Frontotemporal lobar degeneration (FTLD) is the second most frequent neurodegenerative disorder in individuals under the age of 65, accounting for 5–10% of dementia patients (Neary et al. 1998; Ratnavalli et al. 2002). FTLD patients often present with behavioral changes and personality dysfunction, corresponding to the pathology in frontal and temporal lobes (McKhann et al. 2001; Boxer and Miller 2005). Post-mortem analyses of FTLD brains determined that the most common pathological subtype involves intracellular inclusions of ubiquitinated TAR DNA binding protein 43 (TDP-43). In 2006, mutations causing a 50% loss in progranulin protein (PGRN) were found in the progranulin gene (GRN), accounting for nearly 20% of patients affected by this FTLD subtype (FTLD-TDP) (Baker et al. 2006; Cruts et al. 2006; Gass et al. 2006; Gijselinck et al. 2008). More recently, an expansion in a hexanucleotide repeat in the chromosome 9 open reading frame 72 gene was identified as an additional major genetic cause of FTLD-TDP (DeJesus-Hernandez et al. 2011; Renton et al. 2011).
Genetic studies have also served as a successful tool in the discovery of risk factors for FTLD-TDP. In 2010, Van Deerlin and colleagues performed a genome-wide association study in which single nucleotide polymorphisms (SNPs; top SNP rs1990622 T>C) located at the transmembrane protein 106 B gene locus (TMEM106B) on chromosome 7p21 were identified as FTLD-TDP risk factors (Van Deerlin et al. 2010). Moreover, the risk association of these SNPs was greatest in GRN mutation carriers (Finch et al. 2011; Van Deerlin et al. 2010), in which there was a highly significant decrease in the frequency of homozygous carriers of the rs1990622 minor allele, suggesting a protective effect of this allele in these patients. The exact relationship between TMEM106B polymorphisms and TMEM106B regulation and/or function, however, remains poorly understood. Initial studies showed a dose-dependent decrease in TMEM106B mRNA expression associated with the rs1990622 minor allele (Van Deerlin et al. 2010); however, this could not be confirmed in subsequent studies (Cruchaga et al. 2011; van der Zee et al. 2011). Also, even though individuals homozygous for the protective minor allele of rs1990622 showed higher plasma PGRN levels (Finch et al. 2011), only a minor absolute increase in PGRN was observed, suggesting that other disease mechanisms may be at play. Finally, we and others identified that rs1990622 is in perfect linkage disequilibrium with p.T185S (rs3173615), a coding variant in TMEM106B; however, thus far no studies have been reported to elucidate the functional consequence of p.T185S on TMEM106B.
At the time of these genetic discoveries, TMEM106B was a relatively uncharacterized transmembrane protein; however, recent publications have indicated that TMEM106B is a glycoprotein predominantly localized at the lysosomal membrane where it might interact with intracellular PGRN (Brady et al. 2013; Chen-Plotkin et al. 2012; Lang et al. 2012). Though these reports investigated the effect of TMEM106B expression on PGRN levels in vitro, the data are conflicting, and the effect of the coding variant p.T185S was not extensively studied. To further characterize TMEM106B and its role in FTLD-TDP, we investigated the risk (T185) and protective (S185) isoforms of TMEM106B and their effects on TMEM106B. First, we replicated the importance of TMEM106B polymorphisms on FTLD-TDP risk in GRN mutation carriers in a new cohort of patients. We further confirmed that TMEM106B is located in the lysosomes where it might interact with PGRN. Importantly, we showed that the protective (S185) TMEM106B isoform is consistently expressed at lower levels than the T185 TMEM106B isoform because of an increased rate of protein degradation, possibly resulting from changes in TMEM106B glycosylation. Thus, we provide the first insight into a functional difference between the risk (T185) and protective (S185) isoforms of TMEM106B.
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- Materials and methods
- Supporting Information
It has become increasingly clear that variants in or near the TMEM106B gene play a critical role in the risk of developing FTLD-TDP. Strong association was especially apparent in FTLD-TDP patients carrying GRN mutations, suggesting that TMEM106B might modify the disease through regulation of PGRN levels or function (Van Deerlin et al. 2010). In this study, we further confirmed the association of TMEM106B with FTLD-TDP in an additional 29 patients with loss-of-function GRN mutations. Only one patient (3.4% of patients) was homozygous for the minor protective allele of rs1990622. Consistent with our previous findings, the phenotype in this patient is relatively mild, with a diagnosis of mild cognitive impairment at the age of 71 years. In our laboratory, we previously identified 2 of 127 patients (1.6%) to be homozygous for the minor allele of rs1990622. Cumulatively, among unrelated probands of GRN mutation families, we identified only 3 of 156 (1.9%) patients to be homozygous for the minor allele of rs1990622 as compared to 157 of 822 (19.1%) control individuals studied to date (Van Deerlin et al. 2010; Finch et al. 2011, and this study). These genetic findings add to the growing body of evidence confirming TMEM106B as a disease risk factor and potential modifier in GRN-related FTLD-TDP.
