Revisiting the neuronal localization and trafficking of CLN3 in juvenile neuronal ceroid lipofuscinosis

Abstract

Juvenile neuronal ceroid lipofuscinosis, the most common neurodegenerative disease affecting children, is caused by mutations of the CLN3 gene encoding CLN3, a transmembrane protein with so far undefined function. The embryonic expression of the gene has not been studied in detail before. Moreover, the protein CLN3 was mostly localized on the subcellular level to lysosomes but the exclusiveness is still under debate. Here, we analyze the expression pattern of murine CLN3 at different developmental stages by in situ hybridizations. We observe expression maxima in the developing thalamus and cerebral cortex and outside of the central nervous system in the gastrointestinal tract and other peripheral organs. In differentiated primary neurons, the protein CLN3 shows mainly a somatodendritic localization. In primary neurons, we thoroughly revisit the subcellular localization of CLN3 and find a predominant localization in late endosomal-lysosomal compartments. Moreover, we expressed the major mutant form of CLN3 - CLN3deltaExon7/8 - in neurons and demonstrate that it is retained in the endoplasmatic reticulum. Time-lapse microscopy analysis of neurons revealed co-trafficking of CLN3 with the late endosomal marker Rab7, but not with the early endosomal marker Rab5. Furthermore, a constitutive active mutant of Rab7 traps CLN3 in enlarged endosomes. Our subcellular localization study in neurons refines the localization and subcellular targeting of CLN3 to late endosomal-lysosomal compartments and provides information on the velocity of CLN3 in living neurons which has not been investigated before.

NCLs and ER: A stressful relationship

“…A significant localisation of the CLN3 orthologue of fission and budding yeasts, Btn1p, has been shown at the Golgi complex [71] and also reported for GFP-tagged CLN3 in mammalian cells [72]. Moreover, untagged CLN3 has been shown to localise at late-endosomes and lysosomes [73]. However, the functional localisation of CLN3 is still matter of debate.…”

Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis

“…Disturbed synaptic transmission and impaired short-term plasticity are in line with the proposed dysfunction of CLN3 protein, along with the neurodegenerative processes in patients and in the respective mouse models. CLN3 is localized in synaptosomes ( Luiro et al, 2001 ), in axonal compartments, and in synaptic spines ( Oetjen et al, 2016 ). The proposed role of CLN3 in endocytosis and endosomal functions provides a direct link to short-term plasticity and suggests a role of CLN3 in neuronal transmission.…”

in vivo localization of the neuronal ceroid lipofuscinosis proteins, CLN3 and CLN7 , at endogenous expression levels

“…We attempted to reduce the possibility of mis-folding as far as possible by inserting YFP at the extreme N-terminus of both CLN7 and CLN3 coding sequences. Many groups have used N-terminal fusions of GFP to CLN3 in a variety of models without retention of the protein in the ER (Codlin and Mole, 2009, Huber et al, 2014, Oetjen et al, 2016) and GFP-CLN3 is functional in both yeast (Codlin and Mole, 2009) and Dictyostelium (Huber et al, 2014). In contrast, we and others have shown that truncated forms of CLN3—or adding epitope tags or GFP to the C-terminus—does lead to ER retention and a very different sub-cellular staining pattern than that we see here (example studies include Haskell et al, 1999, Jarvela et al, 1999, Tuxworth et al, 2009).…”