Progranulin, TDP-43 and Toxicity
TDP-43 is a principal component of ubiquitin-positive inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U). Under normal conditions, TDP-43 is a nuclear protein but neurons with cytoplasmic inclusions have a substantial loss of nuclear TDP-43. TDP-43 also exhibits a disease-specific biochemical signature; pathologically altered TDP-43 is hyperphosphorylated and cleaved to generate C-terminal fragments of 24-26 kDa in affected brain and spinal cord regions. The molecular basis for TDP-43 proteolysis and the generation of these C-terminal fragments remains largely unknown, although it is reasonable to believe that many TDP-43 truncation products are generated in TDP-43 proteinopathies. We have shown that the in vitro incubation of recombinant TDP-43 with caspase-3 or caspase-7 produces distinct fragments of ~42, 35 and 25 kDa  (Fig.1 C). Furthermore, the activation of caspases in cells by staurosporine treatment leads to the redistribution of TDP-43 from the nucleus to the cytoplasm  (Fig.2). Of interest, null mutations in the gene encoding progranulin (PGRN) are a cause of FTLD-U with TDP-43-positive inclusions [2-5]. When we modeled progranulin haploinsufficiency in cultured cells using progranulin siRNA, activated caspase-3 levels were increased as was the cleavage of TDP-43 into ~25 and ~35 kDa fragments similar in molecular weight to the fragments observed in FTLD-U brain tissue  (Fig.1 D,E). These findings provide a mechanistic link between decreased progranulin expression and abnormal TDP-43 processing which may contribute to TDP-43-mediated toxicity. Indeed, we have shown that, upon overexpression of the 25 kDa C-terminal TDP-43 caspase-cleavage product in mammalian cells, cytoplasmic TDP-43 aggregates are formed that are both ubiquitinated and phosphorylated and which confer cytotoxicity  (Fig.3). Currently, we are undertaking a multi-tiered approach to further investigate the involvement of abnormal progranulin and TDP-43 in neurotoxicity.
Progranulin mutations and neurotoxicity
As mentioned, null mutations in PGRN lead to reduced levels of PGRN and cause FTLD-U with TDP-43 pathology. Missense mutations in PGRN of unknown pathogenicity are also observed in cases of FTLD-U and ALS [2, 3, 7, 8]. PGRN has wide-ranging functions in the periphery as well as in the central nervous system where its expression is limited to microglia and certain neuronal populations. In the adult brain and spinal cord, PGRN may function in neuronal repair and growth. The normal function of PGRN, however, is complex; on the one hand, full-length PGRN has anti-inflammatory and trophic activity but, on the other hand, the proteolytic cleavage products of PGRN, termed granulins, promote inflammatory activity . At present, the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration, and how it relates to TDP-43-mediated toxicity, remains unclear (Fig.4). To gain further insight on this issue, we are using PGRN knockout mice and cell culture techniques. We are also examining the effect of missense PGRN mutations to determine if they, like null mutations, are pathogenic.
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