Watching the brain adapt to light and dark

Written by: Ken Estrellas

Original Article: Wang et al. PLoS Biology 2020
The Gist of It
Throughout the world, animals of all shapes and sizes demonstrate behaviors linked to cycles of light and dark, from sleeping and wakefulness in humans to navigation in butterflies. These patterns are described as circadian rhythms, processes that are governed by external factors such as light and temperature and tend to cycle every 24 hours. Scientists have studied circadian rhythms in a variety of ways, but the researchers in this particular study have developed what might be the most sophisticated method yet – watching individual cells in a living animal turning genes on and off in cycles of light and dark. Wang and colleagues used zebrafish that were genetically modified to emit a fluorescent protein, Venus-NLS-PEST (VNP), in cells that also have the gene nr1d1, a circadian clock gene, turned on. By putting the zebrafish in a special circulating water bath and using a particular microscope known as a “two-photon microscope,” the authors were able to track and film cells that had increasing levels of VNP in the dark and decreasing levels in the light, which meant that the cells had the circadian clock gene turned on and then off. Most of these cells were located in the pineal gland, a part of the brain in most vertebrates that produces melatonin, a hormone which controls sleep patterns. Specifically, these cells were identified as melatonin-secreting cells that were light sensitive – similar to the ones in our eyes! Finally, in order to demonstrate that light/dark cycles were necessary for these cells to develop, a different set of fish were raised under constantly dark conditions. Without the light/dark cycle, the fish showed decreased levels of fluorescence in regions of the brain that would light up in fish raising with normal light/dark cycles. Overall, the authors of this study appear to have successfully created a unique method for studying circadian rhythm and its effects on living creatures.
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With this new system, scientists can watch how certain brain cells react to light and dark – inside of a living animal!

The Nitty Gritty
In this study, Wang et al. report the development of a novel zebrafish-based model to study circadian rhythm. Transgenic zebrafish lines were generated with expression of Venus-NLS-PEST (VNP) linked to both the proximal and distal promoter regions of nr1d1, a key circadian clock gene which expresses a transcription factor linked to the circadian BMAL1/CLOCK axis. Quantitative PCR revealed oscillating expression of both VNP and nr1d1 roughly linked to 12-hour day/night cycles between 3.5-7.5 days post-fertilization (dpf), with highest levels of expression observed just before “dawn” and lowest levels just before “dusk.” This quantitative assessment of expression was visualized by mounting anesthetized live zebrafish in low melting point agarose and a circulating water bath, then monitoring VNP fluorescence using two-photon microscopy. The most prominent reporter expression as measured by fluorescence intensity was observed in the pineal gland, with subsequent development observed in the optical tectum at 5.5 dpf and cerebellum at 6.5 dpf. When nr1d1:VNP zebrafish were crossed with a second line linking expression of aanat2 with mRFP, significant co-localization was observed, indicating that the cells lighting up with nr1d1:VNP were melatonin-synthesizing photoreceptor cells. Further crosses and co-localization analysis with xops:nfsB-mCherry and lws2:nfsB-mCherry lines revealed that these cells also co-express markers for rod-like and cone-like photoreceptor cells, respectively. Rod-like cells were shown to have higher levels of baseline nr1d1:VNP expression than non-rod like cells were, but single-cell analysis revealed that both cell types maintain oscillations linked to light-dark cycles. Finally, raising a subset of fish in continuously dark conditions from 0-3.5 dpf significantly dampened nr1d1:VNP oscillation amplitudes, indicating the necessity of light-dark cycling on the successful development of circadian expression. Conversely, normally-raised fish subsequently transferred into dark conditions maintained their oscillations. Taken together, these results indicate the successful generation of a novel reporter line to study circadian rhythms with live fluorescence imaging at a higher single-cell resolution compared to previously developed bioluminescence-based lines.
Original Research Article: Wang, H., et al. “Single-cell in vivo imaging of cellular circadian oscillators in zebrafish.” PLoS Biol 18.3 (2020): e3000435.

Overcoming barriers: How Zika virus gets to the brain

Written By: Rebecca Tweedell

The Gist of It:
In 2015 and 2016, the world was closely following the outbreak of a mosquito-borne virus called Zika. While these outbreaks have since faded from the forefront, with very few if any cases being detected currently, there are still a lot of unknowns about this virus. One thing we do know is that Zika virus can cause fetal microcephaly, or swelling of the brain in unborn babies. To do this, the virus must somehow be able to get to the brain. This is especially challenging because there are 2 key lines of defense working to keep viruses out, the placenta and the blood-brain barrier. The placenta is formed during pregnancy and allows oxygen and nutrients to be passed from the mother to the baby while blocking the passage of toxic materials and viruses. The blood-brain barrier exists in both babies and adults and serves as the boundary to keep the circulating blood (and any bad things that might be circulating with it) away from the brain. Since we know that the virus can get into the brain, we know these barriers must be failing, but how this happens is still a big question. Researchers at National Yang-Ming University in Taiwan recently took a major step toward solving this mystery. They studied how the virus passed through placental barrier cells and brain-derived barrier cells in the lab. They discovered that the virus actually crosses these barriers in different ways. Zika virus disrupted the ability of the placental cells to form strong junctions between themselves, weakening the physical structure of the barrier. This was not the case with the brain-derived cells, which maintained the physical structure of the barrier in spite of the virus. But the virus was still able to move through both types of barriers by going directly through the cells through a process known as transcytosis (which literally means being transported across the interior of the cell). Excitingly, blocking transcytosis with inhibitors was able to keep Zika virus from getting through the barriers. Understanding how the virus is able to get through these barriers is important for being able to stop it, and this study helped us take a big step in the right direction.

Zika virus can pass through placental barriers by going between cells, breaking their tight junctions, and the virus can pass through both placental and brain barriers by using transcytosis to go directly through the cells to get to the brain.

The Nitty Gritty:
Chiu et al. used the human placenta trophoblast cells JEG-3 and human brain-derived endothelial cells hCMEC/D3 to create an in vitro model to study interactions with Zika virus. They found that Zika virus could infect both of these cell types when the cells were grown in a monolayer. Using a transwell barrier assay, with a monolayer of JEG-3 or hCMEC/D3 cells in the insert and Vero cells in the bottom chamber, the researchers found that Zika virus was able to pass through the barrier formed by the cells, moving from the portion of the well above the JEG-3 or hCMEC/D3 cells to infect the Vero cells below. When passing through the JEG-3 placental cells, Zika virus damaged the integrity of the membrane, allowing FITC-dextran to pass through. However, this did not occur with the hCMEC/D3 cells. This was likely due to tight junction disruption; the researchers found that the expression of ZO-1 and occludin was decreased in JEG-3 cells following exposure to Zika virus, while this did not occur in hCMEC/D3 cells. After treatment with a proteasome inhibitor MG132, the expression of ZO-1 and occludin was rescued, suggesting that the disruption of tight junctions occurs through the proteasomal degradation pathway. Finally, the authors sought to understand whether Zika virus can use transcytosis to traverse the placental and brain-derived barrier cells. Using a fluorescently labeled virus, they measured the fluorescence in the basal chamber of a transwell following culture of the virus with the monolayer barrier in the insert at 4°C and 37°C, as transcytosis is inhibited at 4°C. They found that the amount of virus that traversed the barrier at 4°C was significantly reduced compared to the amount that traversed at 37°C, while there was no difference in the amount of FITC-dextran that could traverse. Based on the finding that the virus is able to traverse the cells by transcytosis, the authors tried to block the virus using inhibitors of endocytosis and intracellular trafficking (Nystatin, chlorpromazine, dimethyl amiloride, and colchicine). Each inhibitor reduced the amount of virus that made it through the monolayer barrier. Overall, Chiu et al. found that Zika virus crosses placental and brain-derived barrier cells in culture by transcytosis and that the virus disrupts the integrity of tight junctions to weaken the placental cell barrier in the transwell system. These findings pave the way for discovering ways to inhibit viral traversal and prevent fetal infection and encephalitis.
Original Research Article: Chiu, C.-F., et al. “The mechanism of the Zika virus crossing the placental barrier and the blood-brain barrier.” Front Microbiol (2020).

