Shining a Light on Microscale Innovations

What comes to mind when you think of light? Perhaps the vision of yourself basking in the sun on a California beach. Or the most recent book of Michelle Obama “The Light We Carry”. Or even the metaphorical light we’re not supposed to follow when our time comes. Here, we are going to talk about a concept even more fundamental: the physics of light.

Light, with its dual nature as both a wave and particles known as photons, exists in different “forms”, each characterized by its unique wavelength or color. For instance, the wavelength of the visible light, which is the electromagnetic radiation detectable by the human eye, ranges from 400 to 700 nanometers.

Image credits: Chiara Trovatello

Fascinatingly, we can manipulate light and change its wavelength using a powerful tool: nonlinear optics. Inside nonlinear materials light behaves in a “nonlinear” manner, meaning that its response is not directly proportional to the input. In such systems, the properties of light change as it interacts with the material, resulting in nonlinear phenomena. For example, photons interacting with a nonlinear material can be transformed, with a certain probability, into new photons with twice the frequency of the initial photons in a process called Second Harmonic Generation (SHG). Another phenomenon, even more intriguing, is the Spontaneous Parametric Down-Conversion (SPDC), in which one photon of higher energy is converted into a pair of entangled photons of lower energy. What makes these entangled photons unique is their intrinsic interconnection, even when they are separated by vast distances, or even placed at opposite corners of the universe. Changing the state of one of them will result in an instant change in the state of the other photon.

Nowadays, SPDC is especially relevant since it’s at the heart of many applications, for instance in quantum cryptography for secure communications and also for testing fundamental laws of physics in quantum mechanics. Inducing these nonlinear behaviors requires crystals with large thickness, typically of the order of a few millimeters. This requirement is due to the relatively low nonlinearity of these materials. Indeed, when the nonlinearity of a material is low, a larger volume of material is required in order to achieve an equivalent level of efficiency. This limitation has been overcome thanks to the innovative work of Chiara Trovatello, a Columbia postdoc from the Schuck Lab. She and her colleagues have successfully developed a method for generating nonlinear processes, such as SHG and SPDC, in highly nonlinear layered materials with remarkable efficiency, down to sizes as small as 1 micrometer – a thousand times thinner than a millimeter! To put this in perspective, the materials that Chiara and her colleagues have realized are 10-100 times thinner than standard nonlinear materials with similar performances.

While materials with a high conversion efficiency were already well-known in the field, achieving macroscopic efficiencies over microscopic thicknesses was still an open challenge. The critical component was to find a way to achieve the so-called phase matching condition in microscopic crystals with superior nonlinearity. Phase matching ensures efficient conversion of light by maintaining the fields of the interacting light waves in-phase during their propagation inside the nonlinear crystal. To this end, Chiara and her colleagues designed and realized multiple layers of crystalline sheets, stacked with alternating dipole orientation. With this procedure, they were able to change the way the material responds to light, meeting the phase matching condition over microscopic thicknesses. This methodology provides high efficiency in converting light at the nanoscale, and unlocks the possibility of embedding novel miniaturized entangled-photon sources on-chip. It paves the way for novel technologies and more compact optical devices, for applications in quantum optics. Projecting ourselves into the future, it’s not hard to envision a world where everyone will carry a sophisticated quantum device in their pockets, this time embracing the physical light rather than running away from it.

Reviewed by: Trang Nguyen

Unlocking the Mysteries of Muscle Contraction: A Breakthrough in Ryanodine Receptor Structure

Ever wonder how our muscles work when we move? It all comes down to calcium—a key player, released from storage spaces within our muscle cells called the sarcoplasmic reticulum (SR), that regulates the muscle contraction. The crucial protein allowing calcium release is the type 1 ryanodine receptor (RyR1), an ion channel of enormous proportions (the largest known so far) that is embedded in the SR membrane.
RyR is more than just a protein; it’s a critical piece of the puzzle in understanding diseases like muscular dystrophy and heart failure. Plus, RyR serves as a potential pharmacological target, opening avenues for therapeutic interventions in muscle-related disorders.
Yet, working with such bulky proteins in its native state – within the cell membrane- is a challenging subject. Typically, the steps needed to resolve the structure of proteins normally involve using detergents and harsh conditions that disrupt the native state of a protein.
However, why is it crucial to comprehend the structure of proteins in their native state?? Because it enables us to mimic the conditions in living cells and gain valuable insights into their physiological relevance, their interaction with other molecules, exploring disease mechanisms, and facilitating drug development, among others.
So far, it has been used cryogenic electron microscopy (cryo-EM), an imaging technique that freezing samples and using electrons instead of light to reveal the protein’s intricate 3D structure (i.e., protein’s shape, how its various parts are arranged, and the interactions between its amino acids). While the 3D arrangement of the RyR1 protein had been solved at a very detailed level (referred to as the “High-resolution structure”), the configuration in its native state was still unknown.
In the pursuit to unravel the structure of proteins within native or near-native membranes, a recent breakthrough has provided a high-resolution view of RyR1.
Departing from traditional detergent-based methods used to solubilize and stabilize membrane proteins, Dr Melville Zephan, CUIMC postdoc, and colleagues adopted a gel-filtration approach with on-column detergent removal, they’ve captured RyR1 within liposomes—tiny bubbles mimicking cell membranes (Figure 1).

