Accumulating evidence suggests that impaired lysosomal protein degradation plays a major role in FTLD-TDP [
15]. Lysosomal dysfunction seems to be specifically associated with FTLD-TDP caused by
GRN haploinsufficiency [
18]. Furthermore total loss of PGRN leads to NCL (CLN11) [
3], a lysosomal storage disease with severe neurodegeneration. However, it is still unknown if and how PGRN affects lysosomal homeostasis. Based on the selective expression of GRN in microglia (Fig.
5b) [
51,
58,
59], we now searched for cell autonomous and non-cell autonomous deficits upon loss of PGRN.
We provide strong evidence that loss of PGRN selectively impairs lysosomal function in microglia. Microglia isolated from 3-month-old
Grn−/− mice showed strongly reduced CatD levels compared to microglia isolated from
Grn+/+ mice, which surprisingly did not result in a significantly reduced in vitro activity. However, impaired maturation might not be reflected by the in vitro activity assay since defective CatD maturation and catalytic activity could be hidden by optimal in vitro conditions. In addition, maturation of CatB, CatL and CatS was impaired. In contrast, in the microglia depleted fraction, isolated from the same 3-month-old
Grn−/− mice, no altered cathepsin expression or maturation was observed. Furthermore, during ageing saposin D and LAMP1 accumulated earlier and to a higher extent in the microglia enriched fraction than in the microglia depleted fraction. Our finding that impaired maturation of lysosomal enzymes in microglia already occurs in early adulthood before other pathological hallmarks suggests that lysosomal dysfunction may be a primary consequence upon loss of PGRN expression. Thus, our findings suggest a cell autonomous reduction of lysosomal function caused by PGRN deficiency in microglia, which as a consequence appears to culminate during ageing in a compensatory upregulation of lysosomal activity selectively in non-microglial cells. Indeed, in the microglia depleted fraction isolated from aged
Grn−/− mice CatD single and heavy chain are 2-fold and CatD in vitro activity is 2.5-fold elevated compared to
Grn+/+ mice. Our findings are supported by the observation that cultured neurons from
Grn−/− mice show enhanced lysosomal proteolysis [
51]. Moreover, in brain tissue of FTLD-TDP patients CatD is accumulating in neurons [
21]. In line with enhanced cathepsin expression in non-microglial cells, mRNA, protein levels and in vitro activities of CatD, CatB, and CatL were increased in total brain of aged
Grn−/− mice. Furthermore, in line with recent findings [
21,
38,
40,
51,
59,
60], a subset of lysosomal proteases and membrane proteins were upregulated in 6- and 12-month-old
Grn−/− mice. In addition to altered cathepsin levels, we demonstrate altered proteolytic processing and maturation of CatD, CatB, and CatL in the microglia enriched fraction, total brain lysates and MEF of
Grn−/− mice. While in microglia proteolytic inactive pro-forms accumulate, potentially active single chain or heavy chain variants accumulate in total brain and MEF in accordance with increased in vitro activity. For example, robustly enhanced levels of the CatD
hc variant are observed in
Grn−/− MEF which is in line with findings by Tanaka et al. [
21]. Previous research revealed enhanced [
51,
59] as well reduced lysosomal enzyme activities [
38,
39] in various
Grn−/− cells types or tissue. Based on our findings, these discrepancies may be explained by different cell types analyzed, difficulties with the determination of specific activities of lysosomal enzymes due to their complex proteolytic processing and consequences for their proteolytic activity [
53,
61]. Single chain variants as well as dimeric variants of heavy and light chain are catalytically active whereas separated heavy and light chains are inactive [
53,
61]. Since we cannot determine the amount of active species, we cannot calculate the specific activity. Indeed, in total brain of
Grn−/− mice the increase of CatD protein is much stronger than the increase in enzyme activity which might indicate reduced specific activity as previously shown for CatD [
38,
39]. However, it is unlikely that PGRN directly affects the specific activity of lysosomal proteases, because in our hands, adding PGRN, elastase digested PGRN, or granulin E to the in vitro activity assays of CatD, CatB, and CatL did not alter their activity. This might indicate that PGRN most likely modulates maturation and turnover of cathepsins.
In MEF, enhanced cathepsin activities are reversible by low expression levels of PGRN. Rescue of the lysosomal phenotype of
Grn−/− by a very minor amount of PGRN is in line with recent data showing that low levels of AAV-expressed neuronal PGRN are sufficient to rescue lysosomal phenotypes of
Grn knockout mice [
62]. Moreover, this also provides additional support for the lack of lysosomal abnormalities in heterozygous, neuronal or incomplete microglial
Grn knockout mouse models [
63,
64]. Finally, elevated catalytic activities of cathepsins result in enhanced protein turnover in
Grn−/− MEF, which indicates enhanced protein degradation in lysosomes. In line with enhanced lysosomal degradation, levels of lysosomal targeted proteins such as mature APP and its CTF are significantly reduced in
Grn−/− MEF while LC3I and LC3II levels are unchanged. Only under cellular stress impaired autophagy or altered autophagic flux has been reported for bone marrow derived macrophages (BMDM) [
40].