Does a tense environment affect behavior? A synthetic extracellular matrix may help answer that question

Written By: Kaitlyn Sadtler

Original Article: Davidson et al. Acta Biomaterialia 2020
The Gist of It:
Just like we take in information from our environment and change our behavior (like putting a coat on when it’s cold outside), cells take in information from their environment – the extracellular matrix. The cues they receive can cause them to change their environment by secreting different proteins, making the matrix stiffer or softer (just like we can turn on the A/C or put another log on the fire to make it cooler or warmer). Stiffening and densifying of the extracellular matrix is called fibrosis, and this process contributes to numerous disorders, including liver cirrhosis, cardiac dysfunction, skin scarring, and lung fibrosis. Understanding how our cells sense changes in the stiffness of the matrix around them and how that affects disease progression is necessary for the development of new therapeutics. Researchers from the University of Michigan Ann Arbor have developed a new material and platform to study this phenomenon in the laboratory. Davidson and colleagues synthesized a fibrous biologic matrix out of dextran vinyl sulfone (DexVS). The researchers were able to spin these fibers onto a plate to create a sort of cellular hammock, where the cells would feel just the stiffness of the material and not the plastic bottom of a petri dish. With these fibers, they were able to look in a highly controllable manner at how cells interacted with stiffer and softer matrices. In contrast to what had been seen when the interaction between cells and the matrix was studied using other methods, like looking at solid synthetic gels (jello-like), they found that stiffer materials made cells less fibrotic than softer materials did. Using these models, scientists will be able to probe interactions between cells and fiber-based matrices that are more similar to the natural extracellular matrix and compare to previous studies which used fully synthetic hydrogels that do not have fibers. Moving forward, these scientists have developed ways to modify these materials with different proteins and change the way that the cells interact with the matrix. Further research like this could expand our understanding of what happens to our cells in stiff environments, which could lead to therapeutics for fibrotic diseases.

New Dextran Vinyl Sulfone (DexVS) fibers are tunable, which means scientists can alter the stiffness of each fiber, along with the stiffness of the overall synthetic extracellular matrix they have created. Using a draped matrix, cells in contact with the fibers will only feel stiffness of those fibers, and not the plastic around them.

The Nitty Gritty:
Previously, researchers had developed an electrospun methacrylated dextran (DexMA) to study matrix stiffness in vivo. However, DexMA fibers degraded in vitro due to hydrolytic cleavage of the ester bonds in the polymeric matrix within days of exposure to cell culture media. Here, the researchers presented a dextran vinyl sulfone (DexVS) that lacks the sensitive ester bonds and can still be functionalized via Michael-type addition and crosslinked with a photoinitiator (in this case, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP). They tested the stiffness of these materials by changing the concentrations of photoinitiator or duration of UV exposure. They further functionalized these materials with cyclic RGD (cRGD) for cell attachment or with methacrylated heparin to promote association of cell-secreted matrix with the synthetic polymer fibers. DexVS fibers were spun onto a multi-well plate to create a suspended surface with mechanical properties independent of the tissue culture plastic. Culturing normal human lung fibroblasts (NHLFs) on stiffer and softer matrices revealed that softer fibrous matrices, in the presence of pro-fibrotic soluble transforming growth factor beta-1 (TGFβ1), induced higher levels of myofibroblast-associated alpha smooth muscle actin (αSMA) than did stiffer matrices. This is contradictory to studies performed with solid hydrogels, such as poly(ethylene glycol) (PEG)-based hydrogels, that suggested stiffer matrices induce more of the fibrotic myofibroblast phenotype. Further research using multiple model systems in vitro and comparison to in vivo samples will hone in vitro modeling to pave the way for understanding mechanistic biology, modeling disease, and screening novel therapeutics.
Original Research Article: Davidson, C.D., et al. “Myofibroblast activation in synthetic fibrous matrices composed of dextran vinyl sulfone.” Acta Biomaterialia (2020).

