spinal-cord-lateral-hemisection-asymmetric-behavioral-assessments

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Indiana University-Indianapolis

Indiana University School of Medicine

We previously reported on the purification and characterization of a functional multi-protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry-out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30-fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7-10 ribonucleotides long and DNA primers that are 20-40 deoxyribonucleotides long using a poly(dT) template of exogenous single-stranded DNA. The synthesome-catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double-stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10-20 nt in length and the primers are covalently link to DNA to form RNA-primed nascent DNA of 100-200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins.

Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome.

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC(50) of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases alpha, delta and epsilon is 15, 45 and 125 muM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC(50)=88 muM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome.

The objective of this study is to determine whether an altered DNA replication process is responsible for some of genetic damage observed in ovarian cancer.

Error-promoting DNA synthesis in ovarian cancer cells.

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. , Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. , targeted delivery of AAV-NT-3  transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.

Remodeling of lumbar motor circuitry remote to a thoracic spinal cord injury promotes locomotor recovery.

Locomotor function, mediated by lumbar neural circuitry, is modulated by descending spinal pathways. Spinal cord injury (SCI) interrupts descending projections and denervates lumbar motor neurons (MNs). We previously reported that retrogradely transported neurotrophin-3 (NT-3) to lumbar MNs attenuated SCI-induced lumbar MN dendritic atrophy and enabled functional recovery after a rostral thoracic contusion. Here we functionally dissected the role of descending neural pathways in response to NT-3-mediated recovery after a T9 contusive SCI in mice. We find that residual projections to lumbar MNs are required to produce leg movements after SCI. Next, we show that the spared descending propriospinal pathway, rather than other pathways (including the corticospinal, rubrospinal, serotonergic, and dopaminergic pathways), accounts for NT-3-enhanced recovery. Lastly, we show that NT-3 induced propriospino-MN circuit reorganization after the T9 contusion via promotion of dendritic regrowth rather than prevention of dendritic atrophy.

Heqiao Dai

Indiana University-Indianapolis

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats