The role of astrocytic RFX4_v3 in demyelination

RFX4_v3 is the brain-specific splice variant of the transcription factor RFX4 (Blackshear et al., 2003; Matsushita et al., 2005). It was shown to play an important role during early brain development and in the genesis of congenital hydrocephalus (Blackshear et al., 2003). To study the astrocytic role of RFX4_v3 in demyelination, GFAP cell specific RFX4 conditional knockout mice were generated that possess an astrocyte-specific disruption of the RFX4_v3 gene by deletion of exon 4. A proportion of these GFAP cell specific RFX4 conditional knockout mice showed a severe brain phenotype. Macroscopical and histological analysis of the brains at time of birth and during adulthood revealed a hydrocephalus with dramatic extension of the lateral ventricle, which is indicative for a congenital, non-communicating hydrocephalus. Homozygous RFX4 conditional knockout mice were more severely affected than heterozygotes pointing toward a gene dosage effect. The subcommissural organ (SCO), an ependymal brain gland that is important for the patency of the Sylvian aqueduct, was altered and dysfunctional, which was evidenced by reduced or absent SCO glycoprotein (Reissner fiber) secretion. Furthermore, astrocytes from RFX4 conditional knockout mice at the time of birth showed differential expression of genes critical for brain morphogenesis such as Wnt signaling pathways that regulate dorsal midline structuring and development of the SCO. These observations were in line with previous findings (Blackshear et al., 2003) and supported the idea that the hydrocephalus in RFX4 conditional knockout animals is caused by maldevelopment and dysfunction of the SCO. My data further show that RFX4_v3 depletion in GFAP cell lineages including astrocytes and ependymal cells is sufficient to induce congenital hydrocephalus. I conclude that astrocytic and ependymal RFX4_v3 is highly relevant for brain development and the pathogenesis of congenital hydrocephalus. Our group previously identified RFX4_v3 as a highly up-regulated factor in MS lesions. IHC of MS lesions showed RFX4 expression in various cell types with a prominent nuclear localization in astrocytes. The aim of the present study was to investigate the potential astrocytic role of RFX4_v3 in MS and a demyelinating mouse model. Therefore, to study the impact of astrocytic RFX4_v3, GFAP cell specific RFX4_v3 conditional knockout mice were subject to cuprizone induced demyelination. RFX4_v3 deficient mice showed increased demyelination, a reduced number of oligodendrocytes and enhanced axonal damage. In addition, remyelination in RFX4_v3 deficient mice was delayed. These alterations were associated with increased microglia activation in RFX4_v3 deficient mice. Further investigations during the early phase of cuprizone treatment indicated that increased microglia activation may be caused by enhanced oligodendrocyte apoptosis and increased transcript levels of pro-inflammatory chemokines and cytokines in RFX4_v3 deficient mice. This study demonstrates that astrocytic RFX4_v3 expression is important for regulation of myelination and activation of microglia during the processes of de- and remyelination in the cuprizone mouse model. In addition these results point towards a control of the inflammatory environment by the transcription factor RFX4_v3 to restrict the activation and recruitment of microglia cells as well as oligodendrocyte apoptosis, which eventually controls the extent of de- and remyelination in the cuprizone mouse model. Altogether these findings indicate that RFX4_v3 plays a protective role in the cuprizone mouse model. The fact that RFX4_v3 has been described as a transcriptional repressor suggests that astrocytic RFX4_v3 in MS might act as a regulator of production and release of inflammatory mediators and exerts a beneficial role in MS by restricting neuroinflammation.