Can moths teach drones to navigate?

Written by: Sayan Roychowdhury

Original Article: Zhu et al. PLOS Computational Biology 2020
The Gist of it:
Autonomous aerial vehicles, such as drones, are carefully programmed and trained to move based on their surroundings. However, these controls sometimes react poorly when introduced to new or unknown environments. So, how can safe and reliable flying strategies be developed for use in unfamiliar territory? To answer this question, a group of scientists from Boston University and the University of Washington turned to hawkmoths for inspiration. Previous research shows that hawkmoths navigate largely based on visual cues just as drones do, making the moths’ flight decision-making very applicable to drones. In this study, the researchers gathered physical flight data from hawkmoths through a virtual forest projected on a screen. The researchers then created a moth decision-making model based on this data; this model could simulate how a hawkmoth would react to seeing a certain visual scenario. The researchers tested this model on a wide variety of forest arrangements differing in size and density to examine how well a hawkmoth would perform when encountering new surroundings. They also compared the performance of the hawkmoth to an optimal flight pattern for each virtual forest.
The researchers found that for each case, the moth decision-making model performed very well, suggesting that the moths’ strategy is quite versatile, even for unfamiliar terrain. In particular, the moth model worked best in dense forest scenarios, possibly indicating that hawkmoths have developed flight strategies best suited to the types of forests they encounter in their natural habitat. These results are exciting because researchers can study biologically evolved flight patterns to help program autonomous vehicles to perform better in new and unfamiliar environments.

Flight data from hawkmoths navigating a virtual forest can be used to train flight decision-making for autonomous drones.

The Nitty Gritty:
To study navigational patterns, the researchers connected eight hawkmoths (Manduca sexta) to a metal rod and placed them in front of a large screen displaying a virtual forest moving at a constant speed of 2 m/s. Each moth’s motion in the horizontal direction was measured through a torque meter, capturing changes in direction based on the view. Data were collected for each moth at 5 varying levels of fog density to simulate varying visibility conditions. The virtual forest and the moth’s flight trajectory were discretized in time and space, and a Markov Decision Process was used to model the flight. Logistic regression was used to estimate the parameters of the moth control policy. This policy was further refined using a reward function based on distance to trees – minimizing distance to simulate hiding from predators in the foliage, while maintaining enough distance to avoid collision. The original moth policy and an optimized one were then compared on a new set of virtual forests with varying size and density, where the optimal policy was tuned on a per forest basis. The original moth control policy was very robust and was within 30% of the performance of the refined policy for all of the dense forests. Thus, this work shows that hawkmoths can serve as a reliable starting point for successful bio-inspired control and decision rules for autonomous aerial vehicles exposed to new terrains.
Original Research Article: Zhu, H., et al. “Learning from animals: How to navigate complex terrains.PLoS Comput Biol 16.1 (2020): e1007452.

Paraquat herbicide poisoning: Antidote on the horizon?

