The inner workings of a rare childhood disease

Vanessa Sanchez was the lead author on this study. As a scientist, Vanessa is passionate about understanding the cell biology of neurons, especially in the context of pediatric neurodevelopmental – degenerative disorders. Outside of lab, you can find Vanessa tending to her garden, trying new recipes, rock climbing, or hanging out with her cat, Jiji!  

or technically,

Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency

[See Original Abstract on Pubmed]

Authors of the study: Yijing Zhou, Vanessa B Sanchez, Peining Xu, Thomas Roule, Marco Flores-Mendez, Brianna Ciesielski, Donna Yoo, Hiab Teshome, Teresa Jimenez, Shibo Liu, Mike Henne, Tim O'Brien, Ye He, Clementina Mesaros, Naiara Akizu

Neurons are special cells in our bodies that communicate with one another to help us do everyday things like eat, think and walk. Amazingly, for most healthy people, the neurons that we are born with will last our lifetimes and support us as we navigate the world. However, in some rare and unfortunate diseases, neurons die prematurely. These kinds of diseases are called neurodegenerative diseases. There are many different types of neurodegenerative disease, each targeting different groups of neurons and resulting in different symptoms. In 2014, a new and extremely rare neurodegenerative disease was discovered called SCAR20. SCAR20 was found to negatively affect newborn children by causing intellectual disability and impairing motor functions, like the ability to walk. Researchers were quickly able to identify the culprit of the disease: the total lack of a protein called SNX14. Since little is known about SNX14 and how its absence causes SCAR20, Vanessa Sanchez, a current NGG student, and her collaborators designed a study to learn more about the nature of this disease, with the hope that one day there might be a cure or treatment.

Figure 1. Key takeaways from Vanessa and colleagues’ experiments investigating the underlying causes of the SCAR20 disease.

To begin their investigation, Vanessa and her collaborators used genetic tools to remove the SNX14 protein from mice. Genetically modified mice are immensely useful in neuroscience research as they allow scientists to study the underlying causes of disease in detail. In this case, since the researchers removed a protein, the genetically modified mice are referred to as a knockout mouse model. After they generated their new knockout mice, Vanessa and her colleagues tested these mice to make sure that they had all of the symptoms that the children experienced. This was an important step in their study because they wanted to be sure that any discoveries that they make using the knockout mouse model are directly relevant for human patients. Vanessa and her colleagues compared the knockout mice to normal healthy mice and found a few convincing results (Figure 1, Healthy mouse vs. Knockout mouse). First, they found that knockout mice had a complete lack of SNX14 in their brains - the direct cause of SCAR20 in humans. Next, they found that knockout mice were smaller in size and had a structurally abnormal face - two known symptoms of SCAR20 in humans. Finally, they found that the knockout mice had worse social memory and motor ability compared to healthy mice - again, a clear-cut sign of SCAR20 in humans. Given these results, Vanessa and her colleagues were convinced that they had developed a good mouse model of the SCAR20 disease and were now able to investigate how the disease develops.

In order to gain insight into the underlying causes of the disease, Vanessa and her colleagues needed to narrow down their focus to a single brain area. In human patients, SCAR20 seems to preferentially kill neurons in a brain area known as the cerebellum. This brain area is typically thought to be involved in motor control and coordination, which might explain why SCAR20 patients have severe motor disability. Vanessa and her colleagues discovered that, just as in human SCAR20, the knockout mouse model also showed a preferential negative effect on the cerebellum of the mice. She found that both the number of neurons and the overall size of the cerebellum were reduced in the knockout mice compared to healthy mice, once again validating the model for the study of SCAR20 and identifying a key brain area to narrow in on.

At this point, Vanessa and her colleagues have all the tools they need to study the inner workings of the disease. They performed a very important experiment where they extracted neurons in the cerebellum of knockout mice before they were killed by the disease, and looked for differences compared to the neurons in the cerebellum of healthy mice (Figure 1, Healthy neuron vs. Knockout neuron). By testing various cell properties, they discovered that there was one key cell property that was disrupted in the neurons of knockout mice compared to the neurons of healthy mice. This key cell property is called lipid homeostasis, which is important for regulating lipids, the building blocks of fat, inside the cell. Despite what you may expect, fats play an essential role in cell biology. Disrupting the total amount of fats inside of the cell can be toxic, resulting in cell death. In fact, Vanessa and her colleagues discovered that knockout neurons had trouble removing fats from the cell, resulting in a build-up. They went on to show that this disruption in lipid homeostasis is most likely the root cause of neuron death in SCAR20, which underlies the known symptoms of the disease.

This important research by Vanessa and her colleagues sheds light on the inner workings of a new disease that severely impacts the well-being of newborn children. Although there is still much to learn about the nature of this disease, such as how it affects neurons in other brain areas, the findings from Vanessa’s experiments offer a strong foundation for the possible development of treatments for this debilitating disease. Finally, research like Vanessa’s is invaluable as it contributes to our basic understanding of how neurons work and what causes them to die prematurely - knowledge that is fundamental for all neurodegenerative diseases.

About the brief writer: [Brief writer’s name here]

Jafar Bhatti is a PhD Candidate in the lab of Dr. Long Ding / Dr. Josh Gold. He is broadly interested in brain systems involved in sensory decision-making.

Interested in reading more? See the full paper here!

A new method for looking through the (cyto)skeletons in the closet

Rachel Dvorak was the lead author on this study. She is interested in studying the biochemical mechanisms by which mutations in gamma smooth muscle actin cause visceral myopathy. Patients with visceral myopathy present with severe abdominal distension, intractable constipation, feeding intolerance, and growth delays. Because there are no targeted therapies for this disease, many patients die in adolescence. She hopes that by understanding how disease-causing mutations alter actin biochemistry we can develop treatments for patients with visceral myopathy and other rare conditions caused by actin mutations.

or technically,

A solution to the long-standing problem of actin expression and purification

[See Original Abstract on Pubmed]

Authors of the study: Rachel H. Ceron, Peter J. Carman, Grzegorz Rebowski, Malgorzata Boczkowska, Robert O. Heuckeroth, and Roberto Dominguez

Take a moment to picture the last evening you spent with friends. Now, think about something that happened over 10 years ago.

Throughout both of those events- in fact, throughout every day of your life- you’ve had the same exact cells living in your brain, helping you navigate each situation. These cells are called neurons, and your body can’t replace them; when they’re gone, they’re gone for good. This means it’s especially important for neurons to be well-built to withstand the everyday challenges that cells face, and well-equipped to respond to emergencies that could threaten their existence.

One contributor to neurons’ ability to last a lifetime lies in how they’re built; much like buildings that are constantly exposed to the elements, neurons need structural integrity to last a lifetime. To do this, there are several types of miniature “skeletons” that exist within the cell. Making up these miniature cellular skeletons, collectively called the cytoskeleton, are proteins. Proteins are large molecules that perform specific jobs intended to keep cells alive. One type of protein is called actin. Similar to links in a chain, actin proteins can attach to each other to form a long structure called a filament. As part of the cytoskeleton, actin filaments are important for helping neurons and many other types of cells keep their shape. Correspondingly, neurons are only as structurally sound as their components; issues within actin filament structures cause severe defects in overall brain structure. This includes lissencephaly, a condition where the brain lacks the grooves that normally sprawl across its surface, leaving it completely smooth. By understanding how actin behaves, both on its own and in the presence of other proteins, scientists hope to develop better treatments for diseases where actin isn’t acting the way it normally would.

Rachel Dvorak, a PhD candidate in Dr. Roberto Dominguez’s lab at the University of Pennsylvania, wanted to develop a robust way to study how actin works. Specifically, she was interested in studying how actin interacts with itself and other proteins- much like people, proteins can interact with each other in ways that influence their behavior in the cell. To do this, she aimed to isolate actin from cells. Isolating a single type of protein is a common method to study the way it functions. The inside of a cell is a bustling place, and it can be hard to tease out the specific interactions that proteins have with one another when they exist within that cellular environment.  

This is far from a simple undertaking. Inside of an actual cell, there are many actin proteins, and they are not all identical. Instead, there are several possible varieties of actin. One way to understand this is by comparing it to different flavors of ice cream. Although chocolate and vanilla are made of slightly different ingredients and paired with different foods, they are both still ice cream. Actin also comes in different “flavors” called isoforms. While each of their structures are slightly different, and each isoform might be best suited for use in different scenarios, they are all still considered actin due to their overall similarities and jobs in the cell. Moreover, even two actin proteins that are the same isoform can be slightly different, because the cell can modify actin by attaching other molecules to it. These attachments are called post-translational modifications, and they also influence actin’s behavior. However, despite their important effect on actin behavior, many of these post-translational modifications are usually lost during the process of isolating actin to study its behavior.

Another challenge of isolating specific isoforms of actin lies in getting cells to produce large quantities of the actin isoform of interest. Making actin is an involved, multi-step process for the cell that requires a lot of molecular machinery. Because of this, most scientists use only one type of actin isolated from muscle cells to conduct experiments outside of the cellular environment. This severely limits our ability to study whether different isoforms of actin behave in slightly different ways. It also means that oftentimes, our out-of-the-cell reconstructions of cellular events are created using actin filaments that lack much of the nuance they would have in cells. This limits the accuracy of this model and makes it impossible to study how actin that is incorrectly produced causes human disease.

To tackle this, Rachel decided to use modified human kidney cells to produce actin. This meant that the cells would already contain the machinery unique to humans that is necessary to carry out production of actin. This is in contrast with existing methods that use cells like bacteria or insect cells; these cell types can also be used to churn out proteins for isolation, but lack the machinery to make some proteins native to human cells or add post-translational modifications the way that human cells would. Rachel was able to introduce genetic instructions that caused the human kidney cells she worked with to essentially become actin-making factories, synthesizing large quantities of whichever actin isoform she was interested in.

However, the actin the kidney cells made based on the genetic instructions that Rachel provided was a bit different than any other actin in the cells; each of these actin proteins was also attached to two other man-made proteins called tags. Tags are helpful because scientists have materials that can grab onto them, allowing for the separation of the tags (and anything attached to them) from everything else in the cell. Most tags don’t occur naturally, so only the protein (or, in this case, the actin isoform) that you’ve instructed the cell to produce will have this unique feature. To further ensure she was isolating only the type of actin she wanted, Rachel also used a different protein her lab had engineered to grab and release actin based on how much calcium is in the environment. By using a combination of materials that grab the tags attached to actin in tandem with the protein that grabs onto actin itself, Rachel was able to isolate specific isoforms of actin with post-translational modifications from the rest of the contents of the kidney cells.

Rachel and her colleagues ultimately invented a new method for isolating actin exactly as it would exist in the cell. This is important because it means scientists now have a new way to study the exact things that are going awry in diseases that involve issues with interactions between actin and other proteins, such as the one that causes lissencephaly. The more we understand about how actin functions differently in diseases like this, the better our ability to develop effective treatments.

About the brief writer: Julia Riley

Julia is a PhD candidate in Dr. Erika Holzbaur’s lab studying the consequences that damaged mitochondria (the powerhouses of the cell) have on the function of astrocytes, a cell type found in the brain. This is important for understanding diseases like Parkinson’s, where we know mitochondrial damage occurs but don’t fully understand how it impacts the health of brain cells.

Interested in reading more about actin? Find the full paper here!