An altered secretome is an early marker of the pathogenesis of CLN6 Batten disease

Abstract

Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited childhood neurodegenerative disorders. In addition to the accumulation of auto-fluorescent storage material in lysosomes, NCLs are largely characterised by region-specific neuroinflammation that can predict neuron loss. These phenotypes suggest alterations in the extracellular environment-making the secretome an area of significant interest.This study investigated the secretome in the CLN6 (ceroid-lipofuscinosis neuronal protein 6) variant of NCL. To investigate the CLN6 secretome, we co-cultured neurons and glia isolated from Cln6 nclf or Cln6 ± mice, and utilised mass spectrometry to compare protein constituents of conditioned media. The significant changes noted in cathepsin enzymes, were investigated further via western blotting and enzyme activity assays. Viral-mediated gene therapy was used to try and rescue the wildtype phenotype and restore the secretome-both in vitro in co-cultures and in vivo in mouse plasma. In Cln6 nclf cells, proteomics revealed a marked increase in catabolic and cytoskeletal-associated proteins-revealing new similarities between the pathogenic signatures of NCLs with other neurodegenerative disorders. These changes were, in part, corrected by gene therapy intervention, suggesting these proteins as candidate in vitro biomarkers. Importantly, these in vitro changes show promise for in vivo translation, with Cathepsin L (CTSL) activity reduced in both co-cultures and Cln6 nclf plasma samples post gene-therapy. This work suggests the secretome plays a role in CLN6 pathogenesis and highlights its potential use as an in vitro model. Proteomic changes present a list of candidate biomarkers for monitoring disease and assessing potential therapeutics in future studies.

Altered protein secretion in Batten disease

“…For example, STRING analysis highlighted a cluster of proteins associated with proteolysis. More specifically, Cln6 nclf conditioned medium contained increased levels of the proteases CTSB, CTSD and CTSL (Best et al, 2021), which is consistent with earlier findings in D. discoideum cln3 − (Huber, 2017) (Fig. 3) and mfsd8 − cells (Huber et al, 2020b), as well as in mammalian models of CLN2 and CLN3 disease (Bennett et al, 1992;Metcalf et al, 2008).…”

“…NRCAM and VCAM1 are members of the immunoglobin gene superfamily of adhesion proteins that regulate cell adhesion in the brain (Sakurai, 2012). In support of their altered levels in blood plasma, D. discoideum cln3 − cells Huber, 2017) and neural cells derived from a mouse model of CLN6 disease (Best et al, 2021) also aberrantly secrete adhesion proteins, as discussed earlier. In addition, loss of cln3 or cln5 (Huber and Mathavarajah, 2018b) in D. discoideum causes adhesion defects.…”

“…Restoring Cln6 expression by using viral-mediated gene therapy partially corrected the increased extracellular amounts of angiotensin (Box 1), high endothelial venule protein (Box 1), 14-3-3 protein zeta/delta (Box 1), cationic trypsinogen (Box 1) and the chaperone heat shock protein 90 (Box 1) in Cln6 nclf conditioned medium, as well as the increased extracellular activity of CTSL (Best et al, 2021) supporting the use of these proteins as biomarkers in short-term therapeutic studies. Overall, Best et al (2021) identified a set of potential biomarkers for CLN6 disease that can be evaluated further.…”

“…As discussed above, previous work in D. discoideum revealed proteins whose secretion is affected by cln3 deficiency ( Huber, 2017 ). Using a similar approach, recent work examined the secretome of a CLN6 disease mouse model ( Cln6 nclf ), by co-culturing primary cortical cell cultures of Cln6 nclf neurons, microglia and astrocytes, followed by an analysis of the protein content of conditioned medium ( Box 1 ) ( Best et al, 2021 ). Conditioned medium from WT cells contained 152 proteins, whereas conditioned medium from Cln6 nclf cells contained 169 proteins.…”

“…The remaining 31% of proteins were predicted to be secreted by unconventional mechanisms or in response to some form of cellular stress (e.g. inflammation) ( Best et al, 2021 ). These results are consistent with the presence of proteins in conditioned buffer of D. discoideum cln3 − cells, which are normally not secreted and do not have a putative secretion-signal peptide ( Huber, 2017 ).…”