Microprotein Atlas Links Brain Immune Cell Dysfunction to Alzheimer’s

Scientists studying Alzheimer’s disease have long focused on genes, proteins, and cells, but one class of molecules has remained largely outside the playbook: microproteins. These small proteins, produced from small open reading frames (ORFs) and typically measuring 150 amino acids or fewer, have been difficult to detect and study. Yet growing evidence suggests they may have important roles in health and disease.

Now, researchers at the Salk Institute have created what they describe as the first microprotein atlas of the human frontal cortex with and without Alzheimer’s disease. The study, “A microprotein atlas of the human frontal cortex in Alzheimer’s disease,” was published in Nature Aging. The resource integrates transcriptomics, mass spectrometry, and deep-learning-predicted spectra across postmortem brain samples to identify microproteins that have been overlooked in standard protein catalogs.

“We still do not fully understand the molecular mechanisms of healthy aging, and that is especially true for microproteins, which have been inadvertently overlooked for decades,” said senior and co-corresponding author Alan Saghatelian, PhD, professor and the Dr. Frederik Paulsen Chair at Salk, in a press release. “Our atlas allows scientists to systemically investigate microproteins in aging and neurodegeneration, which should bring us closer to understanding and tackling diseases like Alzheimer’s or Parkinson’s.”

Saghatelian told GEN the work began with a basic limitation in how proteomes are annotated. “Every reference proteome is built on gene models that exclude smORFs by construction. So the first motivation was straightforward: build a search database that can actually see these sequences, and point it at the deepest human brain proteomics data that exists.”

The atlas was built using data from hundreds of postmortem human frontal cortex samples from individuals with and without Alzheimer’s disease, including samples made available through the Religious Orders Study/Memory and Aging Project cohort. The team analyzed existing native transcriptomic and mass spectrometry data with custom computational tools, including ShortStop, an AI-powered microprotein-finding tool developed in Saghatelian’s lab.

“We were able to take all these technologies and tools and reapply them to existing data from nearly 500 brains to find new microproteins,” said first and co-corresponding author Brendan Miller, PhD, a postdoctoral researcher in Saghatelian’s lab. “We were able to create an entirely new database that researchers can download and use to better interpret functions of genes.”

In total, the researchers identified 1,067 previously uncharacterized microproteins absent from reviewed UniProtKB entries, supported by high-confidence spectral support, according to the paper. Some of these microproteins were expressed differently in Alzheimer’s disease samples compared with non-Alzheimer’s samples, and the paper reports that Alzheimer’s disease cells tended to show higher overall microprotein expression.

The investigators then focused on microglia, which are known to change with aging and neurodegeneration. Their analysis highlighted a small open reading frame at the MKKS locus encoding a 63-amino-acid microprotein that appeared to be the predominant translation product at that locus and is downregulated in Alzheimer’s disease. When the researchers knocked out the microprotein-making gene in microglia, mitochondrial respiration was impaired, suggesting a role for the microprotein in microglial bioenergetics.

The MKKS finding also underscored a broader issue with relying only on canonical protein annotations. “The general implication is uncomfortable: the most abundant and most tissue-relevant protein product at a locus can be the one that isn’t annotated,” added Saghatelian.

He also cautioned that not every microprotein identified in the atlas should be assumed to be functional. “There are two ways to read an expressed microprotein. It may be a marker—evidence that its gene’s transcription or splicing is disrupted—without the peptide itself doing anything. Or it may be a bioactive molecule with biology distinct from the canonical product at that locus.”

The findings point to a possible connection between microproteins and immune cell dysfunction in Alzheimer’s disease. “There is sometimes an assumption that we know everything about our genome, and we know all the genes our cells can make—that’s just not true,” Saghatelian said. “What we know is constantly expanding, and this atlas makes it that much easier to study microproteins in life science research.” Beyond Alzheimer’s disease, the publicly available atlas could serve as a framework for mapping microproteins in other brain regions, tissues, and disease contexts.