Cell-Type Atlas of Brain Lysosomal Proteins May Clarify Neurological Disorders

Functioning brain cells need a functioning system for picking up the trash and sorting the recycling. But when lysosomes, the cellular sanitation machines responsible for those tasks, break down or get overwhelmed, it can increase the risk of Alzheimer’s, disease, Parkinson’s disease, and other neurological disorders.

“Lysosomal function is essential for brain health, and mutations in lysosomal genes are risk factors for neurodegenerative diseases,” said Monther Abu-Remaileh, PhD, a Wu Tsai Neuro affiliate and an assistant professor of chemical engineering in the Stanford School of Engineering and an assistant professor of genetics in the Stanford School of Medicine.

Scientists aren’t sure exactly how lysosomes do their work, or what goes wrong with them that leads to neurodegeneration, or even in which cell types neurodegenerative disease begins. And there might even be other lysosomal disorders yet to be discovered.

Researchers have now laid out the first-ever atlas of lysosomal proteins in the brain, indicating which proteins are most closely associated with lysosomes across different brain cell types. The data, the researchers say, could help scientists better understand lysosomal function and what happens when they break down.

“Now we know which lysosomal proteins are enriched in which brain cell types,” commented Ali Ghoochani, PhD, a research scientist in Abu-Remaileh’s lab. “This allows us to better understand the functions of these proteins and how their dysfunction contributes to Alzheimer’s disease, Parkinson’s disease, and other neurological disorders.” The team was also able to use the atlas to tie a rare neurological disorder, SLC45A1-associated disease, to lysosomal dysfunction, identifying SLC45A1 as a neuron-specific lysosomal protein.

Abu-Remaileh is senior author, and Ghoochani co-first author of the team’s published paper in Cell, titled “Cell-type resolved protein atlas of brain lysosomes identifies SLC45A1-associated disease as a lysosomal disorder.” In their paper the team concluded “Our findings provide a detailed view of the cell-type-specific lysosomal proteomic landscape in the brain and establish a foundation for future studies on lysosomal biology and its roles in neurological diseases.” The team has made its data accessible to scientists via a website.

Lysosomes are membrane-bound organelles responsible for degrading macromolecules and clearing damaged organelles, to maintain cellular homeostasis, the authors wrote. But lysosomes are also key regulators of nutrient and energy-sensing pathways, and serve as reservoirs for nutrients including amino acids, cholesterol, calcium and iron, and are involved in functions such as plasma membrane repair, stress resistance, cell growth, and programmed cell death. “Given their central roles, mutations in lysosomal genes cause lysosomal storage disorders (LSDs) and contribute to a wide range of diseases, in particular neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and frontotemporal dementia.” However, the cell-type-specific composition and roles of lysosomes in the brain hasn’t been clear, nor have scientists known whether specific lysosomal proteins drive specialized functions in different brain cells.

The team’s newly reported study builds on research that started when Abu-Remaileh’s group began applying proteomics—the systematic examination of the proteins present in an organism—specifically to lysosomes. The hard part, they realized, was isolating lysosomes from other cellular machinery in a way that preserved the chemistry within. In a 2017 paper, Abu-Remaileh reported a new method called LysoIP that tags lysosomes so they could be removed from other cellular components and studied independently. In 2022, his lab extended that technique to genetically modify mice so that their lysosomes would express the tag automatically. Drawing on that method, called LysoTag, Abu-Remaileh’s lab discovered the role that a particular protein, CLN3, played in breaking down lipids—and how the loss of CLN3 could lead to Batten disease, a rare childhood disorder in which cells accumulate waste, leading to seizures, problems thinking and moving, and ultimately death.

LysoTag’s success got Ghoochani, Abu-Remaileh, and their colleagues thinking. If they knew more about where proteins like CLN3 shows up the most in the brain they might learn more about their functions and dysfunctions. “What we wanted to do here was to use that technology to be able to see what the composition of the lysosome is in different cell types in the brain,” Abu-Remaileh said. “We thought by doing so, we might be able to tell which lysosomes in which cell types are the culprits in different genetic neurodegenerative diseases.” Figuring that out could help researchers and doctors better understand neurodegeneration and perhaps point toward new therapeutics.

