Lysosome size, motility and stress response regulated by fronto-temporal dementia modifier TMEM106B

https://doi.org/10.1016/j.mcn.2014.07.006Get rights and content

Highlights

  • TMEM106B regulates neuronal lysosome size, number and transport

  • Increased TMEM106B causes TFEB nuclear translocation and lysosomal gene expression

  • Loss of TMEM106B delays Torin-induced TFEB nuclear translocation

  • Neuronal TMEM106B protects lysosomes from oxidative photodamage

Abstract

Fronto-temporal lobar degeneration with TDP-43 (FTLD-TDP) is a fatal neurodegeneration. TMEM106B variants are linked to FTLD-TDP risk, and TMEM106B is lysosomal. Here, we focus on neuronal TMEM106B, and demonstrate co-localization and traffic with lysosomal LAMP-1. pH-sensitive reporters demonstrate that the TMEM106B C-terminus is lumenal. The TMEM106B N-terminus interacts with endosomal adaptors and other TMEM106 proteins. TMEM106B knockdown reduces neuronal lysosomal number and diameter by STED microscopy, and overexpression enlarges LAMP-positive structures. Reduction of TMEM106B increases axonally transported lysosomes, while TMEM106B elevation inhibits transport and yields large lysosomes in the soma. TMEM106B overexpression alters lysosomal stress signaling, causing a translocation of the mTOR-sensitive transcription factor, TFEB, to neuronal nuclei. TMEM106B loss-of-function delays TFEB translocation after Torin-1-induced stress. Enlarged TMEM106B-overexpressing lysosomes maintain organelle integrity longer after lysosomal photodamage than do control lysosomes, while small TMEM106B-knockdown lysosomes are more sensitive to illumination. Thus, neuronal TMEM106B plays a central role in regulating lysosomal size, motility and responsiveness to stress, highlighting the possible role of lysosomal biology in FTLD-TDP.

Introduction

FTLD is amongst the most prevalent neurodegenerative dementias after Alzheimer's. Symptomatology includes pronounced behavioral changes with altered motivation, personality and/or language function, with pathology in the frontal and temporal lobes (Mackenzie et al., 2010). The largest subset of FTLD exhibits neuronal TDP-43 aggregates. Common inherited causes of FTLD-TDP are repeat expansions in C9ORF72, and loss-of-function in the granulin (GRN) gene encoding progranulin (PGRN) protein. PGRN is internalized and concentrated in neuronal lysosomes after binding to Sortilin receptors (Hu et al., 2010). In one family, homozygous GRN mutation causes a lysosomal storage disorder (Smith et al., 2012). Thus, understanding of FTLD-TDP is intimately connected with the function of the neuronal lysosome.

A genome-wide association study of FTLD-TDP risk demonstrated linkage with polymorphisms near the TMEM106B locus (Van Deerlin et al., 2010). Risk association of TMEM106B variants was greatest for GRN mutation carriers, in which there was a decrease in the frequency of homozygous carriers of the rs1990622 minor allele (Finch et al., 2011, Van Deerlin et al., 2010). The most strongly associated SNP, rs1990622, is tightly linked to a missense T185S variant in TMEM106B (rs1042949), suggesting a protective effect of the p.185T form. This association has been replicated and extended to age of FTLD onset (Cruchaga et al., 2011), to clinical cohorts (van der Zee et al., 2011) and to cognitive change in ALS cohorts (Vass et al., 2011). There is evidence that this allele protects against hippocampal sclerosis pathology in those with Alzheimer's disease (Rutherford et al., 2012).

TMEM106B variation alters PGRN level in human serum (Cruchaga et al., 2011, Finch et al., 2011), but the cell biological basis of this effect is unclear. TMEM106B encodes a 274 aa transmembrane protein, and recent publications indicate that TMEM106B is predominantly localized at the endo-lysosomal membrane where it might interact indirectly with PGRN (Brady et al., 2013, Chen-Plotkin et al., 2012, Lang et al., 2012). However, TMEM106B's role in neurons and in lysosomal biology remains poorly defined. A report that appeared online while this manuscript was in preparation (Schwenk et al., 2014), describes TMEM106B interaction with MAP6 and a selective regulation on dendritic retrograde transport.

Recently, there have been advances in understanding lysosomal regulation. The transcription factor EB (TFEB) controls autophagy and lysosomal biogenesis by regulating expression of the Coordinated Lysosomal Expression and Regulation (CLEAR) gene network (Sardiello et al., 2009, Settembre et al., 2013). TFEB colocalizes with the mechanistic target of rapamycin complex 1 (mTORC1) on the lysosome. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB. Conversely, inhibition of mTORC1 or starvation or lysosomal disruption activates TFEB by promoting dephosphorylation and nuclear translocation (Settembre et al., 2012). TFEB acts to sense lysosome state at the lysosome and as an effector of lysosomal function when translocated to the nucleus. Lysosome-to-nucleus signaling allows organelle self-regulation (Settembre et al., 2012).

We sought to determine whether TMEM106B is present in the neuronal lysosome and whether it has a key role in determining organelle size, motility and stability. We report that depletion of neuronal TMEM106B reduces lysosome size and responsiveness to stress, but increases motility. These findings expand our understanding of lysosomal regulation and suggest that these pathways contribute to FTLD-TDP pathophysiology.

Section snippets

TMEM106B protein is localized to neuronal lysosomes

To assess the subcellular localization of TMEM106B, we expressed fluorescently tagged protein in COS-7 cells. In single images of live cells, there was strong co-localization with the lysosomal markers, LAMP-1 and PGRN (not shown). Moreover, these markers moved in concert during time-lapse imaging (Movie 1). These observations match previous reports using fixed samples in immortalized cell lines (Brady et al., 2013, Chen-Plotkin et al., 2012, Lang et al., 2012).

To assess the relevance to FTLD,

Discussion

This study provides multiple lines of evidence that the FTLD-TDP risk-related protein, TMEM106B, plays a crucial role in regulating neuronal lysosomes. In neurons, the TMEM106B protein localizes to lysosomes, where its cytoplasmic domain interacts with endo-lysosomal adaptors and forms multimers. This localization leads to changes in lysosomal size with a direct correlation of neuronal TMEM106B level with lysosome size and number. Active transport of lysosomes in neuronal processes is inversely

Conclusions

These studies show that TMEM106B levels determine the setpoint for lysosomal auto-regulation by TFEB pathways in neurons. Levels of TMEM106B regulate lysosomal size, transport within the neuron and the resilience of the lysosome to photo-oxidative stress. The influence of TMEM106B genetic variation on the risk of FTLD-TDP highlights the potential role of lysosomal dysfunction in this neurodegenerative dementia.

Plasmids and reagents

TMEM106B expression vectors under the chicken beta actin promoter were generated by amplifying the coding sequence of human TMEM106B–WT or TMEM106B–T185S full-length cDNAs (Origene, for both sequences) and cloning into the pCAGG vector via EcoRI sites. From pCAGG–human–TMEM106B–WT or T185S, a Cherry fluorescent protein tag was sub-cloned in frame immediately before the stop codon of TMEM via SmaI to obtain pCAGG–hTMEM106B–Cherry with WT or T185S. From pCAGG–human–TMEM106b, a pHlourin

Acknowledgments

We thank Andrea Ballabio, Roberto Zoncu and Pietro De Camilli for the reagents, and Thihan Padukkavidana for the assistance with acridine orange. This work was supported by grants to S.M.S. from the Association for Frontotemporal Degeneration, the National Institutes of Health R01NS074319 and the Falk Medical Research Trust.

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      TMEM106B consists of a C-terminal domain that projects in the lysosomal lumen, a single-pass transmembrane domain, and an N-terminal domain that extends into the cytosol (Figure 1B) (Lang et al., 2012). The N terminus, TMEM106B(1–96) (Figures 1A and 1B), is involved in lysosomal and endosomal trafficking via interactions with MAP6 and can interact with itself as well as with TMEM106C to form homo- and hetero-multimers at the surface of the lysosome (Schwenk et al., 2014; Stagi et al., 2014). The transmembrane domain, TMEM106B(97–117), is a single-pass α-helix (Figures 1A and 1B).

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    Present address: Institute of Translational Medicine, Liverpool University, Liverpool L693GL, UK.

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