Role of "neuronal" gap junction proteins in regulating post-natal hippocampal neurodegeneration and neurogenesis in vitro and in vivo

Adult neural stem and progenitor cells (NPCs) are located within specific neurogenic niches, such as the subgranular zone of the dentate gyrus. Cell-cell communication through gap junction channels is believed to play an important role in embryonic neurogenesis. Connexins, the protein subunits of gap junctions, are expressed in NPCs and connexin36 (Cx36) is considered the prototypical neuronal connexin although a role in neurogenesis had yet to be assessed. Here, I examined the influence of Cx36 on NPC proliferation, specification, and survival in the hippocampus in vitro and in vivo in both wild type and Cx36-/- mice. Using cultured neurospheres from P0-P3 mice pup hippocampi, I show that Cx36 protein is first expressed upon engagement to a laminin matrix. Moreover, a Cx36 null mutation enhances neurogenesis under certain microenvironment conditions apparently by accelerating differentiation. Using thymidine analogs to label actively proliferating cells in uninjured mice and following kainic acid-induced seizures, I show that Cx36 deletion does not alter cell proliferation in the SGZ. However, the loss of Cx36 is neuroprotective following hippocampal injury without affecting neuroregenerative capacity. Finally, I demonstrate that the gap junction-like protein, pannexin2 (Panx2), is also expressed within mature neurons of the central nervous system (CNS) although functional channel activity and a role in neurogenesis have not elucidated. Here, I characterized the localization and morphology of Panx2 within hippocampal derived neurospheres in vitro and discovered that Panx2 exhibits a unique rod-like morphology with Type I and Type IIa NPCs. Together, these data implicate Cx36 in regulating postnatal NPC fate in the postnatal hippocampus and suggest a distinctive, regulatory role for Panx2 in neurogenesis

ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes

The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2(KA)-7. Moreover, together with the finding that Mcm2(K549A) does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2-7 complexes

Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment*

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

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy