Clark Nelson

Termites and aging

Within one colony, social hymenopteran lifespans can span more than a hundred-fold difference between caste members. In a new study with termites, investigators compared reproductive caste members (kings and queens) to working caste members (majors and minors) using transcriptome sequencing of head tissues. They discovered that majors, who typically have shorter lives due to performing outside duties, showed elevated expression of transposable elements (TE) and TE-related genes in old majors versus young majors. Alternatively, genes known to suppress TE, termed PIWI-interacting RNAs, were reduced in old majors. Reports from other organisms also link elevated TE activity to increased aging, with TE believed to randomly insert into genes and regulatory regions, thereby disrupting function. The authors liken a colony to a superorganism, where the majors are the disposable soma that is not maintained due to its’ reduced longevity.

Novel systemic factor mitigates aging

While CRISPR is the tool of choice for genome editing, it also has off-label applications. In recent years, biologists modified the targetable nuclease to regulate endogenous and exogenous gene expression by coupling it to transcriptional activators, with the complex controlled by illumination. By requiring irradiation, scientists can control spatial and temporal regulation. Most prior studies employed blue light actuation, but this approach suffers from phototoxicity issues. Authors in a current report put a twist on this by engineering a far-red light stimulated CRISPR-dCas9 effector (FACE) system. Using the longer wavelength technique, investigators showed in vivo upregulation of target genes in mouse muscle that significantly exceeded that of blue light activation, with no deleterious effects. As a further demonstration of its utility, FACE allowed illumination-dependent conversion of murine iPSCs to neurons via stimulation of a neuronal transcription factor. CN

Change the ORF, please

While CRISPR is the tool of choice for genome editing, it also has off-label applications. In recent years, biologists modified the targetable nuclease to regulate endogenous and exogenous gene expression by coupling it to transcriptional activators, with the complex controlled by illumination. By requiring irradiation, scientists can control spatial and temporal regulation. Most prior studies employed blue light actuation, but this approach suffers from phototoxicity issues. Authors in a current report put a twist on this by engineering a far-red light stimulated CRISPR-dCas9 effector (FACE) system. Using the longer wavelength technique, investigators showed in vivo upregulation of target genes in mouse muscle that significantly exceeded that of blue light activation, with no deleterious effects. As a further demonstration of its utility, FACE allowed illumination-dependent conversion of murine iPSCs to neurons via stimulation of a neuronal transcription factor. CN

Improved cancer treatment through diet

While CRISPR is the tool of choice for genome editing, it also has off-label applications. In recent years, biologists modified the targetable nuclease to regulate endogenous and exogenous gene expression by coupling it to transcriptional activators, with the complex controlled by illumination. By requiring irradiation, scientists can control spatial and temporal regulation. Most prior studies employed blue light actuation, but this approach suffers from phototoxicity issues. Authors in a current report put a twist on this by engineering a far-red light stimulated CRISPR-dCas9 effector (FACE) system. Using the longer wavelength technique, investigators showed in vivo upregulation of target genes in mouse muscle that significantly exceeded that of blue light activation, with no deleterious effects. As a further demonstration of its utility, FACE allowed illumination-dependent conversion of murine iPSCs to neurons via stimulation of a neuronal transcription factor. CN

Novel treatment for neuropathy

Recording electrical signals via implanted electrodes helps researchers understand what’s going on in the brain. Rodents have been set free with their implants, allowing brain activity to be recorded in unrestrained subjects. Bats are now being set loose too. Cynthia Moss and colleagues at Johns Hopkins were interested in how the midbrain superior colliculus (SC) contributes to spatial orientation. They’ve looked at this before in bats, but not while the animals were freely flying. In their latest paper, they combine neural activity data from wireless electrodes implanted into the SC with audiovisual recordings and a novel echo model to tease apart how big brown bats determined their position while navigating an obstacle-filled space. Though they only observed two bats, the researchers saw dynamic changes in neuron activity as the bats echolocated their way through the room. EPN