Written by: Sravya Kotaru

 Original article: Liu et al. BioMed Research International 2020
The Gist of It:
 Paraquat is a common herbicide used to kill weeds in croplands. It is poisonous if consumed, breathed, or touched. Severe poisoning can cause multiple organ failure including lungs, kidneys, and liver, and can be fatal. Even mild poisoning leads to long-term respiratory issues from lung damage and scarring. The usage of paraquat is highly debated; it is banned in Europe but is still widely used in the United States and other parts of the world. The people at most risk of exposure are those using the herbicide and those living in areas near where it is used, but it is also possible for paraquat to contaminate food and water sources and reach consumers. Once paraquat enters the human body, it produces harmful molecules called reactive oxygen species (ROS), which cause inflammation and tissue damage. Specifically, in the lungs, paraquat damages the tiny air sacs called alveoli, which are like small balloons that fill with air during each breath. In the alveoli, oxygen from the air diffuses into the bloodstream. When paraquat is breathed in and reaches these lung alveoli, it causes inflammation and the build-up of scar tissue around these air sacs. This scarring restricts the alveoli’s ability to expand with each breath, causing shortness of breath and lung disease. So far, there is no antidote or treatment for paraquat poisoning. Excitingly, scientists at North Sichuan Medical College in China recently discovered more details about how paraquat causes lung damage, allowing them to identify a potential target for treatment. They found that the ROS produced by paraquat lung poisoning in rats damaged mitochondria, the energy factories in alveolar air sacs. This damage turns on two proteins called PINK1 and Parkin that are responsible for removal of the damaged mitochondria and cause scar tissue to develop around damaged alveoli, thus causing lung injury. The researchers also found that deleting the protein PINK1 from lung cells grown in the laboratory prevented the production of fibronectin and collagen, two molecules that form scar tissue. These results are good news because they suggest that PINK1 could be targeted to develop a paraquat antidote and prevent lung damage. This possibility must be further explored.

Paraquat is a poisonous herbicide that causes long-term lung injury by damaging alveolar air sacs and scarring surrounding lung tissue via a molecular mechanism involving PINK1 and Parkin proteins

The Nitty Gritty: 
Paraquat, a common herbicide, is a known oxidant that generates ROS and causes mitochondrial damage in alveolar epithelial cells, resulting in alveolitis and pulmonary fibrosis. Independently, it is known that damaged mitochondria accumulate the kinase PINK1 on their outer membrane; PINK1 in turn recruits Parkin to initiate mitophagy. The authors investigated whether PINK1-Parkin signaling and mitophagy are involved in paraquat-mediated poisoning. They observed that in rat models, paraquat exposure resulted in loss of alveolar architecture and a build-up of fibrous extracellular matrix in lungs over time, indicative of fibrosing alveolitis. The poisoned rat lungs also showed a time-dependent increase in the expression of the extracellular matrix fibers fibronectin (FN) and collagen (COL-1), which contribute to the formation of fibrous scar tissue. In addition, there was a time-dependent increase in PINK1 and Parkin expression and loss of mitochondrial membrane potential, indicative of mitophagy. The researchers observed similar results in the cultured A549 lung cancer cell line, where paraquat induced a time- and dose-dependent loss in cell viability and increase in the expression of PINK1, Parkin, FN, and COL-1. Moreover, silencing PINK1 expression in A549 cells negated the effects of paraquat-mediated upregulation of Parkin, FN, and COL-1 expression. Thus, mitophagy and fibrosis due to paraquat poisoning appear to be PINK1-dependent, and PINK1 is a possible molecular target for the development of a paraquat-specific antidote.
Original Research Article: Liu, K., et al. “Paraquat exposure induces pulmonary cell mitophagy by enhancing the PINK1/Parkin signaling.” BioMed Res Int 6 (2020): 1–6. doi: 10.1155/2020/7103105.

