By Julia Simpson

Dr. Daniela Zarnescu, the Associate Dean for Graduate Education and Postdoctoral Training here at Penn State College of Medicine, has artistic renderings of fruit flies pinned to a corkboard in her office, and a stuffed neuron – small, fuzzy, and blue – on her desk. Dr. Zarnescu finds herself pulled in a lot of directions these days due to her leadership role, but her office, and its décor, is an ode to the primary reason she came to Penn State: to research neurodegenerative diseases via the fruit fly model organism.

It’s this research that drew me to her office. In October 2023, the Zarnescu lab published a paper in Acta Neuropathologica Communications describing the development and characteristics of a novel fruit fly model of the neurodegenerative spectrum disorders frontal temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). FTD and ALS – as well as several other dementias, such as Alzheimer’s disease and Parkinsonism-dementia complex – are linked to changes in normal expression of a protein called TDP-43 in neurons. The Zarnescu lab’s new model organism recapitulates disease-related TDP-43 expression at the cellular level and exhibits disease-state behaviors such as impaired memory and disrupted sleep. I spoke to Dr. Zarnescu to learn more about her research and better understand the implications of her recent publication. One of my first questions: why fruit flies?

“There is a high degree of conservation at the genetic level between fruit flies and humans,” Zarnescu explains. “There have been at least three Nobel Prizes awarded to fruit fly geneticists for fundamental discoveries in human biology and disease – most people don’t know that!”

I certainly didn’t. It’s true, though, and the number is actually even higher: the Nobel Prize in Physiology and Medicine has actually been awarded six times for fruit fly discoveries with major implications for human medicine.

Scientists use so-called “model organisms,” such as mice, zebrafish, and fruit flies – in this case, the species Drosophila melanogaster – as a way to study certain conditions or diseases at a level that more closely mirrors the human condition compared to, say, cells in a dish. “You can do that in flies quickly,” explains Zarnescu. “Their generation time is 10-14 days… [and] it is relatively cheaper to work with these animals compared to mice or other models that are more commonly used in biomedical research.” Relevant for her research into neurodegenerative diseases, fruit fly brains also exhibit “system equivalency” with human brains.

“Although it may not look morphologically the same,” Zarnescu clarifies, “When you look at a fly brain vs a human brain, the genetic map – the gene networks, and some of the circuits – have very similar organizations.” She explains that this also encompasses the presence of neural circuits in flies that have no structural counterpart in humans, but which carry out the same function as parts of the human brain. One such region is called the “mushroom body.”

“Humans don’t have anything that looks like it, but the function of the mushroom body circuit… includes behaviors such as learning and memory, sleep, decision making – all of which are altered in human patients with FTD,” Zarnescu emphasizes.

What is the endgame of this type of model-organism-based neuroscience research? “Knowing the pathways that are altered in humans… being able to validate them functionally in the fly… you get a functional readout of a pathway that’s important for neuronal degeneration,” explains Zarnescu.

In other words: say you know that cellular signaling pathway X is messed up in a human disease. Step one, for researchers working in model organisms, is to mess up pathway X the same way in their model organism; if they see symptoms or behaviors that mirror the human disease, that’s “functional validation.” Since they’re working in model organisms, though, researchers can then take a step further, and figure out what cascade of changes happen in the animal due to messing up pathway X – and this can shine light on new details of the disease, details that could potentially be exploited for human treatment.  

“And that,” Zarnescu emphasizes, “Can give you ideas of how to modulate that pathway to slow down or stop the process of neuronal degeneration – but more likely slow down. […] We can hope for slowing down the process and turning this from a fatal disease into a chronic, manageable disease.”

The paper: a deep dive

1. Where is the TDP-43?

The Zarnescu lab’s recent paper took a step-by-step approach to demonstrate that their model was a valid way to study ALS and FTD. The model consisted of generating flies that expressed lots of human TDP-43 protein in the neurons of the mushroom body. The researchers then examined where in the neurons the TDP-43 accumulated using Laser Scanning Confocal Microscopy. In healthy neurons, the TDP-43 protein mainly lives in the nucleus, happily whiling away the days assisting in RNA transcription and splicing, to aid in the development and functioning of the nervous system. In diseases like FTD, however, TDP-43 exits the nucleus and aggregates in the cytoplasm, where it a) cannot do its normal job, and b) disrupts normal cellular functions.  Results showed that in the neurons of their FTD/ALS model fly larvae, TDP-43 is present in the nucleus; however, in young adult flies (1-3 days old), TDP-43 is found in the cytoplasm (Figure 1).

Zarnescu and colleagues were surprised to find that compared to young adults, older adults had more TDP-43 in the nucleus, though still much less than observed in larvae. However, this may be an example of survivorship bias, because neurons with lots of cytoplasmic TDP-43 – as seen in the disease state – could be selectively dying. Seeing that the TDP-43 in their model flies’ brains localizes to the cytoplasm, and that its overexpression causes age-dependent cell loss, were two encouraging results indicating that they had a promising model system for studying TDP-43-related ALS and FTD.