Figure1. Flow diagram illustrating protein-incorporated liposomes.
RyR1 from rabbit skeletal muscle was homogenized and purified using ion-exchange chromatography, a technique that separates compounds based on their electric charge. Liposomes, which are tiny vesicles made of self-assembly of lipid bilayers in water, were formed using gel filtration. This process helped incorporate RyR1 into liposomes and remove any excess or empty liposomes. (Adapted from Melville Z. Cell Press 2021)

This novel technique maintains a more natural environment, allows to purify membrane proteins and remove detergent molecules in excess, offering a clearer glimpse into how RyR1 functions (Figure 2).

Figure2. RyR1 representation in native and near-native membranes incorporated in liposome after gel filtration. (Adapted from Melville Z. Cell Press 2021)

Their study reveals how RyR1 behaves in its natural environment, forming a network of channels within cell membranes. While their investigation in liposomes gives only a partial view of this complexity (without seeing how molecules interact), it sets the stage for future investigations. This approach, extending beyond RyR, offers a blueprint for studying membrane proteins in native conditions.

In summary, this work successfully captured the intricate structure of RyR1 in liposomes opening doors to a better understanding of muscle physiology and offers a sharper lens for exploring the complex world of membrane proteins.

For more details on this research, refer to the article: “High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor”.

https://www.sciencedirect.com/science/article/pii/S0969212621002963?via%3Dihub

Reviewed by: Trang Nguyen, Carlos Diaz, Erin Cullen

 

“super-SOX” hits iPSC research out of the park

The human body contains trillions of cells, which have extremely diverse functions. Remarkably, every one of these cells has the same genetic code. This diversity is possible because of the developmental process of differentiation. As an organism grows, cells take on traits that are useful in a particular context. For example, skin cells produce a protein called keratin that makes skin tough in order to protect the rest of the body from the outside world. Other cells don’t produce keratin because it would hinder their function. Once cells have differentiated and taken on a particular role, they can only generate more of the same kind of cell – skin cells only divide into more skin cells. This is because sections of DNA are hidden away during differentiation and stay hidden after the cell divides, ensuring that all the cells in a particular organ (like the skin) act like they’re supposed to.

To turn one type of differentiated cell into another was once thought to be impossible. The only cells that can generate any type of cell in the body are pluripotent stem cells (PSCs). PSCs have many potential applications in science, medicine, and industry, including disease modeling and drug testing as well as cell-based therapies and biotechnology. The opportunity to study PSCs can also enable scientists to answer basic questions about mammalian development. 

In 2006, Japanese researchers Takahashi and Yamanaka discovered that PSCs actually can be created from differentiated cells. Doing so requires just four key proteins: Oct4, Sox-2, Klf4, and cMyc. These proteins together are referred to as the Yamanaka factors. The discovery that the Yamanaka factors could turn a differentiated cell into a PSC was incredibly exciting to the scientific community, earning Dr. Shinya Yamanaka and a colleague the Nobel Prize in Physiology or Medicine in 2012.

Although this discovery created a broad array of new possibilities in science, it turns out that making PSCs in a laboratory is tough. Making healthy, high-quality PSCs from different species is even tougher. Induced pluripotent stem cells (iPSCs) have only been generated from a few species, and some cell lines create healthier iPSCs than others. A clearer understanding of how the Yamanaka factors interact with DNA and each other could reveal ways to make iPSC generation more robust and consistent, and realize the exciting potential of iPSCs. 

This challenge was taken on by a research group led by Drs. Sergiy Velychko and Caitlin MacCarthy in collaboration with CUIMC postdoc Dr. Vikas Malik and other teammates around the world. The group published a study this month in Cell Stem Cell describing a new protein based on the Yamanaka factor Sox2. They aptly call this protein “super-SOX” based on a number of studies demonstrating its ability to better generate iPSCs. The experiments also uncovered basic principles of pluripotency and development.

While three of the Yamanaka factors can be replaced with other proteins from the same family to generate iPSCs, Oct4 is the only member of its family (the POU family) that can induce pluripotency. The researchers set out to understand this oddity, with the goal of revealing the molecular interactions that lead to pluripotency. They first found that while a different member of the POU family could not generate iPSCs with the Yamanaka factor Sox2, it could generate them with a mutant protein based on another member of the SOX family, Sox17. 

Next, the researchers created a library to test different Sox2-Sox17 chimeras – mutant proteins that include components from both Sox2 and Sox17. Amino acids are the most basic building blocks of proteins, and surprisingly, the researchers found that a chimera with only one different amino acid from Sox2, Sox2AV, stabilizes the interaction between Sox2 and Oct4 and increases DNA binding efficiency. Oct4 is distinguishable from other members of the POU family by a negative charge in its “linker” domain. This negative charge allows Oct4 to form “salt bridges” connecting it to Sox2. Swapping the Sox2 amino acid alanine for the Sox17 amino acid valine promotes the formation of these bridges, encouraging Sox2AV and Oct4 to interact and bind to DNA together. The stronger connection between these two proteins dramatically improved pluripotency. 

Photo of healthy adult mice generated entirely from iPSCs and schematic of how outcomes change dramatically after replacing Sox2 with Sox2AV (Adapted from McCarthy et al., 2024).

The researchers hypothesized that the improvements in DNA binding they observed could promote the development of iPSCs into mature organisms. Live mice and rats have previously been generated from iPSCs, but these animals rarely survive to adulthood. Replacing Sox2 with Sox2AV – a simple change of one amino acid – when generating iPSCs dramatically increased mouse survival. 10/10 different cell lines treated with Oct4, Sox2AV, Klf4, and cMyc were able to produce live all-iPSC mouse pups, which only 3/8 cell lines treated with all the traditional Yamanaka factors were able to achieve. In the best-performing Sox2AV line, 43.3% of all-IPSC mice became healthy adults, compared to only 15.2% from the best-performing Sox2 line.