A surprising discovery of live mitochondria in human blood

Written by: Abby Stahl

Original Article: Al Amir Dache et al. The FASEB Journal 2020
The Gist of It
You may have heard that mitochondria are the “powerhouse” of the cell, but what is DNA from these tiny organelles doing circulating in the bloodstream?  That was the question a team of scientists at the Institut Regional du Cancer de Montpellier in France asked in response to several studies showing that DNA from mitochondria could be found in circulation.
Mitochondria are important producers of cellular energy that evolved millions of years ago through a symbiotic relationship between primitive eukaryotic cells (our ancestors) and free-living prokaryotes (bacteria). Mitochondria have retained many of their bacterial features today, including having their own DNA. The fact that mitochondrial DNA is in the bloodstream has been known by doctors for years, and its presence can even be used to help diagnose diseases and conditions such as diabetes, cancer, and heart disease. Surprisingly, this team of researchers found that there are 50,000 times more copies of mitochondrial DNA in the bloodstream than nuclear DNA, or the genomic DNA at the “core” of the cell. However, the purpose of this mitochondrial cell-free DNA (McfDNA) was still unknown.
By collecting blood plasma from healthy volunteers, the scientists found that McfDNA was longer and more stable than nuclear cell-free DNA (NcfDNA), suggesting the McfDNA might be protected or encapsulated by some larger structure. The researchers went on to find that spinning the plasma at a high speed caused sedimentation of most of the McfDNA. Spinning at high speed can also cause intact mitochondria to be spun down. In fact, using a dye that labels live mitochondria, they found an abundance of the organelles within the centrifuged blood plasma. More powerful electron microscopy confirmed that these mitochondria existed without a cell around them, and metabolic studies showed that these mitochondria were functional at consuming oxygen, an important step in energy production.
These research studies are the first to report the presence of intact and functional mitochondria in blood from healthy individuals. The scientists estimate that there are between 200,000 and 3.7 million cell-free, intact mitochondria per milliliter of blood plasma and that these mitochondria might have crucial biological roles still to be discovered!

Blood serum collected from healthy patients was found to contain live mitochondria capable of converting oxygen (O2) to energy (ATP) and encapsulating stable, circulating, cell-free mitochondrial DNA.

The Nitty Gritty
In this study, the authors began by comparing the stability of DNA found in the blood of healthy individuals. They hypothesized that NcfDNA would be more stable than McfDNA due to the presence of supporting histone proteins that mitochondrial DNA lacks. To their surprise, the McfDNA had a greater DNA Integrity Index, meaning the McfDNA had a mean size greater than 300 base pairs when assessed from 13 blood samples. Sequencing of cell-free DNA from 80 additional individuals showed that McfDNA contained two populations of DNA – small fragments between 30–300 base pairs (approximately 20%) and large pieces greater than 1000 base pairs (approximately 80%); these populations could not be sequenced due to limits of sequencing technology.
To further determine the structure of this stable McfDNA, the researchers centrifuged blood samples at increasing speeds to sediment different components. Interestingly, the McfDNA sedimented at 16,000 × g, whereas NcfDNA was retained in the blood plasma, suggesting McfDNA was larger and denser. Importantly, the researchers also verified these findings in cells that had been cultured in media to confirm that the McfDNA was not being released by platelets during the experimental handling of blood samples, showing an abundance of McfDNA pelleted at 16,000 × g in cell cultures. In all cases, the McfDNA was too large to pass through a 0.22 mm filter, suggesting it might be retained within mitochondria organelles themselves, which are typically 0.4–1.5 mm in size and are known to sediment at 7,000–20,000 × g .
To confirm these suspicions, the scientists used MitoTracker Green dye to visualize live mitochondria and found them in serum samples and cell media supernatants. Western blot analysis also confirmed the presence of membrane transport proteins, TOM22 and TIM23, in these McfDNA fractions. Finally, the researchers used electron microscopy and identified structures with a double membrane and similar size to mitochondria in the samples, although they noted some morphological differences.
After determining that mitochondria were present and surrounding the McfDNA in blood and cell samples, the scientists asked whether these mitochondria were metabolically functional and able to produce energy, a process known as respiration. Using a Seahorse XF flux analyzer, the researchers found that the isolated mitochondria consumed oxygen and were sensitive to inhibition and activation by known regulators of respiration.
These studies show that human blood contains a previously unknown and significant population of mitochondria that can be useful for diagnostics; however, the physiological roles of these cell-free organelles (if any) remain a mystery for now.
Original Research Article: Al Amir Dache Z, A., et al. “Blood contains circulating cell‐free respiratory competent mitochondria.” FASEB J (2020). doi: 10.1096/fj.201901917RR. [Epub ahead of print].

