Neurite extension: starting at the finish line.

The outgrowth of axons and dendrites from neuronal cell bodies to their appropriate targets is the canonical means of creating new processes. Heiman and Shaham (2009) now show that neuronal processes can also be made by anchoring dendrite tips at their target locations while the cell body pulls away, a process termed retrograde extension.

Cellular mechanisms of mtDNA heteroplasmy dynamics.

Heteroplasmy refers to the coexistence of more than one variant of the mitochondrial genome (mtDNA). Mutated or partially deleted mtDNAs can induce chronic metabolic impairment and cause mitochondrial diseases when their heteroplasmy levels exceed a critical threshold. These mutant mtDNAs can be maternally inherited or can arise de novo. Compelling evidence has emerged showing that mutant mtDNA levels can vary and change in a nonrandom fashion across generations and amongst tissues of an individual. However, our lack of understanding of the basic cellular and molecular mechanisms of mtDNA heteroplasmy dynamics has made it difficult to predict who will inherit or develop mtDNA-associated diseases. More recently, with the advances in technology and the establishment of tractable model systems, insights into the mechanisms underlying the selection forces that modulate heteroplasmy dynamics are beginning to emerge. In this review, we summarize evidence from different organisms, showing that mutant mtDNA can experience both positive and negative selection. We also review the recently identified mechanisms that modulate heteroplasmy dynamics. Taken together, this is an opportune time to survey the literature and to identify key cellular pathways that can be targeted to develop therapies for diseases caused by heteroplasmic mtDNA mutations.

Convergent evolution of escape from hepaciviral antagonism in primates.

The ability to mount an interferon response on sensing viral infection is a critical component of mammalian innate immunity. Several viruses directly antagonize viral sensing pathways to block activation of the host immune response. Here, we show that recurrent viral antagonism has shaped the evolution of the host protein MAVS--a crucial component of the viral-sensing pathway in primates. From sequencing and phylogenetic analyses of MAVS from 21 simian primates, we found that MAVS has evolved under strong positive selection. We focused on how this positive selection has shaped MAVS' susceptibility to Hepatitis C virus (HCV). We functionally tested MAVS proteins from diverse primate species for their ability to resist antagonism by HCV, which uses its protease NS3/4A to cleave human MAVS. We found that MAVS from multiple primates are resistant to inhibition by the HCV protease. This resistance maps to single changes within the protease cleavage site in MAVS, which protect MAVS from getting cleaved by the HCV protease. Remarkably, most of these changes have been independently acquired at a single residue 506 that evolved under positive selection. We show that "escape" mutations lower affinity of the NS3 protease for MAVS and allow it to better restrict HCV replication. We further show that NS3 proteases from all other primate hepaciviruses, including the highly divergent GBV-A and GBV-C viruses, are functionally similar to HCV. We conclude that convergent evolution at residue 506 in multiple primates has resulted in escape from antagonism by hepaciviruses. Our study provides a model whereby insights into the ancient history of viral infections in primates can be gained using extant host and virus genes. Our analyses also provide a means by which primates might clear infections by extant hepaciviruses like HCV.

Intramolecular regulation of presynaptic scaffold protein SYD-2/liprin-α.

SYD-2/liprin-α is a multi-domain protein that associates with and recruits multiple active zone molecules to form presynaptic specializations. Given SYD-2's critical role in synapse formation, its synaptogenic ability is likely tightly regulated. However, mechanisms that regulate SYD-2 function are poorly understood. In this study, we provide evidence that SYD-2's function may be regulated by interactions between its coiled-coil (CC) domains and sterile α-motif (SAM) domains. We show that the N-terminal CC domains are necessary and sufficient to assemble functional synapses while C-terminal SAM domains are not, suggesting that the CC domains are responsible for the synaptogenic activity of SYD-2. Surprisingly, syd-2 alleles with single amino acid mutations in the SAM domain show strong loss of function phenotypes, suggesting that SAM domains also play an important role in SYD-2's function. A previously characterized syd-2 gain-of-function mutation within the CC domains is epistatic to the loss-of-function mutations in the SAM domain. In addition, yeast two-hybrid analysis showed interactions between the CC and SAM domains. Thus, the data is consistent with a model where the SAM domains regulate the CC domain-dependent synaptogenic activity of SYD-2. Taken together, our study provides new mechanistic insights into how SYD-2's activity may be modulated to regulate synapse formation during development.

RNA: De-silencing to the rescue.

The fate of transcribed RNA dictates cellular function. A new study finds that mutations in specific RNA processing machinery genes result in de-silencing of a transcript encoding a subunit of the mitochondrial electron transport chain and rescue of a mitochondrial respiratory complex I defect.

Germline status and micronutrient availability regulate a somatic mitochondrial quality control pathway via short-chain fatty acid metabolism.

Reproductive status, such as pregnancy and menopause in women, profoundly influences metabolism of the body. Mitochondria likely orchestrate many of these metabolic changes. However, the influence of reproductive status on somatic mitochondria and the underlying mechanisms remain largely unexplored. We demonstrate that reproductive signals modulate mitochondria in the Caenorhabditis elegans soma. We show that the germline acts via an RNA endonuclease, HOE-1, which despite its housekeeping role in tRNA maturation, selectively regulates the mitochondrial unfolded protein response (UPRmt). Mechanistically, we uncover a fatty acid metabolism pathway acting upstream of HOE-1 to convey germline status. Furthermore, we link vitamin B12's dietary intake to the germline's regulatory impact on HOE-1-driven UPRmt. Combined, our study uncovers a germline-somatic mitochondrial connection, reveals the underlying mechanism, and highlights the importance of micronutrients in modulating this connection. Our findings provide insights into the interplay between reproductive biology and metabolic regulation.

Design and synthesis of L- and D-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC50=7.7 µM) and 2 (IC50=10.6 µM) as represented by hybrid compound 27 (IC50=6.7 µM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC50 of 2.6 µM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the D-isomers 41 (IC50=19.3 µM) and 45 (IC50=5.4 µM) as enantiomers of the l-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.

Synthesis and evaluation of cell-permeable biotinylated PU-H71 derivatives as tumor Hsp90 probes.

The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics.

A role for N6-methyldeoxyadenosine in C. elegans mitochondrial genome regulation.

Epigenetic modifications provide powerful means for transmitting information from parent to progeny. As a maternally inherited genome that encodes essential components of the electron transport chain, the mitochondrial genome (mtDNA) is ideally positioned to serve as a conduit for the transgenerational transmission of metabolic information. Here, we provide evidence that mtDNA of C. elegans contains the epigenetic mark N6-methyldeoxyadenosine (6mA). Bioinformatic analysis of SMRT sequencing data and methylated DNA IP sequencing data reveal that C. elegans mtDNA is methylated at high levels in a site-specific manner. We further confirmed that mtDNA contains 6mA by leveraging highly specific anti-6mA antibodies. Additionally, we find that mtDNA methylation is dynamically regulated in response to antimycin, a mitochondrial stressor. Further, 6mA is increased in nmad-1 mutants and is accompanied by a significant decrease in mtDNA copy number. Our discovery paves the way for future studies to investigate the regulation and inheritance of mitochondrial epigenetics.

Tissue-specific heteroplasmy segregation is accompanied by a sharp mtDNA decline in Caenorhabditis elegans soma.

Mutations in the mitochondrial genome (mtDNA) can be pathogenic. Owing to the multi-copy nature of mtDNA, wild-type copies can compensate for the effects of mutant mtDNA. Wild-type copies available for compensation vary depending on the mutant load and the total copy number. Here, we examine both mutant load and copy number in the tissues of Caenorhabditis elegans. We found that neurons, but not muscles, have modestly higher mutant load than rest of the soma. We also uncovered different effect of aak-2 knockout on the mutant load in the two tissues. The most surprising result was a sharp decline in somatic mtDNA content over time. The scale of the copy number decline surpasses the modest shifts in mutant load, suggesting that it may exert a substantial effect on mitochondrial function. In summary, measuring both the copy number and the mutant load provides a more comprehensive view of the mutant mtDNA dynamics.

Should I stay, or should I go? Gene retention in organellar genomes.