A further study found that a more complex chimera of Sox2 and Sox17 (Sox2-17) could improve iPSC generation in five different species: mice, cows, pigs, monkeys, and humans. These species were chosen for their potential to bring iPSC technologies to either biomedical research or industry – for example, iPSCs from livestock species could be key to producing lab-grown meat. Non-human primates and livestock species are less established in iPSC research, but the invention of Sox2-17, or “super-SOX”, opens many doors for future uses of iPSCs from these species. 

One barrier to creating iPSCs from human cells is age – differentiated cells from adults are highly resistant to becoming iPSCs. Strikingly, the group found that although the traditional Yamanaka factors could not produce iPSCs from skin cells of certain Parkinson’s disease patients, replacing SOX2 with the chimeric SOX2-17 could produce iPSCs from these samples.  Parkinson’s disease is characterized by the selective death of neurons in a brain region important for motor coordination called the substantia nigra, and could theoretically be treated by replacing lost neurons with iPSCs differentiated into neurons with a particular patient’s genetic code. The finding that SOX2-17 improves iPSC generation in cells from Parkinson’s disease patients demonstrates an exciting potential medical application for this discovery. 

The paper goes on to provide an explanation for these exciting findings. Prior to this study, it was known that although Oct4 is necessary for inducing pluripotency, too much Oct4 impairs iPSC generation. Free Oct4 encourages proliferation, which is actually detrimental to inducing pluripotency. Here, the researchers found that the stronger bond between Oct4 and Sox2AV or Sox2-17 lowers the proliferation rate, which improves the health of the cells that are generated. They went on to show that the bond between these two proteins is a main driver of the process of reverting a differentiated cell to a “naive” iPSC, which broadens the possibilities for the cell’s future development. 

This study makes a significant practical step toward realizing the potential of iPSCs. Simultaneously, it answers longstanding questions regarding how exactly the Yamanaka factors induce pluripotency, showing that the interaction between Oct4 and Sox2 has a central role. These findings have exciting implications for iPSC research, a field at the very forefront of science and medicine. 

Reviewed by: Trang Nguyen, Giulia Mezzadri, Carlos Diaz, Vikas Malik



Revolutionizing Cancer Immunotherapy: Unveiling Novel Biomarkers through Longitudinal High-Parameter Spectral Flow Cytometry

Immunotherapy, a groundbreaking approach to treating diseases by harnessing the body’s immune system, has emerged as a cornerstone in cancer treatment. In contrast to chemotherapy, immunotherapy offers a less toxic alternative for cancer patients. Chemotherapy indiscriminately targets both cancerous and healthy cells throughout the body, whereas immunotherapy specifically focuses on attacking cancer cells. However, the challenge lies in the rarity of responses to immunotherapeutic agents, their limited efficacy across tumor types, and the unpredictable nature of outcomes.

Tumor-infiltrating lymphocytes (TILs), immune cells present in and around tumors, signify an active immune response against cancer. The presence of TILs often correlates with improved patient outcomes, making the identification of predictive biomarkers a crucial pursuit in tumor immunology. Yet, achieving this goal remains a formidable challenge.

Key players in the immune system are white blood cells, produced in the bone marrow, patrolling the body for foreign invaders. Lymphocytes (T cells, B cells, and Natural Killer cells), neutrophils, and monocytes/macrophages constitute the most common types of immune cells.

To address the need for biomarkers to guide immunotherapy, a research group led by Dr. Benjamin Izar at Columbia University Medical Center has pioneered a cutting-edge approach. They developed a 34-parameter spectral flow cytometry panel and an advanced data analysis pipeline to explore protein-level immune phenotypes across different cancer phases. Conventional flow cytometry uses dichroic mirrors and band pass filters to select specific bands of the optical spectrum for detection using point detectors. Unlike conventional flow cytometry, spectral flow cytometry captures the entire spectral profile of fluorophores, allowing for a more comprehensive understanding of immune responses (Figure 1). This innovative method enhances signal resolution by subtracting cellular autofluorescence, showcasing its potential to uncover crucial insights into cancer immunotherapy. This allows the use of more existing fluorophores that would otherwise be incompatible on a conventional flow cytometer and the expansion of immunophenotyping panels beyond 40 fluorescent parameters.

Figure 1: Comparison of traditional and spectral flow cytometry detection mechanism. Images from FluoroFinder.

In this groundbreaking study, researchers meticulously profiled various tissues affected by prostate and colorectal cancers in mice undergoing anti-PD-1 immunotherapy. Their investigation revealed a significant correlation between the expression of KLRG1, recognized as a coinhibitory receptor on T cells and NK cells, and tumor-related factors such as burden, progression, and regression in response to anti-PD-1 treatment. KLRG1 emerged as a potential marker indicative of terminal differentiation and/or senescence, influencing key cellular pathways and checkpoints.

The longitudinal high-parameter spectral flow cytometry approach employed in this research showcased its prowess in extracting novel targets and biomarkers from dynamic ‘temporal atlases’ of antitumor immunity. This innovative methodology holds the promise of unraveling the intricate, ever-changing interactions within the tumor microenvironment. By providing a deeper understanding, it aims to enhance the efficacy of immunotherapy in clinical applications. The study particularly emphasizes the need for further exploration of KLRG1+ CD4 T cell subsets as potential targets or prospective biomarkers for advancing cancer immunotherapy.

