Mutations of the PGRN gene cause frontotemporal lobar degeneration accompanied by the appearance of ubiquitinated-inclusion bodies [7, 8]. The formation of ubiquitinated inclusions occurs in other neurodegenerative diseases, in particular in ALS . While mutations of PGRN do not appear to cause ALS [53, 54], recent work suggests that PGRN is neurotrophic for spinal cord motor neurons . Here we confirmed that PGRN mRNA and protein is expressed in mouse spinal motor neurons, both in situ and in primary culture (Figures 1, 2, 3). Other spinal cord neurons, and spinal cord microglia also express PGRN. The expression in microglia (Figure 3), which also has been seen in the brains of Alzheimer's patients , is of interest, given that microgliosis accompanies motor neuron degeneration. Recent evidence demonstrates that in peripheral inflammation, PGRN inhibits the activity of the pro-inflammatory cytokine tumor necrosis factor-alpha [41, 43], and it is possible that PGRN may also regulate inflammatory processes in the spinal cord.
Cells of different origins handle PGRN in different ways. Whereas epithelial cells appear to secrete PGRN constitutively, innate immune cells including neutrophils process this growth factor to yield 6 kDa granulin peptides that are stored within granules . Similarly, PGRN is stored within the acrosomes of guinea pig spermatozoa , which are vesicular storage structures. In addition, yeast-two hybrid studies have identified PGRN as a potential partner for nuclear proteins such as cyclin-T , adding a further level of complexity to the localization and roles of PGRN.
The subcellular localization and fate of PGRN in neurons has not been previously defined. PGRN appears to be a strong candidate for entry into the ER/Golgi pathways since the PGRN gene encodes a signal sequence for co-translational entry into the ER, it is rich in disulfide bridges and N-glycosylation sites both of which form during transit through the ER and Golgi compartments. However, it has been suggested that in nerve cells PGRN may localize with either mitochondria or endosomal-lysosomal-like structures , which, if correct has profound implications with respect to the mechanism of action of PGRN. It was therefore important to clarify the sub-cellular compartmentalization of PGRN in nerve cells.
Punctate PGRN immunofluorescence was observed within the cell bodies and in the axons of primary motor neurons in culture, but not in their nuclei (Figures 4, 5, 6). We were unable to detect any co-localization of PGRN with mitochondria in primary motor neurons in culture (Figure 4B, b). However, we were also unable to detect immunoreactive PGRN in the ER or Golgi apparatus (Figure 5a, b). The absence of PGRN immunoreactivity in the ER/Golgi system despite the structural arguments that suggests it would be likely to enter the secretory pathway, may reflect a genuine dissociation of localization. Given the structural features of mature PGRN this may also be a misleading result due, for example, to low PGRN concentrations in the ER/Golgi resulting from rapid transfer through the secretory pathway. Alternatively the PGRN antibody may not react with immature protein as it transits through the ER/Golgi processing pathways. The monoclonal antibody was raised against recombinant mouse PGRN. It is not known whether it would detect an immature form of the protein, namely the conformation of PGRN found in the ER/Golgi compartment before N-glycosylation and formation of disulfide bridges have been completed. To avoid some of these possible confounding issues we employed an alternate strategy that was independent of immunolocalization to examine the localization of PGRN within neuronal cells. NSC-34 motor neuron-like cells were transfected with a PGRN-eGFP fusion protein (Figures 7, 8) or with an eGFP control alone. The eGFP control exhibited green immunofluorescence that concentrated mostly in the nucleus. In contrast PGRN-eGFP exhibited a punctuate fluorescence in the cell body and axons, but not the nucleus, in a pattern that was also observed for intrinsic PGRN. The PGRN-eGFP fusion protein co-localized with GM130, a marker for the trans-Golgi apparatus, but not with a mitochondrial marker (Figure 8). We conclude, therefore that neuronal PGRN enters the ER/Golgi secretory pathway.
