A recoverable state of axon injury persists for hours after spinal cord contusion in vivo.

Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover. Here we use morphological and molecular in vivo imaging after contusion SCI in mice to show that injured axons persist in a metastable state for hours. Intra-axonal calcium dynamics influence fate, but the outcome is not invariably destructive in that many axons with calcium elevations recover homeostasis without intervention. Calcium enters axons primarily through mechanopores. Spontaneous pore resealing allows calcium levels to normalize and axons to survive long term. Axon loss can be halted by blocking calcium influx or calpain, even with delayed initiation. Our data identify an inherent self-preservation process in contused axons and a window of opportunity for rescuing connectivity after nontransecting SCI.

Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes.

Here we provide a protocol for rapidly labeling different cell types, distinct subcellular compartments and key injury mediators in the spinal cord of living mice. This method is based on the application of synthetic vital dyes to the surgically exposed spinal cord. Suitable vital dyes applied in appropriate concentrations lead to reliable in vivo labeling, which can be combined with genetic tags and in many cases preserved for postfixation analysis. In combination with in vivo imaging, this approach allows the direct observation of central nervous system physiology and pathophysiology at the cellular, subcellular and functional level. Surgical exposure and preparation of the spinal cord can be achieved in less than 1 h, and then dyes need to be applied for 30-60 min before the labeled spinal cord can be imaged for several hours.

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.

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.

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.

BACKGROUND: Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. RESULTS: We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. CONCLUSION: Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.