For more details on this research, refer to the article: “KLRG1 Marks Tumor-Infiltrating CD4 T Cell Subsets Associated with Tumor Progression and Immunotherapy Response.”

Written by: Trang Nguyen

Reviewed by: Erin Cullen

Take a look in the past to foresee the future: the evolution of genome engineering

Transposons, also known as mobile genetic elements, are segments of DNA capable of moving within the genome. This mobility can potentially induce mutations, as transposon “jumping” within a gene may interrupt the gene and trigger its loss. Transposons utilize their own enzymes, transposases, to move in and out of the genome.

Due to the potential harm caused by transposons, cells continually evolve strategies to control them. In response, transposons adapt, arming themselves with new strategies to evade cellular control. Mobilized transposons can hijack adjacent DNA information and transfer it to a new location, sometimes involving a gene that provides a survival advantage, thus increasing the transposon’s chances of propagation. Simultaneously, cells “steal” genes from transposons, repurposing them to function in processes advantageous to the cell. This ongoing arms race between transposons and host defense systems has facilitated a widespread exchange of genes between different life forms and is a major driver of genome evolution across all domains of life. Transposons play a crucial role in this process.

In bacteria, the CRISPR-Cas9 system is fundamental for protection against viral infections. Using a small RNA molecule called guide RNA, the CRISPR-Cas9 system acts as a nuclease, akin to DNA cutting scissors, destroying the viral genome. This system can be modified to edit genes in organisms beyond bacteria. The guide RNA is altered to direct the Cas DNA scissor to a gene of interest instead of a viral genome. This programmable gene-editing system has diverse applications, from basic biology research to disease treatments, and it earned the 2020 Nobel Prize in Chemistry for Emmanuelle Charpentier and Jennifer Doudna. Notably, scientists have recently traced the evolutionary origins of CRISPR-Cas9 to transposons.

In a recent study published in Nature, Columbia postdoc Chance Meers and his colleagues provided further insight into the evolution of the CRISPR-Cas9 nuclease system by examining how transposons use cellular nucleases to proliferate within genomes. They focused on a specific transposon system called insertion sequences (IS), which are bacterial transposons encoding only the genes necessary for transposon excision and movement to a new genomic location. This includes a transposase. However, many IS transposons contain an accessory gene that carries the information for a nuclease which functions similarly to the CRISPR-Cas system as an RNA-guided DNA cleaving enzyme.

The IS element does not need a nuclease for its movement, the transposase is sufficient to mobilize the transposon to a new location. So why are so many of the IS elements carrying those RNA-guided DNA scissors? While the mechanism by which IS elements are mobilized by their transposase is similar to a cut-and-paste process, leaving no copy at the original DNA location, Meers and colleagues discovered that the rate of excision is more efficient than the rate of integration. Without an additional system for transposon proliferation, the transposon would eventually be lost since not every excised copy manages to be reintegrated. The study of the authors revealed that the accessory CRISPR-like functioning nuclease guides a copy of the IS element back to its original location, generating two copies of the element—one at the original site and one at the newly inserted site. This changes the mechanism from cut and paste to cut and copy when the transposase function is complemented by the accessory nuclease, increasing the transposon copy number present in the genome, and thus serving for the transposon selfish proliferation (Figure 1).

Figure 1. RNA-guided nucleases assist transposon survival via guiding specific breaks at donor sites and transposon restoring repair. During DNA replication the two strands of the DNA duplex are separated and each strand serves as a template for the synthesis of a new DNA molecule. Single stranded DNA facilitates the transposition mediated by the transposase of IS elements. The excision of the transposon leads to its loss at one of two DNA strands, while DNA replication restores the transposon on the other DNA strand. Simultaneously, transposon excision restores a target site at the transposon donor location which is specifically recognized by a guide RNA (blue segment). This guide RNA directs the accessory nuclease on the excision location and DNA cleavage occurs. The resulting double strand break (DSB) is lethal for the bacteria and its only chance of survival is to copy the information from the homologous newly synthesized intact DNA molecule that contains the transposon. This leads to reconstitution of the transposon at the donor site on the two newly synthesized DNA duplexes. Transposition at the new target sites will produce new guides, specific for the new insertion site location (orange segment), which will facilitate the future transposon spread and maintenance by identical mechanism. 

By developing powerful assays to track the movement of transposons within bacterial genomes, or other small DNA molecules called plasmids, as well as from one bacterium to another, Meers and his colleagues uncovered how those RNA-guided DNA cutting nucleases work. The authors’ discovery enhances our understanding of how proteins collaborate with RNA guides to target and edit genomes. In addition, the study unveils the original role of CRISPR’s nucleases from an evolutionary perspective before they were repurposed by the bacterial host genome to fight viruses, which was to serve the selfish propagation of transposons. Moreover, given the abundance of transposons in genomes, it is highly likely that other systems different from CRISPR-Cas9 and derived from transposon genes exist, waiting to be discovered and potentially harnessed, expanding the biotechnological tools available for programmable and specific genome engineering. Thousands of ancient transposons in bacterial genomes carry RNA-guided DNA nucleases that can potentially be programmed to cut DNA similarly to the CRISPR-Cas9 system.

