acidic Protein Gene, But Not That of Vimentin, Improves Neuronal Survival and Neurite Growth by Modifying Adhesion Molecule Expression

“…The involvement of GFAP in the formation of astrocytic processes was demonstrated by using antisense inhibition of GFAP expression (Weinstein et al, 1991), thus opening up the possibility of modulating astrogliosis induced by injury (Yu et al, 1991(Yu et al, , 1993Ghirnikar et al, 1994). We have shown previously that the blockage of astrocyte reaction after a spinal cord (SC) lesion via local pharmacological therapy permits some axonal regeneration .…”

Astrocytes as support for axonal regeneration in the central nervous system of mammals

“…The demonstration of the major role of GFAP in the formation of astrocytic processes (Weinstein et al, 1991) opened up the possibility of modulating astrogliosis induced by injury in an in vitro model (Yu et al, 1991).…”