1. How does TDP-43 impact working memory?

Disruptions in working memory – the small amounts of information kept readily available for problem solving and task execution – is a hallmark of FTD and other dementias associated with altered TDP-43. Zarnescu’s team next measured how the working memory of their flies was affected by TDP-43 overexpression by studying how the flies behaved in a Y-shaped maze (Figure 2).

An animal with intact working memory, when placed in such a maze, should be able to remember which “arm” of the maze it just explored, so is less likely to choose to explore the same arm multiple times in a row. Animals with impeded working memory, however, cannot recall which arm they just explored, and are therefore more likely to venture down the same arm several times in a row. Zarnescu’s group found that flies with TDP-43 overexpression explored the same arm of the maze more consecutive times compared to controls, indicating a deficit in working memory.

1. How does TDP-43 affect sleep?

Another notable symptom of neurodegenerative diseases like FTD is sleep disturbances. To determine whether the TDP-43 in their model flies impacted sleep, Zarnescu’s group placed individual flies at specific ages into small tubes called Drosophila Activity Monitors (DAMs), along with a bit of food. The DAMs then monitored the flies’ activity on a minute-by-minute basis for three days via movement-tracking infrared beam arrays. Results showed that flies with overexpressed TDP-43 in their circuits exhibited increased lethargy, and some had more fragmented nighttime sleep patterns – both of which are hallmarks of disease in human patients with FTD.

• What other cellular stuff gets messed up by cytoplasmic TDP-43?

One of the findings that Dr. Zarnescu is most excited about centers around an RNA target of TDP-43.  When something – like, say, an aggregate of cytoplasmic TDP-43 – snags an RNA transcript, the RNA can’t be made into protein, which impacts downstream cellular pathways that utilize that protein.

Zarnescu’s lab group identified a pool of enriched RNA transcripts near TDP-43 clumps in the cytoplasm, but one in particular caught their attention: Dally-like protein (Dlp), a component of the Wg/Wnt cell signaling pathway. This pathway is essential for a wide range of critical functions, such as key steps in embryonic development, as well as maintaining adult tissue homeostasis. 

The lab investigated what would happen, then, if they overexpressed Dlp protein in their TDP-43 flies. To evaluate the effect of excess Dlp on working memory, they used the Y-maze again, and lo and behold: they found that Dlp overexpression in TDP-43 flies reversed the disease-state behavior. In their previous experiment, TDP-43 flies had impaired working memory compared to flies without TDP-43 overexpression; however, when they overexpressed Dlp in the TDP-43 flies, their working memory did not exhibit those deficits.

• Okay, but does this mean anything for humans?

“We leveraged a collaboration that we have,” Dr. Zarnescu shares excitedly. Her group sought to validate their fly model findings with human patient data. They compared RNA datasets from 3 groups: TDP-43 flies, humans with FTD, and humans with FTD + TDP-43 pathology.

Among the overlaps in the fly and human targets was the RNA of human version of Dlp, called GPC6, which was significantly altered in the brains of patients with FTD. In these patients, there was more GPC6 RNA in the nuclei of neurons that lacked TDP-43. Zarnescu’s group speculates that since Dlp protein levels are low in flies with this disease model, maybe these human cells are seeing low protein levels too, and the increase in GPC6 RNA – the precursor for more GPC6 protein – is compensatory. Though the researchers point out that it’s possible for Dlp and its human counterpart GPC6 to be regulated differently, the fact that both are altered during TDP-43-related disease states validates the value of the fly model to identify relevant disease targets. 

“[It’s] really cool!” Zarnescu says of the discovery. “There is a lot of really cool data that’s now emerging from many labs that report changes in gene expression or protein expression in patient brains.” This is important, she says, to help scientists grasp the complexity of these diseases. “But one of the challenges of this approach,” Zarnescu continues, “Is that you end up with a list of genes or changes, and it’s difficult to figure out which ones are the most important. Now, you can start figuring out which ones are the most important by going back to simple options.…In this particular case, we went from fly to human, and found this validation.”

Takeaways

The Dlp finding – combined with the effects on TDP-43 localization, the flies’ working memory, and the flies’ sleep patterns – demonstrate that Dr. Zarnescu’s lab has successfully developed a new, robustly applicable model in which to study neurodegenerative spectrum diseases like ALS and FTD. It’s an exciting accomplishment – and Zarnescu is already turning her eyes to the horizon. With a new grant, a growing lab, and new research collaborations being forged, Zarnescu’s days won’t be getting any less busy any time soon – but she wouldn’t have it any other way. So, what keeps her engaged every day? Why does Dr. Zarnescu find research exciting?

“Because there’s something new every day!” Dr. Zarnescu says, laughing. “Right? It never gets boring. It’s an exciting opportunity to discover something new, maybe not on a daily basis, but frequently. It’s never boring [and] it gives an opportunity to work with a diverse group of people, and I think that has been one of the most satisfying aspects of my job.”

TL;DR

• Fruit flies are a promising model organism for studying neurodegenerative diseases.
• Dr. Zarnescu’s lab has developed a novel fruit fly model of the pathological and behavioral manifestations of frontal-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by over-expressing a protein called TDP-43 in specific neurons.
• An RNA target of TDP-43 identified in the fly model is also altered in human patients, emphasizing the relevance of studying human diseases in fly models.