Stress granule recruitment and deposition of proteins of the FET family and TDP-43 in ALS and FTD

Neurodegenerative diseases such as Alzheimer´s disease, amyotrophic
lateral sclerosis (ALS) and frontotemporal dementia (FTD) are
defined by progressive and selective loss of neurons. With
increasing age the risk of developing a neurodegenerative disease
exponentially rises. To date these diseases are untreatable,
imposing a significant medical, social and financial burden onto
our ageing society. Typical features of neurodegenerative diseases
are abnormal aggregation of a disease characterizing protein and
its deposition in pathological inclusions. A unifying feature in
the majority of ALS cases and several subtypes of FTD is the
pathological deposition of the TAR DNA-binding protein of 43kDa
(TDP-43) or the Fused in Sarcoma (FUS) protein. Furthermore, stress
granule (SG) marker proteins are consistently detected in FUS
inclusions, suggesting that SGs might be involved in the formation
of FUS inclusions. However, whether pathologic TDP-43 inclusions
contain SG marker proteins is still controversially discussed. In
this thesis I demonstrate that cytosolically mislocalized
full-length TDP-43 is recruited into SGs, whereas C-terminal
fragments of TDP-43 (TDP-CTFs) fail to localize to SGs. In
accordance with these cell culture data, spinal cord inclusions in
ALS and FTD patients contain full-length TDP-43 and SG marker
proteins. In contrast, hippocampal inclusions are enriched for
TDP-CTFs but are SG marker-negative. Thus, the protein composition
of TDP-43 inclusions determines whether SG marker proteins are
co-deposited in TDP-43 inclusions or not. By analyzing the
prerequisites for SG recruitment of TDP-43 and FUS, I demonstrate
that cytosolic mislocalization of TDP-43 and FUS is required for
their localization in SGs. Additionally, I found that both proteins
have the same requirements for SG recruitment, as their main
RNA-binding domain and a glycine-rich domain are essential for SG
localization. A detailed analysis of the protein composition of FUS
inclusions in ALS and FTD cases unveiled that FUS inclusions in FTD
cases contain not only FUS, but all FET (FUS, Ewing sarcoma protein
(EWS), TATA binding protein-associated factor 15 (TAF15)) family
proteins. Here, I provide evidence that this cytosolic deposition
of FET proteins can be mimicked in cultured cells by inhibition of
Transportin-mediated nuclear import, which causes cytosolic
mislocalization of all FET proteins and recruitment of these
proteins in SGs. In contrast to FTD cases, FUS inclusions in ALS
cases contain only FUS, but not EWS and TAF15. In line with that, I
show that ALS-associated FUS mutations result in cytosolic
mislocalization of FUS that is upon subsequent cellular stress
sequestered into SGs. These SGs then contain only FUS but not EWS
or TAF15, demonstrating that mutant FUS is unable to co-sequester
EWS or TAF15. In addition, I contributed to two studies that
revealed that nuclear import defects are involved in the
pathogenesis of ALS and FTD. ALS associated FUS mutations are
frequently located within the proline-tyrosine nuclear localization
signal (PY-NLS) of FUS and thus disrupt Transportin-mediated
nuclear import and cause cytosolic mislocalization of FUS. EWS and
TAF15 also contain a PY-NLS and thus are imported into the nucleus
via Transportin. This interaction between Transportin and FET
proteins can be modulated by arginine methylation that reduces
Transportin binding. In FTD patients with FUS inclusions, this
post-translational modification seems to be defective, as FUS
inclusions in these cases contain hypomethylated FUS. Taken
together, these data provide evidence that nuclear import defects
and sequestration of FUS and TDP-43 in SGs are consecutive steps in
the pathogenesis of ALS and several subtypes of FTD.