Fiske, B. | bioRxiv Science

bioRxiv2019

Widespread haploid-biased gene expression in mammalian spermatogenesis associated with frequent selective sweeps and evolutionary conflict

1Mendels first law dictates that alleles segregate randomly during meiosis and are distributed to offspring with equal frequency, requiring sperm to be functionally independent of their genetic payload. Developing mammalian spermatids have been thought to accomplish this by freely sharing RNA from virtually all genes through cytoplasmic bridges, equalizing allelic gene expression across different genotypes. Applying single cell RNA sequencing to developing spermatids, we identify a large class of mammalian genes whose allelic expression ratio is informative of the haploid genotype, which we call genoinformative markers (GIMs). 29% of spermatid-expressed genes in mice and 47% in non-human primates are not uniformly shared, and instead show a confident allelic expression bias of at least 2-fold towards the haploid genotype. This property of GIMs was significantly conserved between individuals and between rodents and primates. Consistent with the interpretation of specific RNA localization resulting in incomplete sharing through cytoplasmic bridges, we observe a strong depletion of GIM transcripts from chromatoid bodies, structures involved in shuttling RNA across cytoplasmic bridges, and an enrichment for 3 UTR motifs involved in RNA localization. If GIMs are translated and functional in the context of fertility, they would be able to violate Mendels first law, leading to selective sweeps through a population. Indeed, we show that GIMs are enriched for signatures of positive selection, accounting for dozens of recent mouse, human, and primate selective sweeps. Intense selection at the sperm level risks evolutionary conflict between germline and somatic function, and GIMs show evidence of avoiding this conflict by exhibiting more testis-specific gene expression, paralogs, and isoforms than expression-matched control genes. The widespread existence of GIMs suggests that selective forces acting at the level of individual mammalian sperm are much more frequent than commonly believed. 2 Authors summaryMendels first law dictates that alleles are distributed to offspring with equal frequency, requiring sperm carrying different genetics to be functionally equivalent. Despite a small number of known exceptions to this, it is widely believed that sharing of gene products through cytoplasmic bridges erases virtually all differences between haploid sperm. Here, we show that a large class of mammalian genes are not completely shared across these bridges, therefore causing sperm phenotype to correspond partly to haploid genotype. We term these genes "genoinformative markers" (GIMs) and show that their identity tends to be conserved from rodents to primates. Because some GIMs can link sperm genotype to function, they can be thought of as selfish genetic elements which lead to natural selection between sperm rather than between organisms, a violation of Mendels first law. We find evidence of this biased inheritance, showing that GIMs are strongly enriched for selective sweeps that spread alleles through mouse and human populations. For genes expressed both in sperm and in somatic tissues, this can cause a conflict because optimizing gene function for sperm may be detrimental to its other functions. We show that there is evolutionary pressure to avoid this conflict, as GIMs are strongly enriched for testis-specific gene expression, testis-specific paralogs, and testis-specific isoforms. Therefore, GIMs and sperm-level natural selection may provide an elegant explanation for the peculiarity of testis gene expression patterns, which are an extreme outlier relative to all other tissues.

genetics↗

bioRxiv2018

LRRK2 kinase inhibitors induce a reversible effect in the lungs of non-human primates with no measurable pulmonary deficits

Putative gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2), resulting in increased kinase activity and cellular toxicity, are a leading genetic cause of Parkinsons disease (PD). Hence, there is strong interest in developing LRRK2 kinase inhibitors as a disease-modifying therapy. Published reports that repeat dosing with two LRRK2 kinase inhibitors (GNE-7915 and GNE-0877) induce histopathological changes in the lung of non-human primates Fuji et al. 2015 (1) raised concerns about potential safety liability of LRRK2 kinase inhibitors. In the present study, we sought to determine whether previously observed effects in the lung: (a) represent on-target pharmacology, but with the potential for margin of safety, (b) are reversible upon drug withdrawal, and (c) are associated with pulmonary function deficits. To this end, we evaluated the histopathological effects, toxicokinetics and target inhibition of three structurally diverse LRRK2 kinase inhibitors, GNE-7915 (30 mg/kg, BID, as a positive control), MLi-2 (15 and 50 mg/kg, QD) and PFE-360 (3 and 6 mg/kg, QD) following 2 weeks of dosing in non-human primates. Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated after 2-week dose-free periods. All three LRRK2 kinase inhibitors induced mild cytoplasmic vacuolation of type II pneumocytes, as reported previously, confirming an on-target effect of these compounds. Interestingly, despite lower doses of both PFE-360 and MLi-2 producing nearly complete inhibition of LRRK2 kinase activity in the brain as assessed by levels of pS935-LRRK2, histopathological changes in lung were absent in animals treated with low-dose PFE-360 and observed only sporadically in the low-dose MLi-2 group. The lung effect was fully reversible at 2 weeks post-dosing of GNE-7915. In a second study of identical dosing with MLi-2 and GNE-7915, no deficits were observed in a battery of translational pulmonary functional tests. In aggregate, these results do not preclude the development of LRRK2 kinase inhibitors for clinical investigation in Parkinsons disease.

pharmacology and toxicology↗

bioRxiv2018

Crystallizing the Parkinson’s Disease Protein LRRK2 Under Microgravity Conditions

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a considerable cause for Parkinsons disease (PD). However, the high- resolution 3D structure of the protein is still lacking. This structure will not only help to understand PD etiology but will also enable rational drug design. We have established a reliable method to produce LRRK2 crystals for the first time. However, the limited resolution of the diffraction data prevented structure determination using crystallographic methods. Herein we describe our efforts to improve the crystal quality by crystallizing under microgravity conditions aboard the International Space Station (ISS). Our method features diffusive sample mixing in capillaries and controlled crystal formation by transporting the samples in a frozen state. The crystallisation was successfully repeated under microgravity conditions. However, comparison of earth-grown and microgravity-grown LRRK2 crystals did not reveal any differences in diffraction quality. Here we present the established protocol and our experience adapting crystallization condition to the requirements necessary for successful crystallization of large and sensitive biomolecules under microgravity.

biochemistry↗