Reviewed by: Trang Nguyen, Giulia Mezzadri

Much Ado About Sleep: the Importance of Getting Adequate Sleep

How was your sleep last night? Were you able to go to bed early and clock in a solid 7 to 9 hours of sleep, or were you tied up with work and had to postpone your bedtime till the draft was written and the email sent?

On average, we human beings spend 8 hours per day sleeping, which amounts to a total of one-third of our lifetime. That is, for a person who spends 75 years on earth, 25 years is spent sleeping. With such a significant amount of time spent in bed, one couldn’t help but wonder, what would happen if we just shift the bedtime later by an hour and a half, while keeping the wake-up time fixed? Surely it will not have much of an impact?

Well, turns out it will.

In a recent study published in Scientific Reports, Columbia postdoc Vikas Malik and colleagues found that for healthy females, after cutting sleep short by an hour and a half for a mere six weeks, cells that protect our blood vessels become negatively impacted. Evidence has shown that sleep restriction introduces a more pronounced risk for females than males, leading the researchers to focus solely on females for this study. With this reduction in sleep, the amount of harmful molecules (oxidants) starts to overpower that of the good molecules (antioxidants) in the cells. Since oxidants are harmful molecules which are responsible for producing the notorious “free radicals” and causing toxic effects in the body, and antioxidants good molecules responsible for detoxifying the body and repairing damages made by oxidants – this imbalance between the amount of oxidants and antioxidants will lead to impaired cell function and detrimental consequences in the form of cardiovascular diseases.

While the significance of sleep is well known, the mechanisms of how lack of sleep could impact human health has remained largely unclear. In fact, this study provides some of the first evidence demonstrating how mild chronic sleep deprivation could impact the health of our heart. In addition, contrary to previous studies that mainly examine sleep deprivation conditions in a compact time frame (e.g., acute sleep deprivation in flies for 10 days), the authors investigated how mild sleep restriction influences vascular cell health in females (i.e., pushing the bedtime 1.5 hours later than what the participants used to, while keeping the wake-up time fixed over the span of 6 weeks). Owing to a more “modern” work/life balance, the results from the authors’ experimental design carry higher relevance to our sleep patterns.

Specifically, the researchers found that after adequate sleep, the imbalance between oxidant (harmful) and antioxidant (good) molecules in the vascular cells can be cleared by a functional antioxidant response. This response is facilitated by a protein called serum response factor (SRF) – when sleep is adequate, the expression level of SRF increases to bring up the expression level of DCUN1D3, another protein which mediates antioxidant response of the cell. After sleep restriction, however, the antioxidant response cannot be turned on because the expression level of SRF is not adequate enough to activate a sequence of transcription of antioxidant genes, therefore hindering the restoration of balance between oxidant and antioxidant molecules in the cells (see illustration below).

Functional vs. impaired antioxidant response under adequate and inadequate sleep. Adequate sleep increases the expression level of SRF, which in turn increases the expression of DCUN1D3, freeing Nrf2 into the nucleus of the cell to activate the transcription of antioxidant genes, which then restores balance between oxidant and antioxidant molecules in the cells. Inadequate sleep, however, perturbs this process and interferes with the activation of antioxidant response. Figure is from the original publication.

These findings carry significant implications on how we should schedule our sleep. While deadlines will always be looming and stress will always be present, maybe we should still make the effort to defend our bedtime. After all, as Shakespeare puts it, sleep is the “balm of hurt minds, great nature’s second course, chief nourisher in life’s feast”. Perhaps, unlike Macbeth, we should all strive to sleep more.


Written by: Linbi Hong

Reviewed by: Vikas Malik, Trang Nguyen, Martina Proietti Onori, Giulia Mezzadri, and Patricia Cooney

 

When mitochondria get stressed, our brain suffers: Linking mitochondrial dysfunction to neurodegeneration.

Imagine It’s a Saturday morning and you decide to drive upstate for a nice hike. While on the trail, you have a too-close encounter with a too-curious bear, leaving you panting, perplexed. This acute stress response (the famous fight-or-flight response) is extremely useful for us to better react to danger. The main hormones released during a stress response, glucocorticoids (GC), help prepare our body for the adverse situation we may encounter, giving us a better chance of survival.

Chronic stress it’s not so beneficial for us. In fact, it has profound detrimental effects on our bodies, and especially in our brain. There is increasing evidence that it can make our mitochondria (the powerhouse of the cell) function poorly, which can lead to damage in our neurons. Another way chronic stress can damage the brain is by inducing accumulation of the protein Tau, which can lead to neurodegenerative diseases such as Alzheimer’s disease. Tau itself poses an interesting biological conundrum: While it is normally found in the neuron’s microtubules (the cell’s scaffolding) in a healthy setting, it can start to form clumps (i.e, oligomerize) when a neuron becomes stressed or damaged, interfering with the neuron’s basic functions such as synaptic transmission and protein transport.

Scientists are still unsure how exactly GC cause Tau to oligomerize. Furthermore, while the impact of GC in mitochondria’s fitness is pretty well studied, the mechanisms driving such dysfunction, and its downstream consequences, are still poorly understood. Overall, to the scientific community, the molecular mechanisms linking GC exposure, mitochondrial dysfunction, and Tau pathology, are still unclear.

 In a recent study published in Brain, Columbia postdocs Dr. Fang Du and Dr. Qing Yu and colleagues investigated the causal relationship between these three important components of brain health, and found that GC directly precipitate mitochondrial dysfunction and drive Tau oligomerization. In a series of elegant experiments in both mouse models (in vivo) and neurons grown in petri dishes ( in vitro),experiments, the group led by Dr. Clarissa Waites demonstrated that found the mechanism by which GC exposure drives mitochondrial dysfunction and Tau oligomerization, and even discovered a potential treatment.

