Astrocytes with enhanced hGFAP expression lead to oligodendrocyte

Oligodendrocyte apoptosis is the first pathological observation during cuprizone-induced demyelination (Hesse et al 2010, Komoly et al 1987, Mason et al 2004).

The present results point out that enhanced hGFAP expression in astrocytes leads to preserved oligodendrocyte numbers during cuprizone treatment (Figure 7). The observed oligodendrocyte loss in wild type mice is in line with earlier studies (Blakemore 1972, Mason et al 2004). Oligodendrocyte apoptosis is accompanied by down-regulation of myelin protein mRNA expression after two and three weeks of cuprizone treatment (Jurevics et al 2002, Morell et al 1998). Additionally, acute axonal damage occurs due to cuprizone-induced demyelination (Lindner et al 2009).

Furthermore, this study demonstrated that enhanced hGFAP expression in reactive astrocytes decreased oligodendrocyte apoptosis by 80% indicated by activated caspase-3 immunostaining (Figure 4) after one week of cuprizone treatment. It was previously shown that oligodendrocytes undergoing apoptosis express caspase-3 during the first week of cuprizone treatment (Goldberg et al 2013, Hesse et al 2010).

The decrease in oligodendrocyte apoptosis is consistent with the stable oligodendrocyte numbers observed in the present study (Figure 20) and suggests that reactive astrocytes with enhanced hGFAP expression provide protection or support for oligodendrocytes. In line with the present results another study showed that the ablation of reactive astrocytes increases oligodendrocyte damage after five weeks of cuprizone treatment by using GFAP HSV-TK transgenic mice (Skripuletz et al 2012). Additionally in line, the ablation of reactive astrocytes caused a significant increase of more than 90% oligodendrocyte loss and more severe demyelination of the white matter after longitudinal stab spinal cord injury (Faulkner et al 2004).

4 | Discussion

Previous studies show that reactive astrocytes contribute to oligodendrocyte survival by providing growth factors. In vitro, astrocytes were shown to express increased mRNA levels of growth factors like PDGF, BDNF or bone morphogenetic protein-2A/3 (Meeuwsen et al 2003). In vivo it was shown that the overexpression of PDGF in astrocytes increased OPCs survival in lysolecithin-induced demyelinated lesions (Woodruff et al 2004). Therefore, reactive astrocytes may support oligodendrocyte survival during demyelination. Furthermore, the function of astrocytes to eliminate ROS for instance via the enzyme catalase (Desagher et al 1996) could also preserve oligodendrocytes. That ROS is selectively toxic to oligodendrocytes was demonstrated in vitro (Griot et al 1990) and cuprizone is known to induce increased oxidative stress on oligodendrocytes (Cammer 1999, Goldberg et al 2013, Hiremath et al 1998, Pasquini et al 2007). Enhanced GFAP expression might influence the ROS elimination function as it was shown that the double-knockout of GFAP and vimentin impaired the astrocyte ROS elimination function and causes increased cell death in vitro (de Pablo et al 2013).

In the present study acute axonal damage was nearly absent in GFAP Tg73.7 mice resulting in a difference of 95% amyloid precursor protein (APP) accumulation between wild type and transgenic mice after six weeks of cuprizone treatment (Figure 8). The accumulation of APP marks disturbed anterograde axonal transport of an injured axon and, by inference, acute axonal damage (Bjartmar et al 2003, Kuhlmann et al 2002). The results of the present study demonstrated that the preserved myelination in GFAP Tg73.7 mice leads to reduced axonal damage observed in these mice (Figure 8). This is consistent with myelin sheaths being essential for maintaining axonal functions, including axonal transport (Brady et al 1999). The increased preservation of myelin in GFAP Tg73.7 mice were further confirmed on the ultrastructural level using EM. Supporting the present results a previous publication demonstrated in mice with ablated astrocytes that viable oligodendrocytes and myelinated axons correlate with the presence of reactive astrocytes whereas areas devoid of reactive astrocytes exhibit degeneration of myelin after crush spinal cord injury (Faulkner et al 2004).

