Biology subjects

Tolentino, R. E.

Publications and source records attributed to Tolentino, R. E..

3 recordsLinked to original sources

bioRxiv2023

Spinal Cord Injury regulates circular RNA expression in axons

IntroductionNeurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. MethodsWe obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that were then sequenced. We used bioinformatics analyses to identify DEGS and map them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. ResultsThe supernatant fraction was highly enriched for axonal mRNA. Using Gene Ontology, the second most significant pathway for all differentially expressed genes (DEGs) was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth. Focusing on Rims2 we used Circular RNA Interactome to predict that several of the miRNAs that bind to circRims2 also bind to the 3UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. DiscussionBy using our method for enrichment of axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 a known regulator of axonal regeneration indicating that axonal mRNA contribute to regeneration.

neuroscience↗

bioRxiv2018

Systems therapeutics analyses identify genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer

The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop genomic signatures for sensitivity or resistance to specific drugs. We analyzed TCGA data for lung adenocarcinoma (LUAD) patients and identified a subset where xanthine dehydrogenase expression correlated with decreased survival. We tested allopurinol, a FDA approved drug that inhibits xanthine dehydrogenase on a library of human Non Small Cell Lung Cancer (NSCLC) cell lines from CCLE and identified sensitive and resistant cell lines. We utilized the gene expression profiles of these cell lines to identify six-gene signatures for allopurinol sensitive and resistant cell lines. Network building and analyses identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 were verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinolsensitive NSCLC lines, and four allopurinol-resistant lines susceptible to combination therapy. We found that drug treatment of all cell lines yielded responses as predicted by the genomic signatures. We searched the library of patient derived NSCLC tumors from Jackson Laboratory to identify tumors that would be predicted to be sensitive or resistant to allopurinol treatment. Both patient derived tumors predicted to be allopurinol sensitive showed the predicted sensitivity, and the predicted resistant tumors were sensitive to combination therapy. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

systems biology↗

bioRxiv2018

Activated Protein C blocks the inhibitory effect on neurite outgrowth by extracellular histones that mediates its inhibition through a retrograde YB-1 signal

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors we have detected elevated levels of histone H3 in human cerebrospinal fluid (CSF) 24 hours after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor YB-1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C (APC) in vitro, and APC treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS. One sentence summaryProteins typically associated with chromatin structure play an unexpected role in limiting axonal regeneration after injury.

neuroscience↗