Cocaine addiction (CA) is a common chronic brain disorder with markedly increased morbidity and mortality (Degenhardt et al, 2011). ~ 1,500,000 adults in the US reported using cocaine in the past month (SAMSHA, 2013). Although twin studies are consistent with a moderate inherited component, it has been difficult to identify risk-increasing alleles by studying peripheral DNA. It is proposed to study neuronal DNA from post-mortem brain of CA patients, to find somatic neuronal mutation associated with CA. One common somatic mutation in the CNS is mediated by LINE1 (L1) retrotransposons (RTPs), which constitute 17% of the human genome (Beck et al, 2010). L1s are 6 kb in length. They encode a reverse transcriptase and an endonuclease, which function in a copy and paste mechanism, in transcriptionally active genomic regions, to spread in the human genome (Richardson et al, 2014). While most of these somatic de novo L1s will have little effect on neuronal function, some may interfere with neuronal activity because they have inserted into a gene needed by that particular neuron for normal function. If functional L1s occur early in CNS development, all the daughter neurons that derive from that neuronal precursor will also carry the L1 insertion, perhaps leading to a dysfunctional population of neurons destined to increase risk for CA. It is also possible that repeated use of cocaine results in waves of CNS neuronal transcriptional activity, creating windows of opportunity for L1s to spread, leading to progressive disruption of neuronal genes by those L1s. Preliminary results in mPFC of CA patients indicate that de novo L1s may disrupt kinase/phosphorylation pathways (Bonferroni corrected p = 10-6-10-7), pathways which are not significantly impacted among controls. This proposal will employ mPFC from 90 CA patients and 90 matched controls. DNA extracted from mPFC neuronal nuclei will be used in hemidegenerate PCR to enrich the DNA for L1 sequences. DNA will be sequenced and aligned to the reference genome to detect de novo L1 RTP insertions into neuronally-expressed genes. In this manner, it is expected that de novo L1 RTPs, associated with CA, will be discovered. These de novo L1s may be a consequence of repeated cocaine use or they may be antecedent to cocaine use, acting as risk factors for CA. If the de novo L1s are a consequence of repeated cocaine use, this would be a paradigm-shifting discovery that a drug of abuse disrupts neuronal genomes. If the de novo L1s are antecedent to cocaine use, and function as CA risk factors, they would identify novel genes and pathways related to CA, which may create targets for drug development and genetic research. Confirmed de novo L1s from CA tissue will be studied for functional impact by re-creation of the L1 insertion in a neuronal precursor cell line, using CRISPR/Cas9 gene editing technology. In this manner neuronal DNA variation associated with CA will be defined.
Cocaine addiction is a serious public health problem with inadequate current treatment options. There are no FDA-approved medications for cocaine addiction. The goal of this project is to examine the genomes of nerve cells in brains of cocaine addicted persons who died of cocaine overdose, to determine whether there are mutations in genes which might make certain nerve cells function improperly, giving rise to risk for cocaine addiction. Identification of such mutatins would create new targets for medication development and would improve our understanding of the origins of cocaine addiction.
| Doyle, Glenn A; Doucet-O'Hare, Tara T; Hammond, Matthew J et al. (2017) Reading LINEs within the cocaine addicted brain. Brain Behav 7:e00678 |