Figure 1- Graphical representation of the findings by Dr Du, Dr Yu, and colleagues. During Chronic stress, Glucocorticoids induce mitochondria dysfunction by increasing its permeability, which turn promotes Tau oligomerization and neurodegeneration. Figure created with Biorender.

 

 First, Dr. Waites’ group established the role of chronic GC exposure in mitochondrial fitness and pathogenic Tau accumulation in vivo. Using the widely used synthetic glucocorticoid dexamethasone (DEX), they demonstrated that GC induce mitochondrial dysfunction (increased permeability and lower respiratory capacity) and oligomeric Tau accumulation. Next, Drs. Du and Yu very cleverly employed in vitro experiments to demonstrate that DEX triggered mitochondrial dysfunction by increasing mitochondrial permeability: their research showed that GC exposure stimulates the opening of the mitochondrial permeability transition pore, a big, non-discriminant, pathological hole in the mitochondrial membrane that when open, greatly compromises its function. Most importantly, they found that GC triggers opening of the pore by activating its key component, the protein cyclophilin D. This key finding led Dr. Waites’ group to block cyclophilin D activation, either genetically (i.e. changing the DNA of the protein) or using drugs, with great success: cells where cyclophilin D activation was prevented were remarkably resilient to GC stress, and showed much lower levels of mitochondrial dysfunction and pathological Tau accumulation.

 Although this was an extremely exciting result by itself, it posed a problem moving forward: neither genetical nor pharmacological inhibition of cyclophilin D are currently viable therapeutic approaches, due to their poor bio-availability or invasive methods required to deliver the treatments. So what other approach could they use to also prevent mitochondrial pore opening, while still being clinically tractable? After some digging, Drs. Du and Yu found apo-cyanin, an orally-available inhibitor of mitochondrial respiration that could tackle GC-driven brain pathology while not directly targeting cyclophilin D. In line with their predictions, this drug was able to restore mitochondrial function and prevent Tau pathology in DEX-treated cells. Most importantly, they were also able to restore neuronal health, neuronal connectivity, and prevent anxious and depressive behavior associated with GC treatment.

 All of these experiments clearly demonstrated that GC-exposure triggers mitochondrial dysfunction and Tau accumulation, which could in turn promote the development of brain pathologies. How could they demonstrate, however, that targeting mitochondrial permeability is a feasible approach to treat common brain pathologies such as Alzheimer’s disease? To tackle this question, Dr Waites’ group made use of an ingenious technique, where they replaced all mitochondria in a test cell with those coming from an Alzheimer’s disease patient. Excitingly, in this model, treatment with mito-apocynin was sufficient to partially reverse mitochondrial dysfunction and Tau oligomerization found in the cells containing mitochondria from Alzheimer’s patients.

 Dr Waites’ group story is a hallmark out-of-the-box thinking, and clinical relevance. While their research clearly highlights mitochondrial damage induced by GC and cyclophilin D activation as a key trigger of Tau accumulation, the implications of this finding could be much broader:  How generalizable is this mechanism to other forms of brain disease such as ischemia, inflammation, or aging? As a matter of fact, cyclophilin D levels are elevated in Alzheimer’s patients, and its inhibition is protective in mouse models of sclerosis, Parkinson’s, and Alzheimer’s disease.

While this story provided incredible insight into the role of mitochondria in controlling brain pathology, many other questions remain unanswered.  For example, What is the exact mechanism by which mitochondrial dysfunction promotes pathogenic Tau accumulation? It is highly likely that these two events are intertwined, and that oligomeric Tau can also drive mitochondrial dysfunction.

 Despite the still-unknowns, it is clear that after this publication from Dr Waite’s lab, we should re-contextualize our middle-school textbooks: The mitochondria is the powerhouse of the cell, and during stress, the gate-keeper of our brain’s health.

Reviewed by: Trang Nguyen, Erin Cullen 

Make new friends or keep the old? A novel brain circuit explains social novelty preference in mice

Researchers have discovered a pathway in the brain that may dictate whether we decide to interact with a friend versus a stranger. “Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum suppresses interaction with familiar mice”, was published this month in the highly regarded journal Cell. The paper emerged from a collaboration between scientists at the Zuckerman Mind Brain & Behavior Institute at Columbia University and colleagues at the University of Washington in Seattle, Washington and the Instituto de Neurociencias CSIC-UMH in Alicante, Spain. Columbia postdoc Ramon Nogueira contributed to the work. 

A diagram demonstrating Social Novelty Preference in mice. Two mice are in enclosures on opposite sides of an arena. A third mouse, free to explore the arena, chooses to spend more time with a mouse it does not know than a mouse it has met before.
Fig. 1 – Diagram of the task to assess Social Novelty Preference in mice. A mouse with typical social memory and an intact connection between CRH-releasing neurons in the ILA and CRH-receiving neurons in the rLS will spend more time interacting with the novel mouse than the familiar mouse. (Adapted from de León Reyes et al., 2023, Figure 3)

Social novelty preference in mice was first described in the scientific literature 20 years ago, and it is regularly used to assess social memory for research purposes. A typical mouse, when presented with a mouse it knows and a stranger, will spend more time with the familiar mouse (Fig. 1). Until this paper was published, it was unclear what brain regions or pathways influence whether mice choose to interact with novel or familiar mice. Scientists theorized that the lateral septum, a relay center that connects regions of the brain associated with thinking and memory, and the prefrontal cortex, which controls decision-making, might play a part in the social novelty preference. Both regions are also known to be involved in social behavior, and evidence suggested that there may be a neural pathway leading from the infralimbic area (ILA) in the prefrontal cortex to the rostral lateral septum (rLS), a specific region within the lateral septum. The researchers set out to determine how these two regions interact with each other and whether this interaction plays a role in social novelty preference. 

Using a comprehensive and elegant set of experiments to manipulate neuron function in the two regions, the researchers discovered that a population of inhibitory neurons in the ILA connects to the rLS, and that this pathway is crucial for the mice to exhibit the typical social novelty preference. The ILA neurons contain the neurotransmitter corticotropin-releasing hormone (CRH), which is best known for its role in stress and anxiety. Neurons in the rLS have receptors for CRH, but it was previously not known that CRH-containing neurons in the ILA communicate with CRH-receiving neurons in the rLS. When the researchers inactivated the CRH-containing neurons in the ILA, they found that mice failed to exhibit the typical social novelty preference. Next, they asked whether the CRH-receiving neurons in the rLS were also necessary for social novelty preference. They found that removing the CRH receptors from these neurons erased the social novelty preference, just like inactivating the CRH-releasing ILA neurons. Finally, they recorded the activity of CRH-releasing ILA neurons and saw that these neurons were more active when interacting with a familiar mouse than a novel mouse, indicating that these neurons act on the rLS to discourage interactions with familiar mice. 

Graphic showing a pathway through which corticotropin-releasing hormone is sent from the infralimbic area to the rostral lateral septum of the mouse brain. When this pathway is active, mice spend more time with novel rather than familiar mice. When this pathway is inactive, mice spend more time with familiar rather than novel mice.
Fig. 2 – Graphical Abstract of “Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum suppresses interaction with familiar mice” by Noelia Sofia de León Reyes et al., 2023.

To further probe the relevance of this pathway to social dynamics, the researchers assessed whether CRH may be responsible for the social novelty preference appearing in young mice. While adult mice prefer to interact with novel mice, mouse pups less than two weeks old prefer to interact with their mothers and littermates rather than unknown adult females or pups from other litters. During this same developmental time period, the population of CRH-releasing neurons that connect to the rLS steadily grows. When CRH was depleted from these neurons, the young mice failed to exhibit the typical shift from a preference for social familiarity to a preference for social novelty. This may be an evolutionary mechanism that encourages young mice to stay in safe, familiar environments, then emboldens adult mice to explore novel environments, collect food, and procreate. 

While the social novelty preference in mice does not have a direct analogue in humans, this research may provide clues for understanding and treating social anxiety disorders and social phobias. The human prefrontal cortex and septal regions also respond to social information, and CRH is known to be involved in anxiety disorders including social phobias. The researchers suggest that low CRH levels in the prefrontal cortex may prevent people from seeking social novelty, and that this mechanism could be responsible for social anxiety disorders. The field of neuroscience is just beginning to understand the biological underpinnings of social interaction, and new methods give scientists ever expanding ability to learn about this exciting field. 

Reviewed by: Trang Nguyen and Martina Proietti Onori

When Numbers Play Tricks: Unraveling The Brain’s Biases

Imagine living in a dark cave, with your entire understanding of the world based on shadows on the wall. Sounds unrealistic and terrifying, right? However, this allegory presented by Plato is an apt metaphor for our brain’s perception of the real world. While we might believe we perceive reality in its entirety, our brain can only provide a shadowy representation of the variables in our environment, and our decisions are based on these shadowy representations.

A comforting thought might be that numbers are a universal language for Western countries and, specifically, for those who use Arabic numerals. For instance, when the price of a good is marked as $14, it conveys an unambiguous and specific value, meaning that one would unequivocally expect to pay exactly that amount. However, experiments show that people make mistakes when dealing with Arabic numerals. For instance, under time constraints, the closer two numbers are in value, the more challenging it becomes for us to rapidly and accurately pinpoint the larger one. These mistakes are very similar to those made in psychophysics tasks involving physical stimuli, such as comparing the length of segments or averaging the orientation of tilted lines. These results, together with neurobiological studies, suggest the existence of a representation system for numbers that is similar to how we interpret physical stimuli.

A popular idea in theoretical neuroscience is that while the brain’s computational abilities have inherent limits, leading to imprecise representations, these representations are optimal within those constraints. This theory, called “efficient coding”, suggests that our brain’s perceptions are influenced by how often these magnitudes are encountered (i.e., the prior). For example, vertical and horizontal orientations are perceived with more clarity than oblique ones, likely because they’re more common in our environment.

A recent study (https://www.nature.com/articles/s41562-022-01352-4), led by Columbia postdoc Arthur Prat-Carrabin and published in Nature Human Behaviour, delves into whether our brain treats numbers the same way it treats physical stimuli. In their experiment, participants were asked to determine which series of numbers, red or green, had a higher average value. For instance, as shown in Figure 1, the number 79.60 would flash in red on the computer screen, followed by 44.92 in green, and so forth. Participants were tasked with rapidly and intuitively calculating the average of the red and green numbers to determine which sequence had the greater average. To investigate how the frequency at which numbers were encountered impacts their representation, numbers were drawn from different distributions: one, in which smaller numbers were more frequent (Downward prior); another, in which all numbers had equal chances (Uniform prior); and a third, in which larger numbers were more frequent (Upward prior).

