Tetraspanin2 in CNS myelin

Tetraspanin2 has been shown to be present in CNS compact myelin (Birling et al., 1999) and was found to be enriched in PLP^null CNS-enriched myelin fractions by immunoblot analysis (Werner H., pers. comm.). Hence, the localization of TSPAN2 was assessed on brain sections. TSPAN2 appeared to be present in all white matter tracts in wild-type mice, and to some extent in OLs of the gray matter. In the PLP^null mice the levels of TSPAN2 were strongly increased, confirming the prior results. TSPAN2 is more abundant in the white matter tracts and much more in the OLs of the gray matter. This upregulation of TSPAN2 in the absence of PLP suggests that, as both are structurally similar tetraspan proteins found in CNS compact myelin, TSPAN2 could structurally and functionally compensate for PLP in its absence.

5.10. Targeted inactivation of the murine Tspan2 gene

The upregulation of TSPAN2 in PLP^null mice and its early onset of expression during OL development (Nielsen et al., 2006; Dugas et al., 2006), suggests that TSPAN2 could be a relevant protein for CNS myelination. To test this hypothesis, I successfully generated TSPAN2^null mice by homologous recombination of the murine Tspan2 gene in ES cells. The absence of TSPAN2 was proven on the genomic DNA by PCR and on the protein level by immunoblot analysis. The TSPAN2^null mice breed normally and give rise to offspring with genotypes according to the Mendelian inheritance rules. Until known so far, TSPAN2^null mice showed no obvious phenotypic abnormalities. The fact that there is no obvious phenotype could probably be explained by some compensatory mechanism of other myelin tetraspanins. Therefore, it would be e.g. interesting to analyze the TSPAN2^null*CD9^null or the TSPAN2^null*CD81^null mice.

Because the function or at least the abundance of TSPAN2 seemed to be correlating with the presence of PLP, TSPAN2^null*PLP^null mice were generated by cross-breeding the single-mutants. These mice were also included in the pursued studies to evaluate the in vivo consequence of the chronic lack of these two tetraspan proteins of compact CNS myelin.

TSPAN2^null*PLP^null mice do breed normally and have no obvious phenotypes, as known so far.

5.11. Weight increase in TSPAN2^null mice

To evaluate if the normal weight increase does occur during early postnatal development in the absence of TSPAN2, the weight increase was measured between P2 and P30 in wild-type, TSPAN2^null, PLP^null and TSPAN2^null*PLP^null mice. During this period, TSPAN2^null weighted similarly to wild-type mice, and both PLP^null and TSPAN2^null*PLP^null mice had a reduced weight increase. This was also the case when assessing their weight at P30 and at ten months of age. Hence, TSPAN2 has no influence on the weight increase during development, as well as on the weight in adult mice. The reduced levels in PLP^null and TSPAN2^null*PLP^null mice probably refer to a minor phenotype in the mice, not yet proven, as the weight is related to the overall health of the animals.

5.12. Protein composition in TSPAN2^null mice

To assess if the lack of TSPAN2 altered the protein composition of CNS myelin, silver staining was performed. These demonstrated no major differences in the protein abundance in CNS-myelin enriched fractions, neither at P30 (TSPAN2^+/- and TSPAN2^null) nor at P75 (TSPAN2^null and TSPAN2^null*PLP^null). PLP/DM20 was, as expected, absent in the lack of PLP (PLP^null and TSPAN2^null*PLP^null).

To perform an examination in more detail, immunoblots on P30 CNS-myelin enriched fractions of wild-type, TSPAN2^null, PLP^null and TSPAN2^null*PLP^null mice were performed. The most related myelin tetraspanin to TSPAN2, CD9, did not show, surprisingly, any abundance difference. This was an unexpected result, as CD9 was the primary candidate to potentially compensate for the absence of TSPAN2, as both are very similar structurally and are found in the same domains of CNS myelin (Birling et al., 1999; Ishibashi et al., 2004). However, the abundance of the myelin tetraspanin CD81, the next close related tetraspanin (Garcia-España et al., 2008), was augmented in TSPAN2^null,PLP^null and TSPAN2^null*PLP^null mice. And the smaller isoform is additionally increased in PLP^null and TSPAN2^null*PLP^null. Therefore, CD81 does seem to compensate for TSPAN2. The myelin tetraspanins CD82 and CD63 revealed no differences at all. CD82 has been involved in the early stages of OL development (Mela & Goodman, 2009) and possibly its abundance levels could be regulated at earlier postnatal stages. CD63 is known to be in CNS myelin (Baer et al., 2009), but until now, the investigations have related CD63 mainly to exosomes and late lysosomes (reviewed in Pols & Klumperman, 2008) and it is unknown if CD63 is actually present at the compact CNS myelin. The two CNS myelin tetraspanins, CD151 and OAP-1 could not be assessed while performing this characterization. CD151 is an important regulator of cell

evidence of its exact localization in the CNS myelin domains, as well as its function in the nervous system. And OAP-1 has been involved in OL proliferation (Tiwari-Woodruff et al., 2001). It is very tempting to suggest that these two, CD151 and OAP-1, that are known to bind integrins (a typical characteristic of tetraspanins, Hemler, 2005), could compensate for the lack of TSPAN2 in the CNS myelin. Further investigation should assess this interesting question.

In addition, the abundance of Fyn, a tyrosine kinase important in OL maturation (reviewed in Krämer-Albers & White, 2011) was also not altered. This was, yet again, an unexpected result, as tetraspanins assemble signalling molecules and Fyn was a major candidate to be regulated by TSPAN2.

Beside, the non-compact myelin protein CNP has also no different abundance levels in the absence of TSPAN2 at P30 and at P75. However, the MBP levels demonstrated a notably augmented abundance in the CNS-myelin enriched fractions of TSPAN2^null, PLP^null, and TSPAN2^null*PLP^null mice. And in the latter two, the abundance of the larger isoform was as well increased. MBP is the second most abundant compact myelin protein and it is involved in myelin compaction and is essential for myelination (reviewed in Boggs, 2006). The augmented MBP levels suggested a compensation for an altered compaction in TSPAN2^null, PLP^null, and TSPAN2^null*PLP^null mice. This would be in accordance to the prior knowledge of

PLP^null mice having a reduced CNS myelin compaction (Klugmann et al., 1997). This

unexpected and promising result should be assessed in more detail, for understanding the manner TSPAN2 and MBP do possibly interact.

Therefore, an obvious abundance difference was found regarding MBP, which is strongly increased in the absence of TSPAN2, at P30 and at P75. MBP could compensate for a reduced CNS compaction in the TSPAN2^null mice. And it is the tetraspanin CD81 that seemed to compensate for the lack of TSPAN2. This altered regulation of MBP and CD81 should be further assessed.