The major finding of this work is that Sortilin is a principal neuronal binding site for the FTLD protein, PGRN. In a stressed nervous system, after production by activated microglial cells, PGRN binds to Sortilin expressed on the neuronal cell surface. Binding occurs via the carboxyl terminus of PGRN, in a manner that may resemble NT binding to Sortilin (Quistgaard et al., 2009
). A dramatic consequence of such binding is the rapid endocytosis of PGRN by Sortilin. In vivo, the absence of Sortilin raises PGRN levels by 2.5- to 5-fold in different compartments. Given that FTLD is caused by a 2-fold reduction of PGRN levels (Baker et al., 2006
; Cruts et al., 2006
; Finch et al., 2009
; Gass et al., 2006
; Ghidoni et al., 2008
; Sleegers et al., 2009
), the magnitude of this change has clear pathophysiological consequences, and PGRN deficiency in GRN
+/− mice is fully normalized by deletion of Sortilin expression (, Suppl. Fig. S6
There are several mechanisms whereby PGRN/Sortilin interaction might alter neuronal function and contribute to FTLD-TDP. Sortilin may function upstream to titrate PGRN levels and/or downstream of PGRN to mediate its effects on cell function. To distinguish the relative importance of Sortilin upstream versus downstream of PGRN will require the creation of FTLD-TDP relevant models in which altering PGRN function changes the outcome. Unfortunately, such an experimental model is not yet established. The changes in brain and serum PGRN levels in Sort1
−/− mice observed here demonstrate that Sortilin can have an upstream effect to regulate levels of PGRN. However, there is ample reason to believe that Sortilin’s primary effect is downstream of PGRN, as a mediator or receptor for functional effects in neurons. Sortilin is reported to possess signalling function in complex with p75NTR for pro-neurotrophin apoptotic signalling (Domeniconi et al., 2007
; Jansen et al., 2007
; Nykjaer et al., 2004
; Teng et al., 2005
). While we did not observe modulation of PGRN binding to Sortilin by p75NTR, pro-neurotrophin action may be altered in a more complex in vivo setting. Specifically, since pro-NGF and PGRN can both bind to Sortilin (), PGRN might rescue cells otherwise subject to pro-neurotrophin-induced cell death through Sortilin/p75NTR. Displacement of PGRN by pro-NGF is of low potency so it may be indirect or allosteric, and therefore highly dependent on cellular context. Further study of pro-neurotrophin-related effects under a range of conditions may be revealing. However, it remains at least equally likely that PGRN signals via Sortilin independently of pro-neurotrophins.
Separate from signalling at the cell surface, PGRN may have intracellular functions dependent on endocytosis by neuronal Sortilin and delivery to lysosomes. Importantly, immunologically intact PGRN accumulates within the lysosomal compartment of COS-7 cells and neurons. In healthy or FTLD-TDP human brain PGRN is most prominent in microglial cells, but is also present in puncta within the cytoplasm of neurons (Mackenzie et al., 2006
), and these appear most consistent with lysosomes. These observations raise the possibility that PGRN functions within autophagosomal/lysosomal pathways. Endocytosis and lysosomal delivery of another Sortilin ligand, prosaposin, serves to enhance lysosomal function rather than simply degrading the ligand (Lefrancois et al., 2003
; Zeng et al., 2009
Under this hypothesis, Sortilin binding is crucial to provide neuronal competence for TDP-43 clearance via autophagosomal/lysosomal mechanisms. Sortilin is a member of the Vps10 family (Willnow et al., 2008
), members of which are required for sorting to the lysosomal vacuole in yeast (Cooper and Stevens, 1996
). Sortilin is known to participate in the delivery of extracellular prosaposin and intracellular Golgi-derived cathepsins to lysosomes (Canuel et al., 2008
; Lefrancois et al., 2003
; Ni and Morales, 2006
; Zeng et al., 2009
). PGRN is co-regulated with lysosomal genes (see Suppl. Data in Reference (Sardiello et al., 2009
)). We and others (Ahmed et al., 2010
) have observed accelerated brain lipfuscinosis in mice lacking PGRN. Of relevance to FTLD, the clearance of cytoplasmic TDP-43 depends at least in part on autophagy (Caccamo et al., 2009
; Ju et al., 2009
; Urushitani et al., 2010
; Wang et al., 2010
). Ubiquitin co-accumulation with TDP-43 aggregates may reflect disrupted balance between proteosomal pathways, macroautophagy and chaperone-mediated autophagy pathway.
In this regard, it may be relevant to consider those mutations in Valosin-Containing Protein (VCP), which are known to cause FTLD plus myopathy and Paget’s disease (Custer et al., 2010
; Ju et al., 2009
; Watts et al., 2004
). The accumulation of TDP-43 and ubiquitin aggregates in brain of these rare cases is similar to that in more common PGRN-mutant cases. Since VCP has a prominent role in autophagy, the PGRN pathway and the VCP pathway may overlap as intracellular constituents are sorted to lysosomes. Both PGRN and VCP might function to clear TDP-43 aggregates via autophagy.
As noted above, PGRN can be converted proteolytically into smaller GRN peptides (Zhu et al., 2002
). In the extracellular space, this conversion can be mediated by elastase, and inhibited by secreted leukocyte protease inhibitor (SLPI). Lysosomal localization of PGRN may also influence conversion from PGRN to GRN, and therefore the balance between PGRN and GRN function. Such conversion may have downstream effects on PGRN biology and the pathophysiology of FTLD. In order to separate these possibilities further in vivo neuronal studies of GRN versus PGRN activity will be required.
It is clear from the present data that the interaction of PGRN with Sortilin-mediated endocytosis significantly reduces steady-state PGRN levels. The magnitude of this reduction is at least as great as that of those PGRN mutations causing FTLD-TDP. Thus, Sortilin binding provides a potential therapeutic site to alter PGRN-dependent pathways and alleviate TDP-43 pathology.
It is clear that microglial cells are a major source of PGRN production. For example, activated microglia in the vicinity of axotomized motoneurons strongly induce PGRN after sciatic nerve injury. From this observation, it follows that characterizing the mechanisms of microglial PGRN induction or the selectivity of PGRN for subsets of microglia may reveal additional means to modulate the course of FTLD-TDP.
Because neither GRN
+/− nor GRN
−/− mice exhibit an FTLD-like phenotype (Kayasuga et al., 2007
; Yin et al., 2010
) (data not shown), this human inherited disease cannot be modelled simply in rodent. Although peripheral axotomy shifts TDP-43 from the nucleus to the cytoplasm of motoneurons (Moisse et al., 2009
; Sato et al., 2009
) and might sensitize to FTLD/ALS-like pathology, neither GRN
−/− nor Sort1
−/− mice have detectable motoneuron loss or ventral root axonal degeneration after this perturbation (Suppl. Fig. S4
). Future functional studies of Sortilin in PGRN biology will require development of robust rodent models for PGRN-dependent neurodegeneration. Nevertheless, our work implicates Sortilin-mediated PGRN endocytosis as a key pathway for further study in FTLD pathophysiology.