Figure 1: Example trial of the task. Participants were presented with 5 red and 5 green numbers and had to choose the color with the higher average.

To analyze the participants’ decisions, the researchers compared their answers with multiple computational models characterized by two components: first, whether or not the numbers are encoded with a bias that depends on the value of the number, and second, whether or not the imprecision (the noise) with which numbers are represented varies with the number. Their results showed that the model that best aligned with the participants’ answers had to include both components. Notably, less common numbers are fuzzier in their perception. So, when using the Downward prior, bigger numbers are encoded with more noise, while with the Upward prior, the smaller numbers are the noisiest ones.

This discovery not only supports the “efficient coding” theory, which posits that the brain encodes and represents information in the most efficient way possible, but also showcases its applicability beyond just physical perceptions. Whether we’re assessing the speed of a car, the talent of a dancer, or the sweetness of a cake, our brain might use a universal mechanism to represent these variables. This mechanism dynamically adjusts to the statistical distribution of numbers that are expected or experienced, which implies that our understanding of numbers and magnitudes isn’t static but can be influenced by our prior experiences and expectations. In the near future, we might be able to design environments that help people refine their perceptions (such as by crafting digital games to enhance the consumer’s responsiveness to certain prices), allowing them to better discern specific value ranges and improve their decision-making.

Reviewed by: Emily Hokett, Trang Nguyen, Martina Proietti Onori

 

Revealing How Cancer Cells Balance Immune Signals

The immune system constitutes a complex network of biological processes that serves to safeguard organisms from various diseases. An important role of the immune system is its ability to distinguish between normal cells in the body and foreign cells such as germs and cancer cells. This discrimination empowers the immune system to target foreign agents while preserving normal cellular integrity. If the immune system malfunctions, it mistakenly attacks healthy cells, tissues, and organs which is called autoimmune disease. These attacks can affect any part of the body, weakening bodily function and even turning life-threatening. Therefore, it is very critical to maintain a balance between combating tumor cells and avoiding autoimmune disease in the body. The immune system maintains such balance by using checkpoint proteins on immune cells. The checkpoints act like switches that need to be turned on (or off) to ensure an optimal immune response. However, cancer cells sometimes find ways to use these checkpoints to avoid being attacked by the immune system.

Medicines known as monoclonal antibodies can be designed to target these checkpoint proteins. These drugs are called immune checkpoint inhibitors. Checkpoint inhibitors don’t kill cancer cells directly, but they help the immune system to better find and attack the cancer cells in the body. Immune checkpoint inhibitors such as anti-PD-1 antibodies, are now used to treat some types of cancers such as metastatic melanoma or brain tumors due to their ability to boost the immune response against cancer cells. It normally acts as a type of “off switch” that helps keep the T cells from attacking other cells in the body. It does this when it attaches to PD-L1, a protein on some normal (and cancer) cells. When PD-1 binds to PD-L1, it basically tells the T cell to leave the other cell alone. Some cancer cells have large amounts of PD-L1, which helps them hide from an immune attack (Figure 1).

 

Figure 1: The interaction between tumor cells and T cells. Tumor cells contain PD-L1 receptors that bind to PD-1 in T cells to hide an immune attack from T cells (left panel). However, through the application of an immune checkpoint inhibitor (anti-PD-L1 or anti-PD-1), this binding is hindered, empowering T cells to eliminate tumor cells (right panel). Image is created via Biorender.

The Izar group at Columbia University Medical Center, led by Patricia Ho identified a mechanism by which CD58 loss or downregulation contributes to cancer immune evasion and immune checkpoint inhibitor (ICI) therapy. CD58 is known as an adhesion molecule facilitating the initial binding of effector T cells. By sequencing the whole genome of patient samples, specifically those who underwent anti-PD-1 antibody treatment and later developed drug resistance, they illuminated the role of CD58. Notably, CD58 facilitates early T-cell infiltration into tumors within a tumor differentiation. Moreover, the study shed light on CD58 loss potentially expediting tumor progression by upregulation of PD-L1, consequently intensifying resistance to ICI therapies.

To better understand the CD58/PD-L1 co-regulation, the research group performed a genome-wide knock out screen to identify the regulator of CD58. Among the top hits of cell population with low CD58 protein, authors identified CMTM6 as an important regulator for the T cell activation and antitumor responses. CMTM6 binds to CD58 and promotes its recycling from cell surface to endosomes, thus preventing its lysosomal degradation (Figure 2). Interestingly, CMTM6 is also known as an important regulator of PD-L1 maintenance. Thus, loss of CD58 results in increased PD-L1 levels by stabilizing CMTM6-PD-L1 interactions and therefore reducing lysosomal PD-L1 degradation (Figure 2).


Figure 2: Both CD58 and PD-L1 require and directly bind to CMTM6. CD58 forms a direct binding with CMTM6, enhancing recycling and curtailing lysosomal degradation (left). CD58 knockout fosters PD-L1 up-regulation by interacting with CMTM6 (right). Figure is adopted from the original paper.

This work provides a molecular basis for clinically relevant roles of CD58 to facilitate initial infiltration into the tumor. Loss of CD58 confers resistance to immune checkpoint inhibitors which may provide important insights and inform rationale for the next generation of combinations of cancer immunotherapies.

Read more about this novel finding here: The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity.

Reviewed by: Martina Proietti Onori and Carlos Diaz Salazar Albelda

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