Rapamycin Rescues TDP-43 Mislocalization and the Associated Low Molecular Mass Neurofilament Instability

Smita Majumder,Salvatore Oddo,Fiona B. Thornton,Janice J. Deng,Antonella Caccamo,Yidong Bai

doi:10.1074/jbc.m109.031278

Abstract

TDP-43 is a nuclear protein involved in exon skipping and alternative splicing. Recently, TDP-43 has been identified as the pathological signature protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis. In addition, TDP-43-positive inclusions are present in Parkinson disease, dementia with Lewy bodies, and 30% of Alzheimer disease cases. Pathological TDP-43 is redistributed from the nucleus to the cytoplasm, where it accumulates. An approximately 25-kDa C-terminal fragment of TDP-43 accumulates in affected brain regions, suggesting that it may be involved in the disease pathogenesis. Here, we show that overexpression of the 25-kDa C-terminal fragment is sufficient to cause the mislocalization and cytoplasmic accumulation of endogenous full-length TDP-43 in two different cell lines, thus recapitulating a key biochemical characteristic of TDP-43 proteinopathies. We also found that TDP-43 mislocalization is associated with a reduction in the low molecular mass neurofilament mRNA levels. Notably, we show that the autophagic system plays a role in TDP-43 metabolism. Specifically, we found that autophagy inhibition increases the accumulation of the C-terminal fragments of TDP-43, whereas inhibition of mTOR, a key protein kinase involved in autophagy regulation, reduces the 25-kDa C-terminal fragment accumulation and restores TDP-43 localization. Our results suggest that autophagy induction may be a valid therapeutic target for TDP-43 proteinopathies.

Highlights

TDP-43 has been shown to be the major pathological protein in a wide range of disorders referred to as TDP-43 proteinopathies (6 – 8)
TDP-43 Is Degraded by the Autophagic System—The accumulation of abnormal TDP-43 in amyotrophic lateral sclerosis (ALS) and FTLD-U patients suggests that alterations of the protein quality control mechanisms may be involved in the disease pathogenesis
After 24 h of rapamycin treatment, unchanged after 10 mM 3-MA administration, suggesting that the LC3-II/LC3-I ratio was significantly increased in the rapablocking autophagy selectively increased TDP-43 levels (Fig. 2). mycin-treated cells compared with control cells, indicating an Overexpression of C199-TDP Leads to Cytoplasmic TDP-43 increase in autophagosomal formation (Fig. 4, A and B)

Summary

To whom correspondence should be addressed

Degeneration with ubiquitin-positive inclusions (FTLD-U), motor neuron disease, and amyotrophic lateral sclerosis (ALS). These last two disorders have been directly linked to mutations in TDP-43 [9, 10]. TDP-43 proteinopathies are characterized by the accumulation of abnormally modified TDP-43, suggesting that dysfunction in the intracellular quality control systems (ubiquitinproteasome system and the autophagy-lysosome system) may be involved in the disease pathogenesis. Whereas macroautophagy and microautophagy involve the “in bulk” degradation of regions of the cytosol [27, 28], chaperonmediated autophagy is a more selective pathway, and only proteins with a lysosomal targeting sequence are degraded [29]. It has been shown that increasing autophagy activation by mTOR inhibitors has beneficial effects in neurodegeneration [33,34,35]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION