The nuclear intermediate filament, lamin A/C (A and C protein isoforms), was originally associated with the pathogenesis of FXTAS by virtue of its presence in the intranuclear inclusions found in the post-mortem brain tissue of a patient with FXTAS (31
). Additional studies suggested that lamin A/C disorganization and aggregation may participate in the pathogenesis of FXTAS, and led us to hypothesize that in FXTAS a broad dysregulation of lamin A/C may result in (or at least be highly correlated with) CNS pathology and may impact other tissues where FMR1
is expressed, even in the absence of intranuclear inclusion formation (37
). The intranuclear inclusions found in CNS tissue in FXTAS also contain several heat-shock response proteins, including αB-crystallin, HSP27 and HSP70 (31
), suggesting that there may be a cellular stress response as a downstream consequence of expression of the expanded CGG-repeat mRNA. There is an extensive literature on the role of HSPs in stabilizing cytoskeletal proteins (46
, reviewed in 47
). In this regard, the upregulation of HSP mRNAs observed both in frontal cortex and in cultured fibroblasts could reflect a stress response to the disordered architecture of the lamin A/C network, rather than to the elevated FMR1
We analyzed the nuclear lamin A/C architecture and cellular stress response in skin fibroblasts of male carriers of premutation FMR1 alleles and explored the possibility that this cellular phenotype would be aggravated in patients with FXTAS. Consistent with these expectations, we observe in subjects with FXTAS a significant reduction in the percentage of cultured fibroblasts with normal ring-like lamin A/C patterns (Fig. ). A similar pattern of dysregulation, involving upregulation of mRNA levels of three HSPs and a decrease in the percentage of fibroblasts displaying normal lamin A/C rings (Figs D and), was observed in cultured skin fibroblasts from asymptomatic male carriers of premutation alleles. We did not find a significant correlation between the percentage of cells displaying lamin A/C rings and the number of CGG repeats in all premutation carriers (data not shown); however, the absence of a significant correlation could be due to the small sample size available for this study.
The levels of lamin A/C expression were also quantified in post-mortem frontal cortex from 10 patients with FXTAS and from three controls. Whereas the group of patients with FXTAS expressed increased levels of LMNA mRNA (Fig. ), the levels of lamin A/C protein in the RIPA-soluble cellular fraction was reduced or undetectable in eight of 10 cases with FXTAS. Part of this reduction in soluble lamin levels appears to reflect repartitioning of the protein isoforms to less soluble, possibly aggregated forms of lamin (data not shown). These changes may contribute to inclusion formation in a small number of cells.
Associated with the elevated levels of the three HSP and of FMR1
mRNAs in FXTAS subjects (Figs and ; Table and Supplementary Material, Table S2A
), it is evident in the box plots (Fig. ) that the range of mRNA levels for each of the four genes is substantially greater in premutation carriers (with or without FXTAS) than for the corresponding controls; this is true for either normalized data (to account for potential systematic experimental effects) or non-normalized data. This variation arises from at least two sources. First, within the premutation groups, we observe upward trends in mRNA levels with increasing CGG-repeat number (data not shown), although these trends do not reach significance, such variation is all captured as intra-group variation. The positive correlation of FMR1
mRNA levels with increasing CGG-repeat number has been reported previously (53
). A second source of added variation in the premutation subgroups is the greater biological variation than that observed for controls. Although the origin of this biological variation is not understood, repeat measurements based on separate blood draws typically display less than ~20% variation (53
). As expected, the expression levels of control genes JUNB
are not elevated in either fibroblast or brain samples from premutation groups, nor do they display increased variation within the premutation groups relative to the corresponding control groups (Figs and ). Therefore, the greater variation of HSP mRNA levels in the premutation subgroups appears to be a biological effect of the premutation FMR1
Despite increased FMR1 and HSP mRNA levels in individuals with premutation alleles, the levels of the soluble forms of the corresponding proteins were not elevated in either CNS tissue or the skin fibroblasts samples analyzed. At present, we cannot explain the absence of significant, parallel increases in protein levels; however, these observations may reflect either redistribution to insoluble forms of these proteins or a marginal shift toward decreased stability. Interestingly, we did find a significant increase in accumulation of the three HSPs in the RIPA-insoluble (SDS-soluble) protein fraction of brain extracts from the FXTAS group relative to the controls (Table and Fig. ). These observations in samples from patients with FXTAS may also reflect changes in protein translocation into the nuclei. Lamin A/C protein levels were also quantified in the soluble protein fraction of brain extracts from patients with FXTAS. However, as with the HSPs, we did not observe any increases in the levels of lamin A or C isoforms that would correspond to the elevated LMNA mRNA levels.
