Abstract
The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.
Highlights
The signaling systems that maintain multipotential NPC populations in their “stem-like” state within the adult hippocampal network have only begun to be elucidated [2]
Pannexins are a family of membrane channel proteins discovered through their homology to the invertebrate family of gap junction proteins, the innexins
Panx3 is found in skin and cartilage [12,13,14]. Given their sequence homology with innexins and their structural homology with connexins, pannexins were originally proposed to form intercellular channels; recent studies suggest that Panx1 and Panx3 more likely form single membrane channels, with Panx1 channels permeable to ions, ATP, and arachidonic acid-related lipids [15,16,17,18,19,20,21,22,23]
Summary
Cell Culture—Primary NPC cultures were isolated from postnatal day 3 (P3) C57BL/6 mouse hippocampus and expanded as neurospheres for 14 days in vitro (DIV) as described [10]. To assess N-linked glycosylation, lysates were further incubated in 0.5% SDS, 40 mM dithiothreitol and denatured by boiling for 10 min This protocol partially depalmitoylates target proteins without significant impact on other post-translational modifications [33, 34]. Palmitoylation was directly assessed by treatment of RIPA lysates with buffer (10 mM PBS) or 1 M hydroxylamine, pH 7.4, a chemical depalmitoylating agent that cleaves thioester linkages, for 20 min at room temperature, followed by Western blot analysis as described [35]. Neurospheres were labeled with 10 M BODIPY FL hexadecanoic acid [37] (Invitrogen/Molecular Probes) for 1 h at 37 °C in 95% O2, 5% CO2, followed by lysis in RIPA buffer and immunoprecipitation with polyclonal anti-Panx C-term (Zymed Laboratories Inc./Invitrogen). Alternative splicing was ruled out as a potential source of the NPC-specific Panx species by Northern blotting (data not shown)
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