In the present paper, we report the in vivo consequence of altered Ca2+ homoeostasis in OPCs/OLs owing to golli overexpression in those cells. Golli overexpression resulted in a delay in myelination of ~6 weeks and a striking neurological phenotype characterized by tremors that increased in severity and body involvement from P14 to P35 and by occasional seizures. Our analysis of the JOE CNS indicates that the underlying cause for the tremors is apparently due to a significant delay in the onset and extent of myelination (see also Martin et al., 2007). Although the entire CNS is affected, the focus of analysis in the present study has been on the perturbations observed in the cerebral cortex and CC.
Use of the classic MBP
promoter permitted us to target overexpression of the golli protein specifically to OLs and their precursors. Although the classic MBPs are generally considered to be expressed in mature OLs, there are numerous reports of the promoter being active in cells earlier in the OL lineage. The classic MBP
mRNAs, if not the proteins, have been reported to be expressed embryonically as well as in early postnatal animals (Mathison et al., 1993; Nakajima et al., 1993
; Hajihosseini et al., 1996
; Peyron et al., 1997
; Zecević et al., 1998
; Campagnoni and Skoff, 2001
; Tosić et al., 2002
; Jalabi et al., 2005
). In the present study, we have confirmed that the MBP
promoter–golli transgene is active perinatally in the brain, long before active myelination. Thus, in addition to mature OLs, the activity of the classic MBP
promoter appeared to drive expression of golli J37 in OPCs, later-stage OL progenitors and immature OLs.
Other mouse models have been generated by transgenes driven by the classic MBP
promoter. These have included oncogenes (Hayes et al., 1992
), MHC molecules (Turnley et al., 1991
), TNFα (tumour necrosis factor α) (Taupin et al., 1997
), c-myc (Orian et al., 1997
), FGFR (fibroblast growth factor receptor) (Harari et al., 1997
), BDNF (brain-derived neurotrophic factor) (Forsberg-Nilsson et al., 2003
) and reporter genes such as lacZ
(Gow et al., 1992
; Miskimins et al., 1992
; Vanderluit et al., 2000
). Surprisingly few of these transgenics have exhibited serious overt neurological phenotypes or major myelination disturbances, suggesting that overexpression of any gene is not damaging to the OL. The overexpression of the c-myc (Orian et al., 1997
) and MHC (‘wonky' mouse) genes in OLs has been reported to cause a ‘shivering' phenotype (Turnley et al., 1991
). In the case of the MHC transgenic mouse line, disturbance of locomotor activity from P11 to P14 was noted, followed by tonic seizures leading to premature death by P15–P22. Another transgenic mouse, called 2-50, has been generated through the expression of the proto-oncogene, myc, under the control of the classic MBP
promoter (Orian et al., 1997
). This mouse exhibits tremors transiently from P15 to P25 and appears to undergo delayed myelination because of transient expression of the transgene in OPCs during embryonic development. Myelination and myelin apparently become completely normal with further postnatal maturation. This mouse does not express the transgene beyond very early postnatal development, unlike the JOE mice, which express the transgene as early as P1.5 (earliest age examined) and continues to at least 13.5 months (latest age examined). Other models, such as transgenic mice expressing BDNF or FGFR under control of the classic MBP
promoter appear to have rather subtle effects on myelin and do not exhibit any neurological symptoms. The JOE mice are also unlike the spontaneously occurring dysmyelinating mutants (e.g. shi, shimld
, jimpy) and the PLP-overexpressers. These animals suffer from dysmyelination, which results in permanent myelin deficits or premature death through seizures. The JOE mice are hypomyelinated and exhibit neurological tremors over a considerable and important period in the developing brain, after which there appears to be significant recovery of myelination.
