Uncovering the Role of Progranulin in Ameliorating Lysosomal Storage Disease

This study investigates the effect of an inflammation modulator, granulin (GRN), and its role in Sandhoff disease. GRN deficiency has been shown to promote neuroinflammation, neuronal death, and leads to a lysosomal storage disorder (LSD) 1-7. Complete loss of GRN causes Batten’s disease (CLN11), which belongs to a class of LSDs1. Interestingly, expression profiles of mice models of LSDs, i.e., Sandhoff, show that GRN expression is increased during pathogenesis. Our lab previously generated and characterized a mouse deficient in both GRN and HEXΒ (Sandhoff) with the prediction that loss of GRN would worsen Sandhoff disease progression in mice due to increased inflammation. Unexpectedly, the double knockout (HEXΒ-/-/GRN-/-) mice outlived Sandhoff (HEXΒ-/- / HEXΒ+/+) mice. The medium lifespan for a HEXΒ-/-/GRN-/- was 200 days compared to 127 for Sandhoff mice (P value = 0.0002). We evaluated neuronal benefit via neurofilament light chain (NF-L) levels and found that levels were decreased. Based on these results we hypothesized that loss of GRN leads to a reduction in inflammation. This hypothesis goes against the accepted role of GRN, as an inflammation modulator, but aligns with the field of neurodegeneration’s understanding of neuroinflammation. Deleting macrophage inflammatory protein-1a (mip-1a) in Sandhoff mice by Wu et al, lead to a significant reduction in neurodegeneration9. To test our hypothesis, we examined differential expression of mice hindbrains at multiple timepoints for the following genotypes, HEXΒ-/-/GRN+/+, HEXΒ+/+/GRN-/-, HEXΒ-/-/GRN-/-, and GRN+/+/HEXΒ+/+. A set of 40 recognized inflammation markers, including CD68, GPNMB, GFAP, and HPSE, showed equivalent or elevated levels in the HEXΒ-/-/GRN-/- mice compared to HEXΒ-/-/GRN+/+(Sandhoff). Inflammation was confirmed through immunohistochemistry staining on brain slices. These results are consistent with GRN’s known role in mitigating inflammation. This suggests that the survival benefit in the HEXΒ-/-/GRN-/- mouse is not due to reduced inflammation but other components in the pathogenesis. Additional studies were performed to address the accumulation of substrate and lysosomal defect in the HEXΒ-/-/GRN-/- model. Livers from all four genotypes at day 65 were analyzed via mass-spec for ganglioside concentration associated with the primary accumulates in Sandhoff disease. There was no reduction in ganglioside accumulation for the HEXΒ-/-/GRN-/-mice. Lastly, immortalized fibroblasts were fixed and stained for lysosomal-associated membrane protein 1 (LAMP1). Results showed a significant decrease of LAMP1 intensity in the HEXΒ-/- /GRN-/-compared to HEXΒ-/- /GRN+/+(Sandhoff) cells. These invitro results indicate that there is an altered lysosomal pathway, either trafficking of vesicles or potential alteration of autophagy in the HEXΒ-/-/GRN-/- model. Further characterization of the in vivo lysosomal status would elicit the mechanism of action for the survival benefit in the HEXΒ-/-/GRN-/- model.