Expression of the expanded CGG-repeat FMR1
mRNA in cultured human neural cells is capable of inducing cellular pathology within a very short time frame (~1 week) (37
), raising the possibility that early in development, when levels of FMR1
mRNA are highest, there may be evidence of FMR1
mRNA-induced cellular dysregulation. To explore this possibility, MEFs were derived from embryos of a female mouse homozygous for the expanded CGG repeat (~180 and ~210 CGG repeats) that was previously reported to have elevated Fmr1
mRNA levels and neuropathological characteristics of FXTAS, including the presence of inclusions in the CNS and non-CNS organs (45
). We observed that cultured MEFs harboring large premutation Fmr1
alleles have both elevated levels of Fmr1
mRNA and altered morphology of the lamin A/C network (Fig. ). Fibroblasts derived from adult KI mice with the expression of normal levels of expanded CGG-repeat mRNA display a lesser degree of lamin dysregulation than their MEF counterparts. This last result suggests that the fundamental processes leading to lamin dysregulation may be at least partially reversible and that severity seems to be correlated with Fmr1
Although the mechanisms that lead to the cytoskeletal abnormalities in FXTAS and some asymptomatic male premutation carriers remain unknown, the impact that lamin A/C has on other cytoskeletal components is well documented (60
). Moreover, the consequences of abnormal cytoskeletal organization in neural cells include impaired neural cell migration during neurogenesis (64
) and impaired neural plasticity (68
). For example, impaired functional connectivity has been demonstrated in fMRI studies of the amygdala in response to fearful faces in young adult men with the premutation who do not have neurological symptoms (7
). This fits with the elevated rates of psychopathology and social deficits that have been reported in the previous studies of premutation carriers, particularly the males, though the females have elevated rates of depression and anxiety (6
Our findings provide evidence for a model in which increased expression of FMR1 expanded CGG mRNA results in a range of molecular consequences, including altered expression and disruption of the nuclear lamin A/C architecture and induction of the expression of stress response genes. Whether these molecular/cellular changes, also observed in asymptomatic carriers, are ultimately useful for early diagnosis and intervention will depend on whether signs of molecular pathology are predictive of clinical involvement, including incipient neurodegenerative disease; proper assessment of any such predictive associations will require both larger sample sizes and/or longitudinal studies. Nevertheless, the current study provides the experimental underpinnings (fibroblast cellular dysregulation) for such future investigations.
Whereas it may be argued that the lack of significant differences between premutation carriers with and without FXTAS is due to the small sample sizes employed in the current study, it is also possible that the basis of incomplete penetrance is due to second gene effects and/or environmental factors (e.g. general anesthesia). Thus, the findings of at least some level of molecular dysregulation in all premutation carriers suggest that premutation status may be a strong risk factor, albeit not sufficient, for developing FXTAS. Similar findings at the cellular level prior to disease onset have been reported for other neurodegenerative disorders including Alzheimer disease, where neuropathology exists many years before the onset of clinical symptoms (72
). In the current instance, the identification of a fibroblast phenotype provides us with a powerful tool to look for additional genetic and/or environmental factors that may give rise to clinical involvement.
As noted above, an important limitation of the current study is small sample size. Moreover, although the ages of the subjects and controls donating tissue lie within a narrow range, it was not possible to precisely age-match the subgroups. An additional limitation of this study is the variability in the length of post-mortem intervals (PMI) of the subjects contributing brain tissue, which may have also contributed to inter-subject variability in the levels of molecules analyzed. However, from the perspective of the inter-group comparisons, we note that the PMIs of the FXTAS group were, on average, smaller than the PMIs of the controls. To account at least partially for the effects of differing PMIs, all mRNA and protein levels were quantified relative to GUS (mRNA) and to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (protein), respectively.