Where old players meet new regulators

In today’s world of cheap, sugary foods, endomorphy has become common with two in three American adults classified as overweight or obese. Glucose and lipid dysregulation are notorious biomarkers of these and other related disorders. However, less well known to the general public, but highly predictive, are changes in circulating branched-chained amino acids (BCAAs), which includes the proteinogenic amino acids leucine, isoleucine, and valine. Evidence of elevated BCAAs in obese humans was first reported half a century ago, and while other studies since then have validated the obesity/BCAA correlation and expanded the collective knowledge of this linkage, many details remain unclear. However, a new paper from Prof. Christopher Newgard’s group at the Duke Molecular Physiology Institute, published in the journal Cell Metabolism, sheds light on the biochemistry of these processes and their regulation. Based on rodent and human studies, it appears a significant portion of this increase in circulating BCAAs is attributable to decreased breakdown of BCAAs in liver tissue. This reduction in catabolism is due to a decline in activity of the protein assembly responsible for BCAA catabolism, known as branched-chain ketoacid dehydrogenase complex (BCKDH). This enzymatic slowdown results from increases in a specific phosphorylation event on one of the complex proteins, acting like a brake on BCAA catabolism. In turn, the BCKDH phosphosite is known to be regulated by two enzymes: the BCKDH kinase (BDK) adds the phosphate moiety, while Protein Phosphatase, Mg2+ /Mn2+ Dependent 1K (PPM1K) turns up enzyme action by removing the phosphate. Because the metabolism of BCAAs in liver tissue is reduced, investigators decided to see what would happen if they reopened the floodgates, by revving up BCAA catabolism. For the study, they selected fatty Zucker rats, which have a missense mutation in the leptin receptor and will eat till corpulent. As such, they are often used in studies of diet-induced obesity, insulin resistance, and metabolic syndrome. Additionally, notes first author Phillip White, Zuckers accumulate BCAAs in blood without dietary supplementation much like overweight humans, something missing from other animal models of obesity. The authors used two independent approaches to increase BCAA breakdown in liver. First, working with collaborators David Chuang and Max Wynn at the University of Texas Southwestern Medical Center, who had developed a small molecule inhibitor of BDK, they pharmacologically retarded BDK action with the intention to decrease phosphorylation of BCKDH, thereby increasing enzyme function. Investigators also used virally mediated liverspecific overexpression of human PPM1K in the fatty Zuckers to alter phosphorylation levels at the key phosphosite. Over a seven day time course, rats receiving the inhibitor had increased BCKDH activity across different tissues, resulting in decreased BCAA and associated metabolite levels. Investigators also observed decreases in hepatic triglycerides, suggesting increased catabolism of fatty acids, and improved responses to glucose tolerance testing. Upregulation of the phosphatase produced the same promising results. Given the significant and diverse effects these treatments had on metabolism in fatty rats, the Duke team speculated that there might be other targets for BDK and PPM1K. To determine this, they conducted phosphoproteomic screens of liver extracts to identify other phosphosites that were altered in liver tissue of animals subjected to either approach. Several phosphosites changed, but besides the original site on BCKDH, only one other changed with both treatments, meaning co-regulation by both BDK and PPM1K. The new phosphosite was a serine residue on ATP-citrate lyase (ACL), a central enzyme involved in lipogenesis. For ACL, phosphorylation on this particular site increases enzymatic activity. Based on phosphosites identified on other proteins, additional experiments, and informatic analysis, the investigators hypothesized that BDK and PPM1K were part of a lipogenic circuit regulated by the transcription factor carbohydrate response element binding protein (ChREBP). To test this hypothesis, the Newgard lab collaborated with Mark Herman’s group at Duke, experts in ChREBP metabolism. Together they overexpressed the ChREBP-β isoform in liver of healthy lean rats; as predicted, BDK levels increased while PPM1K decreased, establishing a link between an important transcriptional regulator of lipogenesis and elevated BCAAs in obesity. Based on this relationship and the glucose activation of ChREBP, it follows that people on a Western diet would experience elevated lipogenesis, associated dyslipidemia, and elevated BCAAs. White, who was a postdoc in Newgard’s group and now runs his own lab at Duke, believes this work opens up a new metabolic regulatory node, saying “We can go in and kind of normalize three of the macronutrients that are altered in obesity and insulin resistant states by going after one specific kinase/phosphate axis,” He adds, “A strength of the study is that it really begins with retro-translation, which is a theme of our institute, where we aim to start with unbiased observations in human populations of disease and then try to work backwards to understand what those observations mean.” He’s cautiously optimistic about the possibility for translation of these findings into a new therapeutic approach because these studies were based on human observations and White and colleagues already have a potent small molecule inhibitor in hand. He acknowledged the need for additional studies to check for longterm toxicity issues and efficacy for the drug within other models and in longer-term treatment paradigms.

