Neurodegenerative Diseases - Leonard Petrucelli

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 [1] (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 [1] (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 [1] (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 [6] (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 [9]. 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.

Fig.1: Proteolytic processing of TDP-43. A. Progranulin knockdown in H4 neuroglioma cell line using siRNA. B. Schematic of the amino acid sequence of TDP-43 (Entrez accession number NP_031401) depicting the three caspase-3 recognition motifs, DXXD (bold and underlined), cleavage of which is predicted to generate fragments of ~42 kDa (red), ~35 kDa (green) and ~25 kDa (blue). C. Recombinant GST-tagged TDP-43 protein was incubated with purified caspase-3, caspase-7, or caspase-8 for 4 h. Samples were separated on 10 percent SDS-PAGE and immunoblotted with rabbit anti-TDP-43 antibody. D. in vitro generation of pathologic TDP-43 by cells treated with PGRN siRNA but not by cells treated with control siRNA. Progranulin deficiency leads to an increase in cleaved caspase-3 activity. Treatment with a pan-caspase inhibitor suppresses progranulin-mediated TDP-43 cleavage and caspase-3 activity. E. Biochemical analyses of TDP-43 in sporadic and familial (PGRN mutant; Gly333ValfsX28) FTLD-U and AD brains. Immunoblots of urea fractions from temporal cortex of FTLD-U patients and PGRN siRNA total cell extract showed a similar pathological profile of TDP-43. F.L., Full-length.

Fig. 2: Cellular localization of caspase-resistant TDP-43. A. H4 cells transfected with either myc-tagged wild-type TDP-43 or myc-tagged double caspaseTDP-43 mutant (D89E, 35 kDa fragment; D219E, 25 kDa fragment) in the presence or absence of staurosporine (6 h, 1µM). Immunoblots of cell extracts probed with mouse anti-myc antibody revealed resistance of the caspase mutant TDP-43 to proteolytic cleavage in cells treated with staurosporine. B. Subcellular redistribution of wild-type and caspase mutant TDP-43. Immunofluorescent staining for myc-tagged wild-type and caspase mutant TDP-43 in H4 cells treated with staurosporine (6 h, 1µM) with mouse anti-TDP-43 (green) and examined by confocal microscopy. The nucleus is stained with DAPI (blue). Caspase mutant TDP-43 is retained in the nucleus in the presence of staurosporine. Scale bar, 20 µm. Stspn, Staurosporine; WT, wild type.

Fig.3: A. The release of LDH into the media was used as an indicator of cell toxicity. LDH levels were measured 72 h after cells were transfected with the indicated constructs. Data from 3 separate experiments was analyzed by one-way analysis of variance followed by Tukey's post-hoc analysis (** P<0.001). B. Increased apoptosis in differentiated M17 neuroblastoma cells expressing GFP-TDP-25 compared to cells expressing GFP alone or GFP-TDP-43. Cultures were fixed and stained with Hoechst to label nuclei (blue) and activated caspase-3 antibody (red). Scale bar = 10 µM.

Fig. 4: PGRN levels affect many biological functions. Elevated PGRN levels stimulate proliferation, survival and motility in a variety of cells. Consequently, PGRN acts as a tumorigenic agent leading to several different types of cancers. Genetic and functional analysis has shown that decreased levels of PGRN due to null mutations within the PGRN gene are a major cause of FTLD-U with TDP-43-positive inclusions. The broad range of age at onset of FTLD-U patients with PGRN mutations indicates that environmental and/or other genetic factors are also likely to play a role in disease pathogenesis.

References cited on this page

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