Mitochondria and plastids retain their own small but essential genomes. However, the evolutionary pressures that determine whether a gene is retained in organellar DNA or exported to the "host" nuclear genome remain unclear. A new study in Cell Systems addresses this knowledge gap using bioinformatic data and modeling to identify universal "rules" that determine organellar gene retention.

A tRNA processing enzyme is a key regulator of the mitochondrial unfolded protein response.

The mitochondrial unfolded protein response (UPRmt) has emerged as a predominant mechanism that preserves mitochondrial function. Consequently, multiple pathways likely exist to modulate UPRmt. We discovered that the tRNA processing enzyme, homolog of ELAC2 (HOE-1), is key to UPRmt regulation in Caenorhabditis elegans. We find that nuclear HOE-1 is necessary and sufficient to robustly activate UPRmt. We show that HOE-1 acts via transcription factors ATFS-1 and DVE-1 that are crucial for UPRmt. Mechanistically, we show that HOE-1 likely mediates its effects via tRNAs, as blocking tRNA export prevents HOE-1-induced UPRmt. Interestingly, we find that HOE-1 does not act via the integrated stress response, which can be activated by uncharged tRNAs, pointing toward its reliance on a new mechanism. Finally, we show that the subcellular localization of HOE-1 is responsive to mitochondrial stress and is subject to negative regulation via ATFS-1. Together, we have discovered a novel RNA-based cellular pathway that modulates UPRmt.

Independent regulation of mitochondrial DNA quantity and quality in Caenorhabditis elegans primordial germ cells.

Mitochondria harbor an independent genome, called mitochondrial DNA (mtDNA), which contains essential metabolic genes. Although mtDNA mutations occur at high frequency, they are inherited infrequently, indicating that germline mechanisms limit their accumulation. To determine how germline mtDNA is regulated, we examined the control of mtDNA quantity and quality in C. elegans primordial germ cells (PGCs). We show that PGCs combine strategies to generate a low point in mtDNA number by segregating mitochondria into lobe-like protrusions that are cannibalized by adjacent cells, and by concurrently eliminating mitochondria through autophagy, reducing overall mtDNA content twofold. As PGCs exit quiescence and divide, mtDNAs replicate to maintain a set point of ~200 mtDNAs per germline stem cell. Whereas cannibalism and autophagy eliminate mtDNAs stochastically, we show that the kinase PTEN-induced kinase 1 (PINK1), operating independently of Parkin and autophagy, preferentially reduces the fraction of mutant mtDNAs. Thus, PGCs employ parallel mechanisms to control both the quantity and quality of the founding population of germline mtDNAs.

Mitochondria are the powerhouses of every cell in our bodies. These tiny structures convert energy from the food we eat into a form that cells are able to use. As well as being a separate organ-like structure within our cells, mitochondria even have their own DNA. Mitochondrial DNA contains genes for a small number of special enzymes that allow it to extract energy from food. In contrast, the rest of our cells' DNA is stored in another structure called the nucleus. Mitochondrial and nuclear DNA are also inherited differently. We inherit nuclear DNA from both our mother and father, but mitochondrial DNA is only passed down from our mothers. During reproduction, maternal DNA (including mitochondrial DNA) comes from the egg cell, which combines with sperm to produce offspring. Defects, or mutations, in mitochondrial genes often lead to mitochondrial diseases. These have a severe impact on health, especially during the very first stages of life. The lineage of precursor cells that gives rise to egg cells is thought to protect itself from mitochondrial mutations, but how it does this is still unclear. Schwartz et al. therefore set out to determine what molecular mechanisms preserve the integrity of mitochondrial DNA from one generation to the next. To address this question, C. elegans roundworms were used, as they are easy to manipulate genetically, and since they are small and transparent, their cells ­ as well as their mitochondria ­ are also easily viewed under a microscope. Tracking mitochondria in the worms' egg precursor cells (also called primordial germ cells, or PGCs) revealed that PGCs actively removed excess mitochondria. The PGCs did this either by internally breaking down mitochondria themselves, or by moving them into protruding lobe-like structures which surrounding cells then engulfed and 'digested'. Further genetic studies revealed that the PGCs also directly regulated the quality of mitochondrial DNA via a mechanism dependent on the protein PINK1. In worms lacking PINK1, mutant mitochondrial DNA remained in the PGCs at high levels, whereas normal worms successfully reduced the mutant DNA. Thus, the PGCs used parallel mechanisms to control both the quantity and quality of mitochondria passed to the next generation. These results contribute to our understanding of how organisms safeguard their offspring from inheriting mutant mitochondrial DNA. In the future, Schwartz et al. hope that this knowledge will help us treat inherited mitochondrial diseases in humans.

