TMEM106B expression is elevated in the brains of frontotemporal lobar

After the discovery and replication of genetic variation at TMEM106B as a risk factor for FTLD-TDP (Finch et al., 2011; Rollinson et al., 2011a; Van Deerlin et al., 2010; van der Zee et al., 2011), initial follow-up studies investigated the potential role of TMEM106B in FTLD-TDP pathogenesis. In the original GWAS, it was observed that TMEM106B mRNA levels were elevated in FTLD-TDP brains, particularly in GRN+ FTLD-TDP cases, compared to neurologically healthy controls (Van Deerlin et al., 2010). This effect, reported initially in prefrontal cortex samples, was independent of TMEM106B genotype, and was later confirmed in temporal and occipital cortex samples (Chen-Plotkin et al., 2012). Elevated TMEM106B protein levels were also confirmed in a small sample of GRN+ FTLD-TDP prefrontal cortex samples, compared to controls (Chen-Plotkin et al., 2012).

These observations led to the hypothesis that increased TMEM106B levels in brain may either contribute to disease, result from disease, or both. While I discuss in detail the potential pathogenic role of elevated TMEM106B expression levels in Chapter 5.3, here I discuss the identification of an important TMEM106B-regulating microRNA that may at least partially explain the increased TMEM106B levels seen in FTLD-TDP patient brains (Figure 5.1).

5.2.1.1 Dysregulation of the microRNA-132/212 cluster contributes to elevated TMEM106B levels in frontotemporal lobar degeneration

The observation that TMEM106B levels are elevated in FTLD-TDP, irrespective of TMEM106B genotype (Van Deerlin et al., 2010), suggests that one or more upstream pathways regulating the expression of TMEM106B is altered in disease. Taking into account the deleterious effects of elevated TMEM106B levels in cell culture-based experiments (Brady et al., 2013; Busch et al., 2016; Chen-Plotkin et al., 2012; Suzuki and Matsuoka, 2016), identifying upstream regulators of TMEM106B may reveal potential therapeutic targets.

At the time the GWAS was published, it was becoming increasingly clear that microRNAs (miRs) are critical regulators of gene expression levels, and play important roles in many cellular

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processes (Ameres and Zamore, 2013; Jonas and Izaurralde, 2015). With regards to the nervous system, miRs have been implicated in neurite outgrowth, neuronal differentiation, synaptic plasticity, and many other pathways. In addition, miR dysfunction has been implicated in several neurodegenerative diseases, including AD, PD, ALS, and Huntington’s disease (Goodall et al., 2013)

My lab performed a microarray-based screen to quantify all known human miRs in FTLD- TDP and neurologically normal brains, and identified miR-132 as the most dysregulated miR. Specifically, miR-132 is expressed at lower levels in FTLD-TDP brains (Chen-Plotkin et al., 2012), and miR-132 expression has been shown to be required for learning, memory, and neuronal dendritic branching (Aten et al., 2016). Furthermore, miR-132 has also been shown to be downregulated in AD, Huntington’s disease, and schizophrenia (Aten et al., 2016); thus, miR-132 may be protective against neurological disease more generally. In addition, two other miRs that are processed from the same primary transcript as miR-132, miR-132* and miR-212 (Vo et al., 2005), were also significantly downregulated in FTLD-TDP brains (Chen-Plotkin et al., 2012), suggesting that the locus from which they arise is less transcriptionally active in disease.

Surprisingly, out of 283 predicted target genes of miR-132 and miR-212 (both miRs have the same “seed”, and thus many overlapping targets), TMEM106B was the top predicted target by TargetScan (Lewis et al., 2003), an online microRNA target prediction tool. I confirmed this prediction using several complementary cell-based assays, specifically demonstrating that miR- 132 and miR-212 repress TMEM106B mRNA and protein levels through two binding sites in its 3’UTR (Chen-Plotkin et al., 2012). Since miR-132 is expressed at ~100-fold higher levels than miR-212 (Chen-Plotkin et al., 2012; Magill et al., 2010), it is likely more functionally important. Therefore, reduced levels of miR-132 in FTLD-TDP may contribute to the elevated levels of TMEM106B seen in disease brains (Figure 5.1).

While the identification of these miRs as regulators of TMEM106B is notable, several questions remain: first, are there other, more important regulators of TMEM106B that contribute to its upregulation in disease? Indeed, the most recent version of TargetScan identifies 287 predicted

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miR binding sites throughout TMEM106B, most of which have different predicted miR regulators (Agarwal et al., 2015). While it is unlikely that all of these predicted target sites are truly functional, this does indicate that there are likely multiple miRs that directly regulate TMEM106B levels. Notably, the therapeutic potential of targeting miR-132 is questionable, since both overexpression and knockdown approaches have impaired neurological function in vivo (Hansen et al., 2010; Magill et al., 2010; Scott et al., 2012; Wayman et al., 2008). In addition, there are likely many transcriptional pathways that may be affected in FTLD-TDP that either directly or indirectly affect TMEM106B levels. In support of this, the ENCODE Project has identified >70 transcription factors that bind (and presumably regulate) the TMEM106B promoter (Gerstein et al., 2012), some of which may themselves be alternatively regulated in FTLD-TDP. Second, does the downregulation of miR-132/212 and upregulation of TMEM106B contribute to disease, result from disease, or both? If this is truly a key pathway in FTLD pathogenesis, animal models may be required in order to determine the specific role of these miRs, as well as TMEM106B levels, in disease pathogenesis. For example, miR-132 is known to be induced by synaptic activity, in which BDNF signaling activates CREB target genes (including miR-132/212) in order to facilitate neuronal plasticity (Vo et al., 2005). Do defects in this signaling pathway contribute to the development of FTLD by altering miR-132-mediated regulation of TMEM106B,and, potentially, other genes? Or, conversely, does the presence of FTLD adversely affect these pathways? It is also possible that increased TMEM106B levels may act upstream of miR-132/212 dysregulation. For example, given the deleterious effects of increased TMEM106B levels in cells (see Chapter 5.3), elevated TMEM106B levels may first accelerate disease pathways leading to neurodegeneration, which then results in impaired miR-132/212 regulatory pathways, possibly due to impaired BDNF signaling and/or synaptic plasticity. In this way, TMEM106B upregulation may be reinforced through a positive feedback loop involving impaired repression by miR-132/212.

In Chapter 5.3, I discuss the evidence supporting a deleterious effect of increased TMEM106B levels on cells. Before that, however, I will discuss evidence that more than one route may lead to the same intermediate step of increased TMEM106B expression (Figure 5.1).

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Specifically, I will discuss the primary data presented in Chapter 4, suggesting that the common variant responsible for the association of the 7p21 locus with FTLD-TDP risk affects TMEM106B expression levels through changes in chromatin architecture.

5.2.2 The TMEM106B risk haplotype is associated with increased TMEM106B levels