Flipping the switch off

In order to better understand how neural networks operate and more generally how the brain works, neurobiologists need to be able to turn neurons on or off. To this end, scientists have been developing new tools and one of these toolkits is optogenetics. With optogenetics, proteins are modified so as to be light-activated. Within this collection of proteins, there are pumps, that transmit one ion per photocycle, and channels, that allow multiple ions to flow per absorbed photon. Both pumps and channels can be either stimulatory or inhibitory, depending on the ions transported. While there are many proteins for activating neurons, there are fewer tools that can successfully turn neurons off. Therefore, inhibitory channels were the focus of a recent Elife article by Mingshan Xue, a professor in the Department of Neuroscience at the Baylor College of Medicine, and his lab. The article focused on improving Guillardia theta anion channelrhodopsin 2 (GtACR2), a lightactivated chloride channel. While prior work found that GTACR2 was not stimulatory, other light-gated anionic channels were reported to conduct cations and be neuron acitvating, meaning it is possible that GTACR2 might be stimulatory as well. To begin, the Hue lab validated that the GTACR2 channel was an anionic transporter with electrophysiology experiments, validating their belief that it transported negative ions. However, the ensuing experimental data was surprising. First author of the study, Jessica Messier, described how, when neurons expressing this transgenic protein were light-stimulated they released neurotransmitters onto neighboring cells, which is the opposite to what you’d expect if GtACR2 were inhibiting the neurons. This phenomenon occurred in both excitatory and inhibitory neurons in the mouse cortex. In addition, the lab observed this phenomenon in three other light-gated chloride channels, GtACR1, iC+ + , and iChloC. Authors speculated that this was the result of differences in the chloride concentration between the soma and distal axon/pre-synaptic terminals, and believed this to be true for a couple of reasons. First, different studies had indirectly shown that other chloride channels (glycine and GABA) also possessed this anomalous pre-synaptic release upon stimulation from brainstem, hippocampus, and cerebellum. Additionally, one other report showed that a high presynaptic terminal chloride concentration was responsible for this paradoxical neurotransmitter release, as activation of GtACR2 stimulated pre-synaptic terminals to release neurotransmitter. Messier added that while they did not have a molecular explanation for the concentration differences, it was possibly due to varying amounts of the proteins that determine chloride concentration in the two different regions of the cell. Because of this contradictory effect of GtACR2 activation in the soma versus pre-synaptic terminals, the utility of GtACR2 as an optogenetic tool is compromised. To try and fix this problem and make the channel purely inhibitory, investigators explored combining the protein with different targeting motifs in the hopes of moving a larger fraction of the GtACR2 protein from the axon towards the soma and dendrites. After trial and error, they found that a novel hybrid fusion targeting motif, Kv2.1Clinker-TlcnC, which combines a motif from the Kv2.1 potassium channel with a motif from the telecephalin protein, was the best at trafficking GtACR2 towards the soma and dendrites. Upon reexamination with electrophysiological assays, the modified protein demonstrated approximately an 80% decrease in the undesired excitatory effect and a 2-3 folds increase in the desired inhibitory effect, commented Xue. Messier and Xue both thought the most significant part of their work was that it represents the best optogenetic tool to date for neuronal inhibition. Future work for the group includes two different research plans. First, Xue’s lab is trying to improve targeting motifs that move the protein more efficiently into the soma and dendrites as opposed to the axons. Additionally, they are trying to introduce a mutation to make GtACR2 a rectifying channel, meaning that the channel would only allow chloride to flow into the cell. This would help reduce the stimulatory effect observed. They are doing this in collaboration with John Spudich’s group at the University of Texas Health Science Center at Houston.

Protein modification analysis in depth

In eukaryotes, small ubiquitin-like modifier (SUMO) is a protein family with four different isoforms (S1 to S4) that has enzymes dedicated to conjugating SUMO to target proteins. SUMOylation impacts enzyme location and function and has been implicated in cell division, nuclear transport, and transcriptional regulation. In a new report, investigators describe a method to isolate S2 and S3 conjugated peptides, which does not rely on engineered cell lines or animals carrying a tagged variant of SUMO to facilitate enrichment. The authors identified more than 14,000 sites in human cell culture and nearly 2000 across eight different mouse tissues. In cell culture, a much larger fraction of SUMO2/3 pools were conjugated to targets and is consistent with other reports of high SUMOylation levels in rapidly dividing tissues. Brain had the fewest sites of any mouse tissue and is surprising given that SUMO has been implicated in some neuropathologies. This dataset will serve as a reference of SUMOylation in normal/healthy tissues. CN

Playing catch-up

In eukaryotes, small ubiquitin-like modifier (SUMO) is a protein family with four different isoforms (S1 to S4) that has enzymes dedicated to conjugating SUMO to target proteins. SUMOylation impacts enzyme location and function and has been implicated in cell division, nuclear transport, and transcriptional regulation. In a new report, investigators describe a method to isolate S2 and S3 conjugated peptides, which does not rely on engineered cell lines or animals carrying a tagged variant of SUMO to facilitate enrichment. The authors identified more than 14,000 sites in human cell culture and nearly 2000 across eight different mouse tissues. In cell culture, a much larger fraction of SUMO2/3 pools were conjugated to targets and is consistent with other reports of high SUMOylation levels in rapidly dividing tissues. Brain had the fewest sites of any mouse tissue and is surprising given that SUMO has been implicated in some neuropathologies. This dataset will serve as a reference of SUMOylation in normal/healthy tissues. CN

New technique for knock-in mice

In eukaryotes, small ubiquitin-like modifier (SUMO) is a protein family with four different isoforms (S1 to S4) that has enzymes dedicated to conjugating SUMO to target proteins. SUMOylation impacts enzyme location and function and has been implicated in cell division, nuclear transport, and transcriptional regulation. In a new report, investigators describe a method to isolate S2 and S3 conjugated peptides, which does not rely on engineered cell lines or animals carrying a tagged variant of SUMO to facilitate enrichment. The authors identified more than 14,000 sites in human cell culture and nearly 2000 across eight different mouse tissues. In cell culture, a much larger fraction of SUMO2/3 pools were conjugated to targets and is consistent with other reports of high SUMOylation levels in rapidly dividing tissues. Brain had the fewest sites of any mouse tissue and is surprising given that SUMO has been implicated in some neuropathologies. This dataset will serve as a reference of SUMOylation in normal/healthy tissues. CN