How Waste Build-Up in the Brain Occurs in Aging and Neurodegeneration

To function properly, neurons need to recycle cellular waste before it becomes toxic. When neurons can no longer do that, either due to aging or harmful genetic mutations, neurodegenerative disease can set in.

One sign that neurons are losing their recycling function is the build-up of pigmented material called lipofuscin that accumulates with age. Understanding how it forms could help illuminate the aging process and, in turn, how age-related neurodegenerative diseases progress.

In a study published in Acta Neuropathologica, Yale School of Medicine researchers set out to better understand lipofuscin, its origins, and where it tends to deposit in the brain.

Specifically, they created a mouse brain map that tracked lipofuscin as the mice aged and their brains’ abilities to recycle cellular waste declined. Then, by closely examining lipofuscin’s content, the researchers uncovered an overlooked process that led to lipofuscin’s build-up in the brain in both aging and neurodegeneration.

The results “are the most quantitative, systemic analysis of lipofuscin in the murine brain,” says Sreeganga Chandra, PhD, professor of neurology and of neuroscience at Yale School of Medicine and senior author of the study.

Lipofuscin forms through the accumulation of unrecyclable trash inside the cell, such as old fats and proteins that have become damaged by the wear and tear of normal cell activity. Typically, it builds up as people age, likely a result of the reduced efficiency of lysosomes, the recycling centers of the cell.

Rapid accumulation of lipofuscin is also caused by mutations in the gene that encodes the enzyme palmitoyl protein thioesterase (PPT1). PPT1 removes fatty acid chains from proteins, which helps the body break down and recycle proteins. Mutations in PPT1 cause a disease called ceroid neuronal lipofuscinosis type 1 (CLN1), one of the few fatal neurodegenerative disorders that affect children.

“The problem is the disease is so aggressive and rare that it’s very hard to get human samples or a brain autopsy,” Chandra says. So, her team turned to the brains of mice.

The researchers examined 425 fine brain regions and 13 broad regions in mice using the publicly available Allen Brain Atlas, visualizing lipofuscin at various stages in neurodegenerative disease and in normal aging. Thanks to lipofuscin’s ability to autofluoresce, or emit light, its presence is easy to track under the microscope.

The brain atlas revealed that lipofuscin accumulates in specific regions both shared and unique to aging in typical mice and those lacking the PPT1 gene, underscoring areas vulnerable to lipofuscin accrual.

“Once lipofuscin accrues, it cannot be degraded,” Chandra says. This accrual is most pronounced in the neurons of the cortex, hippocampus, and cerebellum, potentially contributing to neurodegenerative effects.

Curious about PPT1’s role in lipofuscin production, the researchers isolated lipofuscin from aging mice and mice without PPT1 and examined its contents using electron microscopy.

In both groups of mice, they observed that lipofuscin was a collection of cellular junk—proteins, lipids, and fragments of degraded mitochondria and lysosomes. Notably, most of the proteins within lipofuscin contained fatty-acid groups that are normally removed by PPT1, not only in mice lacking PPT1 but also in regular, aging mice. Without PPT1 actively removing these fatty acids, these proteins likely had no way to be recycled, contributing to the formation of lipofuscin, say the researchers.

Interestingly, the researchers found that PPT1 activity wanes over time in the brains of aged mice.

“This really underscored the fact that as mice age, and maybe even as people age, the activity of PPT1 decreases,” says Alexander Esqueda, a graduate student in Chandra's lab and a co-author of the study. “This loss of activity is likely an unappreciated cornerstone of aging, which allows accumulation of lipidated proteins that ultimately lead to the formation of lipofuscin.”

Furthermore, the similarities between the protein and lipid makeup of lipofuscin in aged mice and in those without PPT1 suggest that the origin of lipofuscin that drives aging and neurodegenerative diseases is indeed the same, he adds.

To determine whether their findings translate to humans, the researchers obtained cortical samples from four human individuals aged 67 to 96. Their analysis showed that lipofuscin from human tissue shared more than 3,800 proteins with that of aging mice, indicating that what the researchers saw in the animal is happening in humans as well.

Beyond CLN1, lipofuscin has been shown to accumulate in other neurodegenerative diseases like Alzheimer’s and Huntington’s, implying a broad applicability of the findings in the paper.

The presence of degraded mitochondria and lysosomes in lipofuscin supports the notion that aging begins with the breakdown of these two organelles, leading to irreversible damage in non-dividing cells such as neurons.

“This study will hopefully spur a new area of aging research focusing on the role of protein lipidation,” Chandra says.

The researchers are now looking to expand their atlas to these other types of neurodegenerative diseases.