Axonal integrity can also be preserved by remyelination. Here, damaged oligodendrocytes are replaced by newly generated oligodendrocytes from OPC (Blakemore 1973, Franklin 1993). In contrast, the present results indicate that

enhanced astrocytic hGFAP expression did not affect OPCs and oligodendrocyte numbers during cuprizone treatment, except during the first week of treatment (Figure 7). Moreover, myelin sheaths in EM do not appear thinner which would indicate remyelinated axons (Dubois-Dalcq et al 2005). Together with the relatively stable oligodendrocyte numbers in GFAP Tg73.7 mice, these findings argue against a replacement of oligodendrocytes and are consistent with the theory that oligodendrocyte loss is prevented and myelin remained present. After 12 weeks of cuprizone treatment OPC and oligodendrocyte numbers were more in GFAP TG73.7 mice compared to wild type mice. This leads to the question whether increased hGFAP expression might affect remyelination which was not investigated in the present study but would be interesting for future research.

The expression of myelin proteins is required for the formation of intact myelin (Brady et al 1999, Fitzner et al 2006, Griffiths et al 1998) and, for instance, mice lacking MBP are unable to form compact myelin sheaths (Brady et al 1999).

Consequently, the results of the present study show an up-regulation of MBP, PLP1 and MAG mRNA expression in GFAP TG73.7 mice after three weeks of cuprizone treatment normalised to wild type mice (Figure 13). Since a pronounced oligodendrocyte loss was observed in wild type mice from week four of cuprizone treatment leading to reduced mRNA expression of myelin genes, the measured myelin gene expression in transgenic mice represents the higher number of preserved oligodendrocytes during cuprizone treatment. Furthermore, the high levels of myelin mRNA in transgenic mice might further indicate that preserved oligodendrocytes were intact. The study by Griffiths (1998) point out that histologically visible myelin might be non-functional. PLP-deficient mice show myelin but disturbed oligodendrocyte support as the mice develop axonal swellings and degeneration (Griffiths 1998). Furthermore, in this study myelin protein expression was regulated in wild type mice before pronounced myelin loss was observed on the histological level which emphasizes that oligodendrocytes die before myelin is lost. In line, previous studies demonstrated that regulation of MBP, MAG and PLP mRNA expression starts during the first week of cuprizone treatment (Buschmann et al 2012, Hesse et al 2010). MBP and MAG had the lowest expression levels after three weeks of cuprizone treatment and then expression increased again until week six (Morell et al 1998) whereas PLP decreases over treatment duration (Groebe et al 2009). In line, in the present study MBP, MAG but

4 | Discussion

also PLP mRNA expression is less up-regulated after six weeks compared to after three weeks of cuprizone treatment.

In contrast to preserved oligodendrocyte numbers in the cuprizone mode, in lysolecithin-induced focal lesions oligodendrocyte numbers were similarly decreased in the demyelinated lesion area of transgenic mice compared to wild type mice (Figure 11). Lysolecithin is a potent detergent lysing cell membranes including myelin (Gregson 1989, Hall 1972, Hall & Gregson 1971). It acts rapidly within 30 minutes, after two days swollen tongues of oligodendrocytes were observed and after three days also oligodendrocyte damage (Hall 1972). The present results demonstrated that the number of oligodendrocytes in the lesion areas were not different between GFAP Tg73.7 mice and wild type mice. However, compared to the corpus callosum of naïve mice oligodendrocyte numbers were strongly decreased per square millimetre in the lesion area in GFAP Tg73.7 mice by 90%

and in wild type mice by 93% indicating an extensive oligodendrocyte loss seven days after lesion induction. This suggests that most oligodendrocytes in both lesions died due to the detergent function of lysolecithin. The enhanced hGFAP expression in astrocytes seems to be prevent oligodendrocyte death in the cuprizone model but not in the lysolecithin model. However, demyelination was reduced in both models indicating different beneficial effects of reactive astrocytes during cuprizone- and lysolecithin-induced demyelination.

4.3.3 Altered chemokine expression of astrocytes may be beneficial for