Missense Mutations in the Progranulin Gene Linked to Frontotemporal Lobar Degeneration with Ubiquitin-immunoreactive Inclusions Reduce Progranulin Production and Secretion

Abstract

Loss of function mutations in progranulin cause tau-negative frontotemporal lobar degeneration with ubiquitin-positive inclusions. A major protein component of these inclusions is TDP-43, which becomes hyperphosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments, which apparently translocate from nuclei to the cytoplasm. Most progranulin mutations are nonsense mutations resulting in nonsensemediated mRNA decay and consequently reduced progranulin protein levels. However, some missense mutations are described that occur within the signal sequence and mature progranulin. We now demonstrate that a progranulin mutation located within the signal sequence (PGRN A9D) results in cytoplasmic missorting with extremely low expression. In contrast, two other progranulin mutations (PGRN P248L and R432C) are expressed as immature proteins but are inefficiently transported through and partially degraded within the secretory pathway, resulting in a significantly reduced secretion. Thus apparently all progranulin mutations cause reduced protein expression or secretion, although by different cellular mechanisms. To investigate a putative relationship between reduced expression of progranulin and TDP-43 relocalization and deposition, we down-regulated progranulin in human cell lines and in zebrafish. Upon reduction of progranulin, neither a major redistribution of TDP-43 nor proteolytic processing to disease-characterizing C-terminal fragments could be observed.Dementias are a major health problem in our aging society. The most frequent forms of dementia, namely Alzheimer disease, frontotemporal lobar degeneration (FTLD), 3 as well as dementia with Lewy bodies and related disorders are associated with selective neuronal cell loss. In these neurodegenerative disorders, proteins, which are normally soluble are known to misfold because of proteolytic processing and/or abnormal posttranslational modifications. Such insoluble amyloidogenic proteins are often deposited and may form reservoirs for neurotoxic oligomers (1). FTLD, which accounts approximately for 15% of all dementias, is characterized by two different types of cellular inclusions. About 40% of FTLD cases have tau-positive inclusions (2, 3). Genetic linkage led to the identification of more than 40 different mutations in the microtubule-associated protein tau gene locus on chromosome 17 (4). However, a number of familial FTLD cases failed to exhibit mutations in the tau gene, although strong linkage to chromosome 17 was observed (5). These cases were characterized by tau-and ␣-synuclein-negative, ubiquitin-positive cytoplasmic and intranuclear inclusions (3). The inclusions are observed in the frontotemporal cortex, the temporal neocortex, and the hippocampus and define the frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U). FTLD-U is the most frequent neuropathological form of FTLD and presents with progressive social, behavioral symptoms, and language dysfunction. Patients may also develop typical symptoms of motoneuron...

Granulin mutations associated with frontotemporal lobar degeneration and related disorders: An update

“…Similar loss of function effects of missense mutations have been suggested in the angiogenin gene (ANG) leading to ALS [Greenway et al, 2006]. Our predicted data were recently confirmed by showing a significant reduction of the GRN secretion for two missense mutations, p.P248L and p.R432C, supporting the loss of function character of GRN missense mutations [Shankaran et al, 2008].…”

The granulin gene family: from cancer to dementia

“…(59) Other mutants may affect the translated protein, for example, by changing one of the cysteines to another residue and therefore disturbing disulfide bridge formation, or by impeding secretion through mutations of the signal peptide. (60,61) Insufficient production of PGRN protein as the underlying cause of disease was confirmed with the identification of FTD associated with allelic loss of the entire GRN locus. (62) Although neuronal death is the hallmark of FTLD, the GRNand MAPT-dependent phenotypes are strikingly different at the cellular level.…”

“…(68) The fragmentation of TDP-43 appears to be an initial step in the formation of ubiquitin/ TDP-43 inclusions, although it should be pointed out that other investigators found no alteration in TDP-43 localization or stability following depletion of PGRN mRNA. (61) The 25 kDa carboxyl-terminal fragment of TDP-43 accumulates in neurodegenerative tissue, and, when over-expressed in cells, it was phosphorylated, ubiquitinated, and cytotoxic. (69,70) Among its several functions, TDP-43 is a specific mRNAbinding protein for human neurofilament mRNA, (71) and may be involved in the response to neuronal injury since its levels increased and it was translocated from the nucleus to the cytoplasm in injured motor neurons.…”

Abnormal phosphorylation of Ser409/410 of TDP‐43 in FTLD‐U and ALS

“…These results suggest that the sarkosyl-insoluble, urea-soluble TDP-43 of 43 kDa and the 35 kDa fragment detected in brains of human controls and patients with FTLD-U or ALS may have been artifacts generated postmortem. Zhang et al reported that the amounts of the 35 and 25 kDa fragments were increased in cells treated with staurosporin or PGRN siRNA and in brains of patients of FTLD-U [20], whereas Shankaran found that these fragments of TDP-43 were generated independently of PGRN knock-down in cell lines and zebrafish [21]. Our results show that a 35 kDa N-terminal fragment of TDP-43 is generated during postmortem incubation and recovered in insoluble fractions, but this is irrelevant to the pathology of TDP-43 proteinopathy.…”