Summary It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of fifteen normal individuals (aged 4 months to 82 years) as well as nine individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and Xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age—which we term genosenium—shows age-related, region-related, and disease-related molecular signatures, and may be important in other human age-associated conditions.

One-Sentence Summary:Somatic single-nucleotide variants accumulate in human neurons in aging with regional specificity and in progeroid diseases. Summary: (125 words max)It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of fifteen normal individuals (aged 4 months to 82 years) as well as nine individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and Xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age-which we term genosenium-shows age-related, region-related, and disease-related molecular signatures, and may be important in other human age-associated conditions. peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/221960 doi: bioRxiv preprint first posted online Nov. 21, 2017; Main Text:Aging in humans brings increased incidence of nearly all diseases, including neurodegenerative diseases (1). Markers of DNA damage increase in the brain with age (2), and genetic progeroid diseases such as Cockayne syndrome (CS) and Xeroderma pigmentosum (XP), both caused by defects in DNA damage repair (DDR), are associated with neurodegeneration and premature aging (3). Mouse models of aging, CS, and XP have shown inconsistent relationships between these conditions and the accumulation of permanent somatic mutations in brain and non-brain tissue (4-7). While analysis of human bulk brain DNA, comprised of multiple proliferative and non-proliferative cell types, revealed an accumulation of mutations during aging in the human brain (8), it is not known whether permanent somatic mutations accumulate with age in mature neurons of the human brain. Here, we quantitatively examined whether aging or disorders of defective DDR results in more somatic mutations in single postmitotic human neurons.Somatic mutations that occur in postmitotic neurons are unique to each cell, and thus can only be comprehensively assayed by comparing the genomes of single cells (9). Therefore, we analyzed human neurons by single-cell whole-genome sequencing (WGS). Since alterations of the prefrontal cortex (PFC) have been linked to age-related cognitive decline and neurodegenerative disease (10), we analyzed 93 neurons from PFC of 15 neurologically normal individuals (Tables 1, S1, S2) from ages 4 months to 82 years. We further examined 26 neurons from the hippocampal dentate gyrus (DG) of 6 of these individuals because the DG is a focal point for other age-related degenerative cond...

The ability of cocaine to produce lasting neural adaptations in mesocorticolimbic brain regions is thought to promote drug seeking and facilitate addiction in humans. The Ras-controlled Raf-MEK-ERK protein kinase signaling cascade has been implicated in the behavioral and neurobiological actions of cocaine in animals. However, these pharmacological studies have not been able to determine the specific role of the two predominant isoforms of ERK (ERK1 and ERK2) in these processes. We report here that deletion of the ERK1 isoform, which leads to increased ERK2 stimulus-dependent signaling, facilitates the development of cocaine-induced psychomotor sensitization and the acquisition of a cocaine conditioned place preference. Conversely, pharmacological blockade of ERK signaling attenuates the development of psychomotor sensitization to cocaine. Finally, cocaine-evoked gene expression in mesocorticolimbic brain regions is potentiated in ERK1-deficient mice. Thus, alterations in ERK signaling influence both the neurobiological impact of cocaine and its ability to produce enduring forms of drug experience-dependent behavioral plasticity. Our results suggest that enhanced ERK2 signaling following repeated drug exposure may facilitate the development of forms of cocaine-induced plasticity that contribute to addiction.

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Both cis-and trans-regulatory mutations contribute to gene expression divergence within and between species. To estimate their relative contributions, we examined two yeast strains, BY (a laboratory strain) and RM (a wild strain), for their gene-expression divergence by microarray. Using these data and published ChIP-chip data, we obtained a set of single-regulator-regulated genes that showed expression divergence between BY and RM. We randomly selected 50 of these genes for further study. We developed a step-by-step approach to assess the relative contributions of cisand trans-variations to expression divergence by using pyrosequencing to quantify the mRNA levels of the BY and RM alleles in the same culture (co-culture) and in hybrid diploids. Forty genes showed expression divergence between the two strains in co-culture, and pyrosequencing of the BY/RM hybrid diploids showed that 45% (18/40) can be attributed to differences in trans-acting factors alone, 17.5% (7/40) mainly to trans-variations, 20% (8/40) to both cis-and trans-acting factors, 7.5% (3/40) mainly to cis-variations, and 10% (4/40) to cis-acting factors alone. In addition, we replaced the BY promoter by the RM promoter in each of 10 BY genes that were found from our microarray data to have expression divergence between BY and RM, and in each case our quantitative PCR analysis revealed a cis effect of the promoter replacement on gene expression. In summary, our study suggests that trans-acting factors play the major role in expression evolution between yeast strains, but the role of cis variation is also important.

The subthalamic nucleus (STN) is traditionally thought to be involved in motor control, and dysfunction of the STN is thought to contribute to movement disorders. Here, we show that the STN also plays an important role in motivational processes and the response to drugs of abuse. Specifically, bilateral STN lesions produced a dose-dependent increase in the psychomotor-activating effects of cocaine, the rate at which animals acquired cocaine self-administration, and the motivation for cocaine assessed using a progressive ratio schedule. Furthermore, bilateral STN lesions enhanced the ability of cocaine to induce gene expression in the nucleus accumbens and caudate-putamen, two structures known to be involved in mediating the psychomotor-activating and incentive motivational effects of drugs of abuse. These findings suggest that engagement of the STN serves to dampen the psychomotor-activating and incentive motivational effects of drugs of abuse. Thus, the STN may serve as a novel target for therapeutic interventions aimed at treating drug dependence.

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Both cis and trans mutations contribute to gene expression divergence within and between species. We used Saccharomyces cerevisiae as a model organism to estimate the relative contributions of cis and trans variations to the expression divergence between a laboratory (BY) and a wild (RM) strain of yeast. We examined whether genes regulated by a single transcription factor (TF; single input module, SIM genes) or genes regulated by multiple TFs (multiple input module, MIM genes) are more susceptible to trans variation. Because a SIM gene is regulated by a single immediate upstream TF, the chance for a change to occur in its trans-acting factors would, on average, be smaller than that for a MIM gene. We chose 232 genes that exhibited expression divergence between BY and RM to test this hypothesis. We examined the expression patterns of these genes in a BY-RM coculture system and in a BY-RM diploid hybrid. We found that trans variation is far more important than cis variation for expression divergence between the two strains. However, because in 75% of the genes studied, cis variation has significantly contributed to expression divergence, cis change also plays a significant role in intraspecific expression evolution. Interestingly, we found that the proportion of genes with diverged expression between BY and RM is larger for MIM genes than for SIM genes; in fact, the proportion tends to increase with the number of transcription factors that regulate the gene. Moreover, MIM genes are, on average, subject to stronger trans effects than SIM genes, though the difference between the two types of genes is not conspicuous.