Hepatocyte mARC1 promotes fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the in vivo impacts of MTARC1. Results: We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and in vivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. Impact and implications: We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 for NAFLD.

Cellular basis of brain maturation and acquisition of complex behaviors in salamanders.

The overall bauplan of the tetrapod brain is highly conserved, yet significant variations exist among species in terms of brain size, structural composition and cellular diversity. Understanding processes underlying neural and behavioral development in a wide range of species is important both from an evolutionary developmental perspective as well as for the identification of cell sources with post-developmental neurogenic potential. Here, we characterize germinal processes in the brain of Notophthalmus viridescens and Pleurodeles waltl during both development and adulthood. Using a combination of cell tracking tools, including clonal analyses in new transgenic salamander lines, we examine the origin of neural stem and progenitor cells found in the adult brain, determine regional variability in cell cycle length of progenitor cells, and show spatiotemporally orchestrated neurogenesis. We analyze how maturation of different brain regions and neuronal subpopulations are linked to the acquisition of complex behaviors, and how these behaviors are altered upon chemical ablation of dopamine neurons. Our data analyzed from an evolutionary perspective reveal both common and species-specific processes in tetrapod brain formation and function.

Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration.

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.

Environmental changes in oxygen tension reveal ROS-dependent neurogenesis and regeneration in the adult newt brain.

Organisms need to adapt to the ecological constraints in their habitat. How specific processes reflect such adaptations are difficult to model experimentally. We tested whether environmental shifts in oxygen tension lead to events in the adult newt brain that share features with processes occurring during neuronal regeneration under normoxia. By experimental simulation of varying oxygen concentrations, we show that hypoxia followed by re-oxygenation lead to neuronal death and hallmarks of an injury response, including activation of neural stem cells ultimately leading to neurogenesis. Neural stem cells accumulate reactive oxygen species (ROS) during re-oxygenation and inhibition of ROS biosynthesis counteracts their proliferation as well as neurogenesis. Importantly, regeneration of dopamine neurons under normoxia also depends on ROS-production. These data demonstrate a role for ROS-production in neurogenesis in newts and suggest that this role may have been recruited to the capacity to replace lost neurons in the brain of an adult vertebrate.

Progenitor cell dynamics in the Newt Telencephalon during homeostasis and neuronal regeneration.

The adult newt brain has a marked neurogenic potential and is highly regenerative. Ventricular, radial glia-like ependymoglia cells give rise to neurons both during normal homeostasis and after injury, but subpopulations among ependymoglia cells have not been defined. We show here that a substantial portion of GFAP(+) ependymoglia cells in the proliferative hot spots of the telencephalon has transit-amplifying characteristics. In contrast, proliferating ependymoglia cells, which are scattered along the ventricular wall, have stem cell features in terms of label retention and insensitivity to AraC treatment. Ablation of neurons remodels the proliferation dynamics and leads to de novo formation of regions displaying features of neurogenic niches, such as the appearance of cells with transit-amplifying features and proliferating neuroblasts. The results have implication both for our understanding of the evolutionary diversification of radial glia cells as well as the processes regulating neurogenesis and regeneration in the adult vertebrate brain.

Expression of medium and heavy chain neurofilaments in the developing human auditory cortex.

Neurofilament medium (NF-M) and heavy (NF-H) chain proteins have been used as markers for maturity in the developing brain since their accumulation in axons leads to an increase in conduction velocity. Earlier studies have demonstrated immunoreactivity of neurofilaments in Layer I of the human auditory cortex at 22 gestation weeks (GW), whereas that in other layers developed between 1 and 12 postnatal years, suggesting a gradual increase in the processing of sounds. However, third trimester fetuses and infants are fairly sophisticated in their ability to discern different aspects of complex sounds. Given these contradictory findings, we decided to study the expression of neurofilaments in human auditory cortex between 15 GW and adulthood. We found that mRNA and protein for both NF-M and NF-H were present in the presumptive human auditory cortex in the second trimester and during the postnatal period (1 year--adulthood). Axons in all layers of the auditory cortex were immunoreactive for neurofilaments by 25 GW and the density of the neurofilament-rich plexus in the cortical wall became adult-like during the first postnatal year in humans (9 postnatal months). Our results suggest that in terms of neurofilament expression, axons within the preterm human auditory cortex may be more mature than previously thought.

Opioid modulation of cell proliferation in the ventricular zone of adult zebra finches (Taenopygia guttata).

Besides modulating pain, stress, physiological functions, motivation, and reward, the opioid system has been implicated in developmental and adult mammalian neurogenesis and gliogenesis. In adult male songbirds including zebra finches, neurons generated from the ventricular zone (VZ) of the lateral ventricles are incorporated throughout the telencephalon, including the song control nuclei, HVC, and area X. Although the endogenous opioid met-enkephalin is present in neurons adjacent to the VZ and is upregulated in song control regions during singing, it is not known whether the opioid system can modulate adult neurogenesis/gliogenesis in zebra finches. We used quantitative RT-PCR and in situ hybridization to demonstrate that µ- and δ-opioid receptors are expressed by the VZ of adult male zebra finches. Treating cultured VZ cells from male birds with the opioid antagonist naloxone led to an increase in cell proliferation measured by 5-bromo-2-deoxyuridine incorporation, whereas administering met-enkephalin had the opposite effect, compared with saline-treated cultures. Systemically administering naloxone (2.5 mg/kg body wt) to adult male zebra finches for 4 d also led to a significant increase in cell proliferation in the ventral VZ of these birds, compared with saline-treated controls. Our results show that cell proliferation is augmented by naloxone in the VZ adjacent to the anterior commissure, suggesting that the endogenous opioids modulate adult neurogenesis/gliogenesis by inhibiting cell proliferation in songbirds.

Opioid modulation of song in male zebra finches (Taenopygia guttata).

Endogenous opioids are known to modulate motivated behaviors. Male zebra finches produce a highly motivated behavior (directed song) to court females and also sing in isolation (undirected song). We found that adult male zebra finches sang significantly fewer directed and undirected songs after administration of low doses (2.5 mg/kg body weight) of the general opioid antagonist naloxone, even though the order of syllables in songs was not altered. Surprisingly, high doses of naloxone (10 mg/kg body weight) dramatically decreased the production of undirected songs but had no significant effects on directed songs. There were no changes in the number of calls during directed or undirected song, movement, stereotyped behaviors including pecking and preening, feeding or drinking behaviors after naloxone administration. We also found that treating zebra finches with naloxone led to a decrease in tonality (goodness of pitch), frequency and amplitude modulation and an increase in the length of intersyllable intervals. Our results suggest that the opioid system can differentially modulate directed and undirected song as well as the acoustic characteristics of birdsong, perhaps by acting on different components of the song control system.