Neurons induce GFAP gene promoter of cultured astrocytes from transgenic mice.

In order to investigate the influence of neuron-glia interaction on astrocyte differentiation, we used a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP linked to the beta-galactosidase (beta-gal) reporter gene. Addition of embryonic cerebral hemisphere (CH) neurons to transgenic CH astrocyte monolayers increased by 50-60% beta-gal positive cell number. Such event was dependent on the brain regional origin of the neurons and was followed by an arrest of astrocytes from the cell cycle and induction of glial differentiation. Time-course assays demonstrated that maximum effect was observed after 24 h of coculture. Addition of conditioned medium (CM) derived from CH neurons also increased beta-gal positive CH astrocytic cell number. However, such CM had no effect on midbrain and cerebellum astroglia. Together, these data suggest that neurons secrete brain region-specific soluble factors which induce GFAP gene promoter, as measured by beta-gal expression, thus suggesting that neuron-glia interaction might induce the astrocytic differentiation program.

Doxycycline control of prion protein transgene expression modulates prion disease in mice.

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at approximately 50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies.

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection.

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2b or H-2d Tg beta-gal mice) expressed the mHAg like beta-galactosidase (beta-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tg beta-gal skin by a beta-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous beta-gal shed by keratinocytes. Indeed, our results showed that 1) H-2b Tg beta-gal skin was rejected by H-2bxs and H-2bxd recipients; 2) rejection was mediated by beta-gal-specific CD8+ CTL effectors; and 3) H-2bxd mice having rejected H-2b Tg beta-gal skin generated beta-gal-specific CTL restricted by H-2b and H-2d class I molecules and rejected subsequently grafted H-2d Tg beta-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous beta-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.

Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.

Normal and pathological expression of GFAP promoter elements in transgenic mice.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.