It has become apparent that golli proteins are important in OPC/OL process extension and retraction. For example, when golli proteins are overexpressed in OL cell lines, they induce the elaboration of processes and sheet-like structures (Reyes and Campagnoni, 2002
; Paez et al., 2007
) that do not have the composition of myelin. In transfected cells, golli co-localizes with F-actin (filamentous actin) in the plasma membrane along the extending processes and at the leading tips (J.M. Feng and A.T. Campagnoni, unpublished data), suggesting that golli may influence process motility. It is likely that this results from the role that golli proteins play in modulating Ca2+
influx at the plasma membrane (Jacobs et al., 2005
; Paez et al., 2007
From what we know about the cellular localization of golli and its influence on process formation in vitro, one might predict that, in the presence of increased golli expression, OLs in the JOE mouse would remain in a less mature state, extending processes, but not differentiating further into mature OLs. The results from the present study support this hypothesis. Specifically, the data from our in vitro studies show a clear reduction in the ability of purified JOE OLs to produce myelin-like sheets in comparison with WT OLs in culture. Instead, these JOE 01+ cells were characterized by highly ramified processes, but without extensive sheet formation. These results with isolated OLs are different from golli overexpression in cell lines. Isolated JOE OLs primarily elaborate processes rather than sheets, revealing an interesting difference in cell lines compared with isolated OLs. Interestingly, impeded maturation of OLs in vivo was greater in the outer cortical layers (I–III), than in the deeper cortical regions and CC in the JOE compared with WT brains.
Pre-myelinating OLs are a transient population of cells that extend elaborate processes over a significant area (80 μm in the cortex and 40 μm in the CC) and largely disappear after P21 (Trapp et al., 1997
). The surviving cells are thought to be those that contact and ensheathe axons and differentiate further into mature myelin-forming OLs. The greater numbers of pre-myelinating cells in the JOE mouse, particularly between P14 and P28, and their continued presence in the adult suggest that overexpression of golli impedes and/or delays the development of OLs.
Whereas the number of pre-myelinating OLs was greater in the JOE brains, some OLs did differentiate into a more mature phenotype, particularly those in the lower cortical layers (III–VI). These OLs tended to have fewer processes than the pre-myelinating OLs, and these processes had a more organized and vertical appearance, suggesting that they were aligning along side adjacent axons. However, results from our PLP immunohistochemical studies and ultrastructural analysis indicated that these cells were not able to myelinate nearby axons. In the CC, many of the OLs had typical intrafascicular morphology and were able to myelinate axons. However, the amount of myelin (number of wraps per axon) was significantly less than that produced in the WT.
In WT brains, pre-myelinating OLs in the upper cortical layers did not stain for PLP very well, but the OLs in the lower cortical layers and the CC did. The same pattern was roughly true for the JOE mouse. However, the PLP+
OLs in the lower cortical layers that appeared to mature beyond the pre-myelinating stage produced PLP, but the majority of the protein remained in the cell body. In contrast, OL processes in the JOE CC were stained more heavily for PLP. This difference may explain why, in contrast with the cortex, myelination, albeit reduced, occurred in the JOE CC. It is possible that regional differences in rates of maturation of OLs or the originating (embryonic) sources of the OLs in these areas of the cortex and subcortical white matter (Kessaris et al., 2006
) account for these differences.
Our finding that OLs in the CC and cortex respond differently to overexpression of golli J37 suggests that there may be innate differences in sensitivity to increased golli expression among various subpopulations of OLs. This conclusion is consistent with increasing evidence in the literature that OPCs and OLs are heterogeneous populations of cells. Their heterogeneity has been documented with respect to their temporal and spatial sites of developmental origin (Kessaris et al., 2006
; Ivanova et al., 2003
; Noble et al., 2003
) as well as their genetic (reviewed by Nicolay et al., 2007
) and morphological phenotype (Del Rio-Hortega, 1928
; Weruaga-Prieto et al., 1996
). Pre-myelinating OLs in the CC have also been shown by Trapp et al. (1997)
to have significant morphological and structural differences than those found in the cerebral cortex. This well-documented heterogeneity among OL populations may underlie the differences that we noted in the OPC/OL populations in the layers of the cortex and subcortical white matter in the JOE brain.