To provide insight in the molecular mechanisms associated with TMEM106B variants, we focused our studies on the coding variant p.T185S, which is in complete linkage disequilibrium with rs1990622, as a potential functional variant implicated in FTLD-TDP risk. As the TMEM106B variants were associated most strongly with FTLD-TDP risk in GRN mutations carriers, TMEM106B might confer risk by directly affecting PGRN levels or function. Here, we show that both isoforms of TMEM106B (T185 and S185 TMEM106B) colocalize, in part, with lysosomal compartments. Immunofluorescence analyses of T185 and S185 TMEM106B also showed that both TMEM106B isoforms colocalize with PGRN. This is in agreement with two recent reports that also observed significant overlap of the TMEM106B and PGRN (Brady et al. 2013; Chen-Plotkin et al. 2012). Moreover, we found that PGRN levels were significantly increased as compared with control-transfected cells in the media and lysates of T185- and S185-transfected cells at 2 days. Even though the first characterization of TMEM106B did not reveal TMEM106B-induced changes in PGRN (Lang et al. 2012), we are now the third group to observe an increase in PGRN with TMEM106B over-expression (Brady et al. 2013; Chen-Plotkin et al. 2012). However, similar to findings by Brady et al. (Brady et al. 2013) both TMEM106B T185 and S185 isoforms similarly affected PGRN levels. These results, therefore, fail to explain the decrease in FTLD-TDP risk in people who are homozygous for the rs3173615 TMEM106B minor allele.
In previous reports discerning the association between TMEM106B and FTLD-TDP, attention has been drawn to the potential effect of the TMEM106B SNPs on TMEM106B mRNA expression levels (Van Deerlin et al. 2010; Brady et al. 2013; Chen-Plotkin et al. 2012). This began with the observation that individuals homozygous for the protective C-allele of rs199022 had lower TMEM106B RNA levels in brain tissue (Van Deerlin et al. 2010). However, this finding was performed using a small subset of samples and has not been replicated by other groups (Cruchaga et al. 2011; van der Zee et al. 2011). Although we cannot exclude that rs1990622 or variants in linkage disequilibrium with rs1990622 located in non-coding regulatory regions of TMEM106B might contribute to difference in TMEM106B expression levels in vivo, our study now provides strong evidence implicating p.T185S as a functional TMEM106B variant modulating TMEM106B protein levels. Using multiple cell lines and expression vectors, we consistently showed that the risk T185 TMEM106B isoform was expressed nearly two-fold greater than S185 TMEM106B. Subsequent analyses showed that the difference in expression resulted from a more rapid degradation of the S185 TMEM106B isoform in our cell culture system. Brady et al. did not report differences between these two TMEM106B isoforms; however, discrepancies might result from their use of a mouse cell line (Brady et al. 2013). In addition, the transfection levels were not reported for each isoform (Brady et al. 2013). Our findings support the hypothesis that higher TMEM106B protein levels are, at least in part, contributing to the risk differences between the T185 and S185 TMEM106B isoforms and provide the first variant-related difference in the post-translational regulation of TMEM106B.
The p.T185S coding variant of the TMEM106B protein is a part of the N-X-T/S glycosylation consensus sequence for N-glycosylation at position 183 (Lang et al. 2012). Protein glycosylation is a critical post-translational modification that enhances functional diversity as well as biological activity and expression level of a wide-range of glycoproteins. Even though a T or S residue at position 185 is expected to be sufficient for N-glycosylation at TMEM106B N183, we speculate that glycosylation may be different between the two isoforms with a slight change in the glycan composition and/or complexity at N183 in TMEM106B T185 compared with S185. In support of this hypothesis, it has been previously shown that T and S amino acids can affect N-glycosylation transfer rates in vitro, with less efficient interaction between glycotransferase enzymes in S-containing consensus sequences (Bause and Legler 1981). A previous study showed that complete loss of glycosylation at N183 results in retention of TMEM106B to the endoplasmic reticulum (ER) (Lang et al. 2012). Based on our results, cells over-expressing S185 TMEM106B still show TMEM106B localized to the lysosomes suggesting that more subtle changes in glycosylation may be at play. Subtle differences in S185 versus T185 TEM106B N183 glycosylation could explain why no gross differences in the molecular weights or EndoH digestion products of T185 versus S185 TMEM106B were observed. Confirming differences in the composition of complex N-glycans at TMEM106B amino acid 183 would require extensive mass spectrometry analyses and/or specific high performance liquid chromatography beyond the scope of this study. Thus, while our current data do not specifically confirm N183 glycosylation as the functional process involved in regulating T185 versus S185 TMEM106B expression levels, abnormal glycosylation of TMEM106B S185 could explain the enhanced degradation of this TMEM106B isoform. In line with this hypothesis, introduction of an artificial N183S glycosylation-defective mutant within the TMEM106B T185 and S185 isoforms ablated the observed differences in protein expression between these two isoforms.
In conclusion, our study is the first to demonstrate that the p.T185S coding variant, genetically associated with FTLD-TDP risk, acts as a functional variant to regulate TMEM106B protein levels, which we speculate are because of changes in glycosylation. As TMEM106B levels have been shown to be important for determining proper endolysosomal homeostasis, these results support a critical role for lysosomal dysfunction in the development of FTLD-TDP. We also confirmed an effect of TMEM106B expression on PGRN levels. However, in contrast to what would be expected from an FTLD risk factor, all published studies observed an increase (not decrease) in PGRN after over-expression of TMEM106B. As this increase in PGRN may be the consequence of lysosomal dysfunction it remains unclear how this finding has to be interpreted and whether it has any relevance with regards to FTLD-TDP disease risk. One possibility for the strong association of TMEM106B SNPs in patients with GRN mutations may be that these patients merely reflect a patient population vulnerable to additional genetic modifying factors such as TMEM106B. The recent observation of an effect of TMEM106B genotypes on cognition in amyotrophic lateral sclerosis patients and the presence of TDP-43 pathology in Alzheimer's disease patients, two neurodegenerative diseases in which patients are thought to have normal PGRN levels, is of interest in this regard (Vass et al. 2011; Rutherford et al. 2012).
Together, our findings support a critical role for p.T185S in FTLD-TDP risk by regulating TMEM106B protein levels providing a promising novel avenue for disease intervention in FTLD-TDP and related TDP-43 proteinopathies.