There are three major exit pathways from the Golgi apparatus; namely, to the lysosome, to the regulated secretory pathway, which is characterized by dense core secretory granules often containing neuropeptides, or to the constitutive secretory pathway. We detected only limited colocalization of PGRN with lysosomes (Figure 6a), suggesting that this is at most only a minor destination for PGRN. The vesicle-like PGRN structures in the motor neurons did not colocalize with chromogranin A (Figure 5c), indicating that PGRN does not enter the regulated secretory pathway. There was, however, evidence of some co-localization between PGRN and synaptophysin (Figure 6c), although further investigation using higher resolution techniques are necessary to confirm this. PGRN is unlikely to be secreted primarily from synaptic junctions since it did not co-localize with the SNAP-25 marker for neurotransmitter vesicle docking and release sites (Figure 6b). The use of the NSC-34 PGRN-eGFP system in conjunction with confocal microscopy of primary motor neuron cultures may be useful in defining some aspects of PGRN secretion by neurons.
We used the NSC-34 cell line to investigate the effects of PGRN upon cell growth and survival. Using species-specific reverse-transcription PCR, the expression of human PGRN was confirmed in the NSC-34/PGRN cells, and there was no compensatory alteration in the expression of the murine PGRN mRNA (Figure 9A). PGRN elicited a change in the appearance of the NSC-34 cells, causing a more flattened cell shape and more prominent neuritic extensions (Figures 10, 11). Serum deprivation was employed as an apoptotic challenge. Upon more prolonged incubation in serum-free medium the number of NSC-34/vector cells declined, becoming statistically significantly different from NSC-34/PGRN between days 12 and 15 (Figure 12A). This was due to reduction in apoptosis in NSC-34/PGRN cells since the number of TUNEL-positive cells was significantly lower in cultures of NSC-34/PGRN cells compared to cultures of NSC-34/vector cells (Figure 12C). The overall cell number did not significantly increase in NSC34 cell deprived of serum and, except at the earliest time points, the percentage of BrdU positive cells was not significantly changed (Figure 12B), suggesting that PGRN is cytoprotective rather than proliferative for NSC-34 cells in the absence of serum. The ability of exogenous PGRN to increase the survival of NSC-34 cells was confirmed by incubating the wild type NSC-34 cells with purified PGRN in serum free medium (Figure 12D). In other non-neuronal cells, such as dermal fibroblasts, PGRN is strongly protective against acidosis , suggesting that it may play a widespread role in protecting cells against metabolic shocks in their microenvironment.
In order to assess the effect of reduced endogenous PGRN expression upon proliferation, NSC-34 cells were stably transfected with shRNA for PGRN and validated in terms of RNA and protein expression (Figure 13A, B, C). In the presence of 10% serum, the NSC-34 cells actively proliferate. Under these conditions the reduction of PGRN mRNA expression using shRNA silencing decreased cell proliferation by about 50% (Figure 14A), but had no significant effect on apoptosis (Figure 14C). PGRN added back to the culture medium prevented the inhibition of proliferation brought about by PGRN shRNA (Figure 14B). This confirms the specificity of PGRN shRNA and suggests that PGRN acts primarily through an extracellular mechanism. Therefore, PGRN supports both cell survival (Figure 12) and proliferation of NCS-34 cells (Figure 14) and these activities can be functionally separated depending on growth conditions.
The enhanced synthesis of PGRN provided prolonged trophic support for NSC-34 cells in the absence of serum for periods of at least 60 days (Figure 11D). The extended serum-free cultures continued to show a mixed population of rounded and more-differentiated cells, however neuron-like cells were maintained throughout. In these cultures the projections became highly elongated, and were dynamic structures that displayed structural rearrangement over a period of a few hours (Figure 11A). PGRN may promote neurite extension in cortical neurons , and in short-term NSC34-PGRN cultures (Figure 10), however we cannot exclude the possibility that the extended length of projections in the long-term serum-deprived NSC-34/PGRN cultures may be due to improved overall cellular health rather than the direct stimulation of neurite outgrowth.