One consequence of golli overexpression in vivo
appears to be an inhibition or delay of myelination. This delay may be due to the extensive cell death of OPC/OLs that we observed in the JOE mouse during the first two postnatal months. This increased cell death is likely to be a result of a perturbation in Ca2+
homoeostasis owing to increased production of golli. Our previous work showed that golli enhances Ca2+
uptake into OLs and OPCs and this can lead directly to cell death (Jacobs et al., 2005
; Paez et al., 2009a
). It is well-documented that increased influx of Ca2+
can be lethal in many cell types including OLs (Orrenius et al., 2003
; Benjamins and Nedelkoska, 1996
; Smith and Hall, 1994
). Thus these in vivo
findings are consistent with in vitro
studies by Paez et al. (2009a)
, who found an increase in apoptotic cell death in JOE OPCs cultured under basal conditions. Moreover, when cultures of OPCs from golli-knockout mice were treated with high levels of K+
influx), Paez et al. (2009a)
found that they were more resistant to apoptotic death than controls.
At a cellular level, early stages of OPC maturation may actually be accelerated by overexpression of golli. For example, we know that migration is increased in OLs both in vivo
and in vitro
(Paez et al., 2009b
). However, we believe that golli has no role in the elaboration of myelin sheets and may in fact inhibit that process. In JOE mice, increased golli expression may enhance this inhibition and account for the delay in further OL maturation. Eventually, other factor(s) intervene and override the inhibition. This factor could include, but is not limited to, lower expression of the MBP
promoter with development and/or the interaction with other molecules.
One of the most interesting phenotypic characteristics of the JOE mouse is its delayed recovery of near WT levels of myelin and loss of neurological systems by ~60 days. In vitro
, increases in internal Ca2+
have been shown to increase proliferation of JOE OPCs in culture (Paez et al., 2009a
). In the JOE mouse, there was an ~20% increase in the number of GFP+
OLs in JOEplp/gfp
brains compared with controls at P28 and P60, but the results were not statistically significant. It is possible that this small difference over time could result in a ‘rebound' effect that eventually leads to the myelination occurring at later ages. However, another interpretation is that the small increase of histone H3+
proliferating cells in older animals (P28–P60) suggests that remyelination is unlikely to be due to the production of newly generated OPCs. Rather, the continued presence of pre-myelinating OLs suggests a greater pool of immature OLs that could be recruited in JOE animals that are able to differentiate and form myelin. These cells are likely to be able to do this only when the activity of the MBP
promoter declines (after ~21–28 days), reducing the intracellular levels of the golli J37 protein.
The lack of a difference in proliferating OLs between the JOE and WT mice is surprising given the increase in proliferation of JOE OPCs observed in vitro
(Paez et al., 2009a
). However, although we did not detect an increase in proliferating histone H3+
cells between P14 and P60, we did detect a greater number of GFP+
OLs at P14 in the JOEplp/gfp
cortices. One explanation for this difference may be that increased proliferation occurred in the JOEplp/gfp
mice before P14. Furthermore, increased rates of OPC migration in the JOEplp/gfp
may also explain the presence of a greater number of GFP+
cells in the cortices of JOEplp/gfp
mice. This would be consistent with Paez et al. (2009b)
, who found increased rates of migration in JOE OPCs compared with controls.
In summary, the present paper indicates that increased levels of golli delays myelination, probably due to significant cell death of OLs particularly in white matter tracts such as the CC. These findings are consistent with in vitro studies that indicate that golli acts by influencing Ca2+ influx into OPCs/OLs through store-operated Ca2+ channels and voltage-gated Ca2+ channels. The results provide in vivo evidence for a significant role of the golli proteins in the regulation and maturation of OLs and normal myelination. The JOE mouse also provides an unique model to examine the longer-term effects of hypomyelination imposed during a critical period of brain development during which neural networks are being established, axonal sprouting and synaptic connections are being made and neuronal–glial relationships are being established.