For their study outlined in Cell, and with support from the Knight Initiative and the Metabolomics Group at the Nucleus at Stanford’s Sarafan ChEM-H, Ghoochani and colleagues first extended LysoTag’s capabilities, combining it with a method for targeting it to specific cells. That way, they could add LysoTag to specific cell types and extract lysosomal proteins specifically from each of the four major brain cell types—neurons, astrocytes, oligodendrocytes, and microglia. “Using the LysoIP method coupled with high-resolution mass spectrometry, 13 we created a comprehensive lysosomal protein atlas across major brain cell types, including neurons, astrocytes, microglia, and oligodendrocytes,” they wrote. “This approach thus provides a robust method for studying cell-type-specific lysosomal composition.”

Working with Julia Heiby, PhD, and Allesandro Ori, PhD, at the Leibniz Institute on Aging, the researchers then developed data analysis tools to identify which proteins were most closely associated with lysosomes from each of the four brain cell types. With those tools in hand, the team built their atlas, as a catalog of 790 proteins associated with lysosomes—that is, more likely to be found in lysosomes than elsewhere—along with data on which proteins were more likely to be found in which types of brain cells.

That atlas presented the researchers with an unusual opportunity to explore connections between lysosomal proteins, the genes that encode them, and neurodegenerative disease. “Among the brain LysoIP-enriched proteins, 303 were previously known lysosomal components, whereas 487 had not been associated with lysosomes before … potentially representing uncharacterized lysosomal proteins or tissue-specific cargoes targeted for degradation,” the investigators stated. In particular, the team found 67 lysosomal proteins associated with Alzheimer’s-related dementia, Parkinson’s disease, and lysosomal storage disorders (LSDs).

Further analyses indicated that a few of those proteins were expressed more in one cell type than in others. For example, a protein called GRN associated with frontotemporal dementia was more common in microglia, a kind of immune cell that has been linked to neurodegeneration, than others. That, Ghoochani and Abu-Remaileh suggested, meant they had an unusual opportunity to study links between disease and lysosomal dysfunction.

The researchers knew that a protein known as SLC45A1 helps transport sugars across cell membranes, and that mutations in the protein cause intellectual disabilities with neuropsychiatric symptoms, but no one had previously connected it to the lysosome or lysosome dysfunction.

Their new data showed that SLC45A1 was specific to neurons. “The identification of SLC45A1 as a neuron-specific lysosomal protein highlights the effectiveness of LysoIP in uncovering cell type-specific lysosomal components,” the investigators pointed out. “To our knowledge, SLC45A1 is the only lysosomal protein currently annotated as neuron specific.

The studies also showed that the loss of SLC45A1 disrupts lysosomal degradation—the process of breaking apart waste—which makes it harder for lysosomes to clear that waste out. Experiments indicated a key role for SLC45A1 in clearing hexose sugars from neuronal lysosomes, “… shedding light on its essential function in maintaining lysosomal homeostasis and energy metabolism in neurons,” the authors noted.

The results strongly indicated SLC45A1-associated disease as a lysosomal storage disorder. “Altogether, our results demonstrate that SLC45A1 loss induces lysosomal dysfunction consistent with LSD and further implicate a pathological mechanism through mitochondrial dysfunction in SLC45A1-associated disease.” The finding, the researchers believe, is just the start. One project, Ghoochani said, aims to better understand the function of SLC45A1 in lysosomes, which could eventually inform approaches to therapeutics.

There are other possibilities as well, Ghoochani suggested. “Now that we have the atlas, there are many proteins with potential disease relevance that we plan to investigate further.”

The authors commented, “In conclusion, our lysosomal atlas provides a valuable resource for understanding the diverse roles of lysosomes across brain cell types and how cell-type-specific lysosomal functions contribute to brain diseases … These findings open opportunities for studying lysosomal biology in the brain and its implications for neurological diseases and LSDs.”

Other labs are getting excited Abu-Remaileh said, thanks to the website where researchers can scour the atlas for ideas. “People are already using our approach to look at disease in different cell types, and there are plenty of labs following in Ali’s footsteps in doing cell-type specific work on the lysosome in disease models.”