Health and wellness for disadvantaged older adults: The AFRESH pilot study.

Introduction: Older adults are unaware of the biological mechanisms that contribute to the development of disabilities, chronic conditions, and frailty, yet, when made aware, desire to employ lifestyle changes to mitigate these conditions. We developed the AFRESH health and wellness program and report on pilot testing undertaken in a local older adults apartment community. Materials and methods: After program development, pilot testing was conducted. Participants: Older adults (N = 20; age 62+) residing in an apartment community. Procedures: Collection of baseline objective and self-report measures with a focus on physical activity; administration of the 10-week AFRESH program via weekly sessions; collection of follow-up data 12 and 36 weeks after baseline data collection. Data analysis: Descriptive statistics, growth curve analyses. Results: Significant increases were observed for grip strength (lbs) (T1:56.2; T2:65.0 [d = 0.77]; T3:69.4 [d = 0.62], p = .001), the 6-min walk test (meters) (T1:327m: T2:388.7 m [d = 0.99]; T3:363.3 m [d = 0.60], p = .001), the Rapid Assessment of Physical Activity (RAPA) strength and flexibility score, and the Pittsburg Sleep Quality Index (PSQI) global score. These effects showed some attenuation by the final time point. Conclusion: By combining novel educational content (bioenergetics), facilitation of physical activity, and habit formation, AFRESH is a multicomponent intervention that shows promise for future research.

Elevated mitochondrial DNA copy number found in ubiquinone-deficient clk-1 mutants is not rescued by ubiquinone precursor 2-4-dihydroxybenzoate.

Inside mitochondria reside semi-autonomous genomes, called mtDNA. mtDNA is multi-copy per cell and mtDNA copy number can vary from hundreds to thousands of copies per cell. The variability of mtDNA copy number between tissues, combined with the lack of variability of copy number within a tissue, suggest a homeostatic copy number regulation mechanism. Mutations in the gene encoding the Caenorhabditis elegans hydroxylase, CLK-1, result in elevated mtDNA. CLK-1's canonical role in ubiquinone biosynthesis results in clk-1 mutants lacking ubiquinone. Importantly, clk-1 mutants also exhibit slowed biological timing phenotypes (pharyngeal pumping, defecation, development) and an activated stress response (UPRmt). These biological timing and stress phenotypes have been attributed to ubiquinone deficiency; however, it is unknown whether the mtDNA phenotype is also due to ubiquinone deficiency. To test this, in animals carrying the uncharacterized clk-1 (ok1247) mutant allele, we supplemented with an exogenous ubiquinone precursor 2-4-dihydroxybenzoate (DHB), which has previously been shown to restore ubiquinone biosynthesis. We measured phenotypes as a function of DHB across a log-scale range. Unlike the biological timing and stress phenotypes, the elevated mtDNA phenotype was not rescued. Since CLK-1's canonical role is in ubiquinone biosynthesis and DHB does not rescue mtDNA copy number, we infer CLK-1 has an additional function in homeostatic mtDNA copy number regulation.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Comparative study of the binding characteristics to and inhibitory potencies towards PARP and in vivo antidiabetogenic potencies of taurine, 3-aminobenzamide and nicotinamide.

