Mapping of Cbln1‐like immunoreactivity in adult and developing mouse brain and its localization to the endolysosomal compartment of neurons

Abstract

Cbln1 is a secreted glycoprotein essential for synapse structure and function in cerebellum that is also expressed in extracerebellar structures where its function is unknown. Furthermore, Cbln1 assembles into homomeric complexes and heteromeric complexes with three family members (Cbln2-Cbln4), thereby influencing each other's degradation and secretion. Therefore, to understand its function, it is essential to establish the location of Cbln1 relative to other family members. The localization of Cbln1 in brain was determined using immunohistochemistry and cbln1-lacZ transgenic mice. Cbln1-like immunoreactivity (CLI) was always punctate and localized to the cytoplasm of neurons. The punctate CLI colocalized with cathepsin D, a lysosomal marker, but not with markers of endoplasmic reticulum or Golgi, indicating that Cbln1 is present in neuronal endosomes/lysosomes. This may represent the cellular mechanism underlying the regulated degradation of Cbln1 observed in vivo. Outside the cerebellum, CLI mapped to multiple brain regions that were frequently synaptically interconnected, warranting their analysis in cbln1-null mice. Furthermore, whereas CLI increased dramatically in the cerebellum of cbln3-null mice it was unchanged in extracerebellar neurons. This opens the possibility that other family members that are coexpressed in these areas control Cbln1 levels, potentially by modulating processing in the endolysosomal pathway. During development of cbln1-lacZ mice, beta-galactosidase staining was first observed in proliferating granule cell precursors prior to synaptogenesis and thereafter in maturing and adult granule cells. As cbln3 is only expressed in post-mitotic, post-migratory granule cells, Cbln1 homomeric complexes in precursors and Cbln1-Cbln3 heteromeric complexes in mature granule cells may have distinct functions and turnover.

Characterization of trans-neuronal trafficking of Cbln1

“…The localization of the CLI was consistent with Purkinje cells and/or Bergmann glia, although neither cell types express cbln 1 mRNA (Pang et al, 2000; Hirai et al, 2005; Miura et al, 2006) or (β-galactosidase driven from the cbln 1 promoter in transgenic mice (Hirai et al, 2005; Wei et al, 2007). To confirm the cell types expressing CLI, sections were triple labeled for DAPI and Cbln1 and either the Purkinje cell marker, calbindin D-28K (Figure 2A–D) or the glia marker S100 (Figure 2E–H).…”

“…The punctate pattern of CLI in Purkinje cells is reminiscent of its localization to vesicular structures in neurons elsewhere in brain (Wei et al, 2007). To study its sub-cellular localization in Purkinje cells, cerebellar sections were labeled by triple immunofluorescence for Cbln1, calbindin-D28K, and markers of endoplasmic reticulum (KDEL, Morishita et al, 2005) (Figure 3A–D), Golgi apparatus (GM130, Kerov et al, 2005) (Figure 3E–H), late endosomes (mannose-6-phosphate receptor, Morishita et al, 2005) (Figure 3I–L) and lysosomes (cathepsin D, Fukaya et al, 2003) (Figure 3M–P).…”

“…al., 1988; Mugnaini et al, 1988; Miura et al, 2009). We recently developed an antiserum to a distinct epitope in the C-terminus of Cbln1 that uniquely detects Cbln1 in endolysosomal vesicles of neurons in many brain regions (Wei et al, 2007) that also express cbln 1 mRNA (Miura et al, 2006). This suggested that Cbln1 undergoes intracellular vesicular trafficking and as it is a secreted protein that is present at PF-PC synapses (Miura et al, 2009), potentially transneuronal trafficking involving secretion and endocytosis into the endosome pathway in Purkinje cells.…”

Cbln and C1q family proteins – New transneuronal cytokines

“…Thus, Cbln1 proteins synthesized in granule cells may be rapidly released and taken up by postsynaptic Purkinje cells. In contrast, in a very recent report, Cbln1-like immunoreactivity was not observed in Purkinje cells but was localized in the lysosomal compartment of cerebellar granule cells [15]. Since Cbln1 undergoes extensive proteolysis [9], like several other C1q family proteins, the discrepant results may reflect different antibodies recognizing various proteolytic fragments of Cbln1.…”

“…However, since full-length Cbln1 is released into a medium from cultured granule neurons and heterologous cells [9], proteolytic cleavage is not required for the secretion of Cbln1. Since Cbln1-like immunoreactivity is localized in the lysosomal compartment of cerebellar granule cells [15], proteolysis by TACE-like enzymes may represent a mechanism to regulate the amount of Cbln1 secretion in presynaptic neurons. Further studies are warranted to clarify how Cbln subfamily proteins are released from neurons.…”