Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10. Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

The mechanistic target of rapamycin complex 1 (mTORC1) is a eukaryotic cell growth regulator that responds to nutrient and growth factor availability. Under nutrient-replete conditions, mTORC1 licenses anabolic processes while inhibiting catabolic ones. Given the myriad of stimuli that mTORC1 responds to, it is no surprise that a diverse set of proteins, many as part of large complexes, act in a coordinated manner to regulate mTORC1 activity.The heterodimeric Rag GTPases (RagA/B and RagC/D) play a central role in the control of mTORC1 by nutrients. In response to amino acids, as well as glucose and cholesterol, GTP-bound RagA/B and GDP-bound RagC/D mediate the recruitment of mTORC1 to the lysosomal surface (1–5). Once at the lysosome, GTP-bound Rheb, which is under the control of growth factors through the TSC complex pathway, binds to mTORC1 and stimulates its kinase activity (6–13). Together, Rheb and the Rags form a GTPase-based coincidence detector at the lysosomal surface that ensures that mTORC1 becomes activated only when nutrient and growth factor conditions are optimal. Given that the Rag and Rheb GTPases are central arbiters of mTORC1 activation, the regulation of their respective nucleotide states is of great interest.Dozens of proteins have been shown to modulate mTORC1 activity, many acting indirectly through one of several key effectors. However, the relative contributions of these proteins to the regulation of mTORC1 activity has not been systematically interrogated. Additionally, the majority of the proteins that regulate mTORC1 were identified using proteomic approaches. While fruitful, these studies leave open the possibility that proteins that play a role in mTORC1 regulation through transient or indirect interactions with pathway components, or are not easily detected with mass spectrometry-based proteomics, have not been identified.Advances in CRISPR-Cas9-based screening have generated large catalogs of gene essentiality data in numerous cell lines, which can be leveraged to identify genes that are coessential with those encoding components of the mTORC1 pathway (14, 15). Though this type of analysis reveals many established mTORC1 regulators, a caveat is that it relies on cell fitness rather than mTORC1 activity as a readout. A recent study utilized a gene-trap approach to identify mTORC1 regulators in haploid cells and define new relationships among established components (16). The CRISPR-screening strategy we present here expands this toolbox by enabling screening in a large set of genetically diverse cell lines of different lineages and allows for the identification of genes that regulate mTORC1 signaling but whose loss is not tolerated long term. We carried out a genome-wide CRISPR-Cas9 screen and a series of focused sublibrary screens to identify positive regulators of mTORC1. The hits from these screens ultimately led us to study how mTORC1 senses mitochondrial dysfunction. We find that two kinases (AMPK and HRI) act in a coordinated fashion to mediate the inhibition of mTORC1 caused by mitochondrial stress.     

Building social capital among rural,older Australians through information and communication technologies: A review article

This paper undertakes a comprehensive review of the growing international literature on the adoption and use of information and communication technologies (ICTs) among older people. Issues associated with access and adoption of ICTs among older people living in rural communities will be explored, drawing on social capital as a theoretical lens through which to identify how these new technologies can build healthy ageing. ICTs as bridging social capital can address some of the challenges of service provision in rural Australia and provide access to more extensive information and resources. ICTs can also contribute to bonding social capital through access to other forms of communication to build on local connectedness. However, rural, older people face particular challenges of access, which may exacerbate the cycle of rural social exclusion. In the context of the Australian National Broadband rollout, it is timely to consider how some of these disparities can be addressed.     

The concept of social dignity as a yardstick to delimit ethical use of robotic assistance in the care of older persons

Medicine, Health Care and Philosophy - With robots being introduced into caregiving, particularly for older persons, various ethical concerns are raised. Among them is the fear of replacing human...     

Outbreaks of Foot‐and‐Mouth Disease in Libya and Saudi Arabia During 2013 Due to an Exotic O/ME‐SA/Ind‐2001 Lineage Virus

Foot‐and‐mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O‐Ind‐2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real‐time RT‐PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co‐circulating in North Africa and western Eurasia.     

Rabies virus strains circulating in Bhutan: implications for control

We report a molecular epidemiological study of rabies virus (RABV) strains circulating in animal populations in Bhutan, and investigate potential origins of these viruses. Twenty-three RABV isolates originating from dogs and other domestic animals were characterized by sequencing the partial nucleoprotein (N) gene (395 bp). Phylogenetic analysis was conducted and the Bhutanese isolates were compared with rabies viruses originating from other parts of the world. Phylogenetic analysis showed that Bhutanese isolates were highly similar and were closely related to Indian strains and South Asian Arctic-like-1 viruses. Our study suggests that the rabies viruses spreading in southern parts of Bhutan have originated from a common ancestor, perhaps from the Indian virus strain.     

Is the 2015 eye care service delivery profile in Southeast Asia closer to universal eye health need!

Purpose

The year 2015 status of eye care service profile in Southeast Asia countries was compared with year 2010 data to determine the state of preparedness to achieve the World Health Organization global action plan 2019.

Methods

Information was collected from the International Agency for Prevention of Blindness country chairs and from the recent PubMed referenced articles. The data included the following: blindness and low vision prevalence, national eye health policy, eye health expenses, presence of international non-governmental organizations, density of eye health personnel, and the cataract surgical rate and coverage. The last two key parameters were compared with year 2010 data.

Results

Ten of 11 country chairs shared the information, and 28 PubMed referenced publications were assessed. The prevalence of blindness was lowest in Bhutan and highest in Timor-Leste. Cataract surgical rate was high in India and Sri Lanka. Cataract surgical coverage was high in Thailand and Sri Lanka. Despite increase in number of ophthalmologists in all countries (except Timor-Leste), the ratio of the population was adequate (1:100,000) only in 4 of 10 countries (Bhutan, India, Maldives and Thailand), but this did not benefit much due to unequal urban–rural divide.

Conclusion

The midterm assessment suggests that all countries must design the current programs to effectively address both current and emerging causes of blindness. Capacity building and proportionate distribution of human resources for adequate rural reach along with poverty alleviation could be the keys to achieve the universal eye health by 2019.