BACKGROUND: Poly(ADP-ribose) is a NAD+-requiring, DNA-repairing, enzyme playing a central role in pancreatic beta-cell death and in the development of endothelial dysfunction in humans and experimental animals. PARP activation is also relevant to the development of complications of diabetes. Hence, agents capable of inhibiting PARP may be useful in preventing the development of diabetes and in slowing down complications of diabetes. METHODS: PARP inhibition was assessed with a colorimetric assay kit. Molecular docking studies on the active site of PARP were conducted using the crystalline structure of the enzyme available as Protein Data Bank Identification No. 1UK1. Type 2 diabetes was induced in male Sprague-Dawley rats with streptozotocin (STZ, 60 mg/kg, i.p.). The test compounds (3-aminobenzamide = 3-AB, nicotinamide = NIC, taurine = TAU) were given by the i.p. route 45 min before STZ at 2.4 mM/kg (all three compounds) or 1.2 and 3.6 mM/kg (only NIC and TAU). Blood samples were collected at 24 hr after STZ and processed for their plasma. The plasma samples were used to measure glucose, insulin, cholesterol, triglycerides, malondialdehyde, nitric oxide, and glutathione levels using reported methods. RESULTS: 3-AB, NIC and TAU were able to inhibit PARP, with the inhibitory potency order being 3-AB>NIC> or =TAU. Molecular docking studies at the active site of PARP showed 3-AB and NIC to interact with the binding site for the nicotinamide moiety of NAD+ and TAU to interact with the binding site for the adenine moiety of NAD+. While STZ-induced diabetes elevated all the experimental parameters examined and lowered the insulin output, a pretreatment with 3-AB, NIC or TAU reversed these trends to a significant extent. At a dose of 2.4 mm/kg, the protective effect decreased in the approximate order 3-AB>NIC> or =TAU. The attenuating actions of both NIC and TAU were dose-related except for the plasma lipids since NIC was without a significant effect at all doses tested. CONCLUSIONS: At equal molar doses, 3-AB was generally more potent than either TAU or NIC as an antidiabetogenic agent, but the differences were not as dramatic as would have been predicted from their differences in PARP inhibitory potencies. NIC and TAU demonstrated dose-related effects, which in the case of TAU were only evident at doses > or =2.4 mM/kg. The present results also suggest that in the case of NIC and TAU an increase in dose will enhance the magnitude of their attenuating actions on diabetes-related biochemical alterations to that achieved with a stronger PARP inhibitor such as 3-AB. Hence, dosing will play a critical role in clinical studies assessing the merits of NIC and TAU as diabetes-preventing agents.

Structure-based virtual screening, synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening, synthesis and structure-activity relationship (SAR) optimization approach. Virtual screening of 260,000 compounds from the ChemBridge database against the tetracyclic indole inhibitor binding pocket of NS5B (allosteric pocket-1, AP-1), sequentially down-sized the library by 4 orders of magnitude to yield 23 candidates. In vitro evaluation of the NS5B inhibitory activity of the in-silico selected compounds resulted in 17% hit rate, identifying two novel chemotypes. Of these, compound 3, bearing the rhodanine scaffold, proved amenable for productive SAR exploration and synthetic modification. As a result, 25 derivatives that exhibited IC50 values ranging from 7.7 to 68.0 µM were developed. Docking analysis of lead compound 28 within the tetracyclic indole- and benzylidene-binding allosteric pockets (AP-1 and AP-3, respectively) of NS5B revealed topological similarities between these two pockets. Compound 28, a novel rhodanine analog with NS5B inhibitory potency in the low micromolar level range may be a promising lead for future development of more potent NS5B inhibitors.

Hierarchical assembly of presynaptic components in defined C. elegans synapses.

The presynaptic regions of axons accumulate synaptic vesicles, active zone proteins and periactive zone proteins. However, the rules for orderly recruitment of presynaptic components are not well understood. We systematically examined molecular mechanisms of presynaptic development in egg-laying synapses of Caenorhabditis elegans, demonstrating that two scaffolding molecules, SYD-1 and SYD-2, have key roles in presynaptic assembly. SYD-2 (liprin-alpha) was previously shown to regulate the size and the shape of active zones. We now show that in syd-1 and syd-2 mutants, synaptic vesicles and numerous other presynaptic proteins fail to accumulate at presynaptic sites. SYD-1 and SYD-2 function cell-autonomously at presynaptic terminals, downstream of synaptic specificity molecule SYG-1. SYD-1 is likely to act upstream of SYD-2 to positively regulate its synaptic assembly activity. These data imply a hierarchical organization of presynaptic assembly, in which transmembrane specificity molecules initiate synaptogenesis by recruiting a few key scaffolding proteins, which in turn assemble other presynaptic components.