Delivering Drugs with Sound, Part 2: Treating muscles to treat the brain

Written by: Ken Estrellas

Original Article: Li et al. Molecular Therapy Methods & Clinical Development 2020
The Gist of It
You may recall that in my last post, I presented a study that used ultrasound waves, high-frequency sound waves typically undetectable by human hearing, to deliver drugs to cartilage. A recent study published by Li and colleagues extended this concept to a different area of the body – the brain – and applied it to a significant clinical issue that my fellow contributor Abel Cortinas wrote about in the final post of 2019: Alzheimer’s disease (AD). As Abel mentioned, a protein called amyloid beta (Aβ) clumps up in the brain of patients with AD, forming plaques associated with the disease. Previous studies have shown that there is less of an enzyme called neprilysin in the brains of patients with AD; also, getting rid of neprilysin increases the amount of Aβ made. Consequently, increasing the amount of neprilysin might help reduce the presence of these clumps, potentially relieving the symptoms of AD. However, increasing levels of neprilysin in the brain can be difficult from a practical standpoint, since administering the enzyme itself to the brain or using viral gene therapy to produce the enzyme both come with a significant risk of complications. In this study, the authors take an interesting approach: using ultrasound to inject modified genetic material known as plasmids that can lead to the production of neprilysin. However, instead of injecting this gene therapy directly into the brain, the leg was selected as an injection site instead. Since muscles contain a large number of blood vessels, the injected plasmids are able to circulate throughout the body, including the brain. The authors tested this approach in mice with a form of AD and observed that mice treated with ultrasound injections of plasmid had both more neprilysin and less Aβ protein in the brain one month after injection. These treated mice were also better able to navigate a complex water maze than untreated AD mice, suggesting that cognitive decline (a symptom of AD) had slowed. Importantly, the treated mice showed no signs of damage to the injected muscles or systemic damage in the kidneys. Overall, this study presents an alternative approach to the treatment of AD that appears promising and deserves further investigation.
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Ultrasound was used to deliver a gene therapy to muscle, with the effects observed in the brain

The Nitty Gritty
In this study, Li et al. explored the potential of ultrasound-assisted administration of 11,090 base pair plasmids to hindlimb muscles to more effectively overexpress neprilysin in the brain tissue of APP/PS1 mice, a model of AD, decreasing local concentrations of Aβ protein. The plasmids, which mediated overexpression of human neprilysin (hNEP) under the control of a cytomegalovirus (CMV) promoter, were delivered via microbubbles into the gastrocnemius muscles of N = 20 APP/PS1 mice with the simultaneous administration of 1.7 MHz, 1.0 W/cm2 ultrasound irradiation for one minute. One month after injection, increased levels of neprilysin were observed in the brains and skeletal muscles of hNEP-treated APP/PS1 mice compared with levels in untreated APP/PS1 mice, as determined by western blot. Significantly decreased levels of Aβ were detected in the brains of treated mice via western blot after one month compared with the levels in untreated mice, and this was visually observed via immunohistochemistry. A Morris Water Maze was used to compare potential functional improvements in treated vs untreated mice; treated mice showed a significantly decreased time to find a hidden platform in the shallow water maze (latency) and traveled a significantly shorter distance to do so (distance) after one month of treatment, whereas wild type C57/BL control mice and untreated APP/PS1 mice did not demonstrate any differences. Histological assessment of skeletal muscle, lung, and kidney tissue from treated mice using hematoxylin and eosin (H&E) staining revealed no signs of significant damage after one month. These results appear to present an initially promising strategy to overexpress neprilysin specifically in brain tissue without the need to cross the blood-brain barrier with recombinant enzyme or the complications associated with virally mediated gene therapy. Nonetheless, the authors acknowledge the need to assess the half-life of overexpressed hNEP, and the potential for long-term functional improvements – or declines – remain unknown.
Original Research Article: Li, Y., et al. “Expression of neprilysin in skeletal muscle by ultrasound-mediated gene transfer (sonoporation) reduces amyloid burden for Alzheimer’s disease.” Mol Ther Methods Clin Dev (2020) [Epub ahead of print].