Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS

Abstract

Nuclear to cytoplasmic mislocalization and aggregation of multiple RNA-binding proteins (RBPs), including Fused in sarcoma (FUS), are the main neuropathological features of the majority of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobular degeneration (FTLD). In ALS-FUS these aggregates arise from disease-associated mutations in FUS, whereas in FTLD-FUS the cytoplasmic inclusions do not contain mutant FUS, suggesting different molecular mechanisms of FUS pathogenesis in FTLD that remain to be investigated. We have previously shown that phosphorylation of the C-terminal Tyr526 of FUS results in increased cytoplasmic retention of FUS due to impaired binding to the nuclear import receptor Transportin 1 (TNPO1). Inspired by the above notions, in the current study we developed a novel antibody against the C-terminally phosphorylated Tyr526 FUS (FUSp-Y526) that is specifically capable of recognizing phosphorylated cytoplasmic FUS, which is poorly recognized by other commercially available FUS antibodies. Using this FUSp-Y526 antibody, we demonstrated a FUS phosphorylation-specific effect on the cytoplasmic distribution of soluble and insoluble FUSp-Y526 in various cells and confirmed the involvement of the Src kinase family in Tyr526 FUS phosphorylation. In addition, we found that FUSp-Y526 expression pattern corelates with active pSrc/pAbl kinases in specific brain regions of mice, indicating preferential involvement of cAbl in the cytoplasmic mislocalization of FUSp-Y526 in cortical neurons. Finally, the pattern of immunoreactivity of active cAbl kinase and FUSp-Y526 revealed altered cytoplasmic distribution of FUSp-Y526 in cortical neurons of post-mortem frontal cortex tissue from FTLD patients compared with controls. The overlap of FUSp-Y526 and FUS signals was found preferentially in small diffuse inclusions and was absent in mature aggregates, suggesting possible involvement of FUSp-Y526 in the formation of early toxic FUS aggregates in the cytoplasm that are largely undetected by commercially available FUS antibodies. Given the overlapping patterns of cAbl activity and FUSp-Y526 distribution in cortical neurons, and cAbl induced sequestration of FUSp-Y526 into G3BP1 positive granules in stressed cells, we propose that cAbl kinase is actively involved in mediating cytoplasmic mislocalization and promoting toxic aggregation of wild-type FUS in the brains of FTLD patients, as a novel putative underlying mechanism of FTLD-FUS pathophysiology and progression.

Overlapping Neuroimmune Mechanisms and Therapeutic Targets in Neurodegenerative Disorders

“…Finally, a possible explanation for coaggregation, along with different aggregation profiles in NDDs, could also be related to posttranslational modifications (PTMs) of proteins involved in neurodegeneration [53]. PTM homeostasis is disrupted in NDDs, and there is growing evidence that PTMs may be related to the phenotypic diversity of NDDs [53][54][55][56]. For instance, some TDP-43 PTMs are specific to FTLD-TDP type A (associated with GRN mutations) and some are specific to type B (associated with C9orf72 mutations) [55], phosphorylated Tyr526 FUS is present in the FTLD-FUS pathology [54,56], and the acetylation of K280/K281 in tau increases aggregation in AD.…”

The Role of c-Abl Tyrosine Kinase in Brain and Its Pathologies

“…Finally, increased c-Abl activity has been found in other ND as well. This way increased c-Abl activity was noted in cortical neurons of FTLD-FUS patients [ 12 ], whereas both total and phosphorylated c-Abl were found upregulated in the temporal neocortex of patients with temporal lobe epilepsy compared to nonepileptic controls [ 41 ]. In the temporal neocortex of model rats treated with pilocarpine, upregulation of total and phosphorylated c-Abl begins 6 h after seizures, with relatively high levels persisting for 60 days, whereas in the hippocampus, elevated c-Abl levels persist for 30 days after seizures and then return to normal [ 41 ].…”

“…Non-receptor protein tyrosine kinases of the Src family (c-Src, c-Fyn, c-Yes, and c-Abl) are associated with ND as they are involved in axonal and dendritic outgrowth during central and peripheral nervous system development and regeneration [ 8 ]. In this context, aberrant c-Abl activation was shown to cause early neuroinflammation and loss of neurons in the forebrain of Niemann–Pick type C (NPC) transgenic mice [ 11 ], and increased c-Abl activation has been reported in neurodegenerative pathologies of PD, AD, ALS and FTD by us and others [ 11 , 12 , 13 ]. In the brains of patients with AD, c-Abl activity is associated with the formation of neuritic plaques and insoluble neurofibrillary tangles [ 14 ], whereas in FTD-FUS cases, increased c-Abl activity was associated with C-terminal Tyr phosphorylation of FUS protein and its aggregation in cortical neurons [ 12 ].…”

“…In this context, aberrant c-Abl activation was shown to cause early neuroinflammation and loss of neurons in the forebrain of Niemann–Pick type C (NPC) transgenic mice [ 11 ], and increased c-Abl activation has been reported in neurodegenerative pathologies of PD, AD, ALS and FTD by us and others [ 11 , 12 , 13 ]. In the brains of patients with AD, c-Abl activity is associated with the formation of neuritic plaques and insoluble neurofibrillary tangles [ 14 ], whereas in FTD-FUS cases, increased c-Abl activity was associated with C-terminal Tyr phosphorylation of FUS protein and its aggregation in cortical neurons [ 12 ]. This suggests that abnormal activation of c-Abl may contribute to nonspecific posttranslational modifications of ND-related proteins, which may then promote the occurrence of features associated with ND, such as the accumulation of insoluble protein aggregates and impaired mitochondrial function, both of which are accompanied by synaptic damage.…”

“…Inversely, depending on the oxidation level in the cell, glutathione peroxidase can also be activated via phosphorylation at Tyr96 by c-Abl [ 22 ]. Although the constitutively active form of c-Abl, Bcr-Abl, has a long history in myeloid and lymphoblastic leukemia, aberrant activation of c-Abl has emerged as a link between various triggers of oxidative stress relevant to PD, AD, FTLD and α-synucleinopathies [ 12 , 15 , 23 ]. Inhibition of c-Abl kinase activity by small molecule compounds used in the clinic to treat human leukemia showed neuroprotective effects in cell and animal models of PD [ 24 ].…”