Abstract
Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219–414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease that commonly strikes people between 40 and 60 years of age
Each inhibitor was used at a concentration of 10 mM, except where indicated, and coverslips were examined for the number of cells containing TDP43- and/or human antigen R (HuR)-positive stress granules
These findings indicate that while many kinase inhibitors can prevent the initial formation of TAR DNA binding protein 43 (TDP-43)-- positive stress granules induced under the prolonged stress induction of paraquat, cyclin-dependent kinases (CDKs) inhibitors were able to successfully reverse the pre-accumulated TDP-43 positive stress granules
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease that commonly strikes people between 40 and 60 years of age. In 2006, TAR DNA binding protein 43 (TDP-43) was identified as a major protein constituent within ubiquitinated neuronal inclusions in a large proportion of ALS and FTLD cases [3,4]. This has led to the re-classification of many ALS and FTLD-ubiquitin cases as primary TDP-43 proteinopathies. In TDP-43 proteinopathies there is substantial clearance of nuclear TDP-43 together with accumulation of ubiquitinated and hyperphosphorylated Cterminal fragment (CTF-TDP-43) in cytoplasmic inclusions [2,3] Recapitulation of these effects in cells transfected with CTF-TDP-43 supports a role for cytosolic TDP-43 accumulation in subsequent neuronal cell death [6]. Little is known about the mechanisms that control translocation of TDP-43 to the cytosol or how TDP-43 becomes accumulated in these diseases
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