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Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

ABSTRACT: Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity.

SUBMITTER: Beel S

PROVIDER: S-EPMC5886064 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

Beel Sander S Moisse Matthieu M Damme Markus M De Muynck Louis L Robberecht Wim W Van Den Bosch Ludo L Saftig Paul P Van Damme Philip P

Human molecular genetics 20170801 15

Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleu ...[more]

PMID: 28453791

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Project description:Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1-/-) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects.As the first group to evaluate the effect of PGRN loss in Grn knockout primary neuronal cultures, we show neurite outgrowth and branching are significantly decreased in Grn-/- neurons compared to wild-type (WT) neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However, the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties of PGRN. In addition, we also present evidence that cleavage of full-length PGRN into granulin peptides is required for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sort1-/- cultures.We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth.

Project description:BackgroundMicrotubule (MT) regulators play essential roles in multiple aspects of neural development. In vitro reconstitution assays have established that the XMAP215/Dis1/TOG family of MT regulators function as MT 'plus-end-tracking proteins' (+TIPs) that act as processive polymerases to drive MT growth in all eukaryotes, but few studies have examined their functions in vivo. In this study, we use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons.ResultsHere, we show that XMAP215 is required for persistent axon outgrowth in vivo and ex vivo by preventing actomyosin-mediated axon retraction. Moreover, we discover that the effect of XMAP215 function on MT behavior depends on cell type and context. While partial knockdown leads to slower MT plus-end velocities in most cell types, it results in a surprising increase in MT plus-end velocities selective to growth cones. We investigate this further by using MT speckle microscopy to determine that differences in overall MT translocation are a major contributor of the velocity change within the growth cone. We also find that growth cone MT trajectories in the XMAP215 knockdown (KD) lack the constrained co-linearity that normally results from MT-F-actin interactions.ConclusionsCollectively, our findings reveal unexpected functions for XMAP215 in axon outgrowth and growth cone MT dynamics. Not only does XMAP215 balance actomyosin-mediated axon retraction, but it also affects growth cone MT translocation rates and MT trajectory colinearity, all of which depend on regulated linkages to F-actin. Thus, our analysis suggests that XMAP215 functions as more than a simple MT polymerase, and that in both axon and growth cone, XMAP215 contributes to the coupling between MTs and F-actin. This indicates that the function and regulation of XMAP215 may be significantly more complicated than previously appreciated, and points to the importance of future investigations of XMAP215 function during MT and F-actin interactions.

Dataset Information

Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

Publications

Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

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