The research, conducted by Klaudia Walczak and colleagues at the University of Gdańsk and published in the journal Acta Biochimica Polonica, examined fibroblasts — connective tissue cells grown in the laboratory — obtained from patients with eleven distinct mucopolysaccharidosis subtypes, ranging from the relatively common types I, II, and III to the rarer types IV, VI, VII, and IX. The team purchased these patient-derived cell lines from the Coriell Institute for Medical Research in New Jersey, alongside a control line of healthy fibroblasts, and grew them under standard laboratory conditions. The approach allowed the researchers to compare mitochondria across an unusually wide spectrum of the disease in a single, systematic analysis.

To visualize mitochondria, the team used a fluorescent dye called MitoTracker Green, which lights up these organelles under the microscope regardless of their functional state. Using this technique, the researchers measured two key parameters: the total area occupied by the mitochondrial network within each cell, and the number of discrete fluorescent objects — which the researchers interpreted as individual mitochondria — identified after computer-based image analysis. The results were surprisingly uniform. Across all eleven disease subtypes, neither the size of the mitochondrial network nor the apparent number of mitochondria differed significantly from the healthy control cells. In other words, at the level of structure, mitochondria in these patient cells looked essentially normal.

This structural normality was unexpected, because earlier studies using electron microscopy had reported changes in mitochondrial appearance in fibroblasts from patients with several mucopolysaccharidosis subtypes, including increases in mitochondrial number and alterations in mitochondrial shape. The discrepancy may reflect differences in technique. Electron microscopy can resolve individual organelles with far greater precision, while the fluorescence-based approach used in the new study relies on thresholding fluorescent objects after image segmentation — a process that may merge or split mitochondria in ways that do not perfectly reflect their true biology. The researchers themselves acknowledged this limitation, noting that the parameter they measured may not perfectly correspond to the actual number of isolated mitochondria within the cell.

But the study’s most interesting findings came not from the microscope but from the genetic analysis. The team extracted total DNA from each cell line and used a quantitative polymerase chain reaction technique to measure the relative abundance of mitochondrial DNA — the small circular genome housed within mitochondria — compared with nuclear DNA, using a gene called β-globin as the nuclear reference. This ratio, known as the mtDNA/nDNA ratio, is a widely used proxy for mitochondrial content within a cell, and changes in this ratio can signal alterations in mitochondrial biogenesis, the process by which cells build new mitochondria. The results were strikingly different from the structural findings.

In several disease subtypes, the mtDNA/nDNA ratio was significantly lower than in the healthy control cells. The most pronounced decreases appeared in types I, II, IIIA, and VI — a pattern that did not align neatly with the structural normality observed under the microscope. In the remaining disease subtypes, the ratio did not differ significantly from the control. This dissociation between structure and genetic content is biologically meaningful, because it suggests that in some mucopolysaccharidosis subtypes, mitochondria may be present in normal numbers and normal organization but may carry less mitochondrial DNA per cell — a change that could reflect alterations in mitochondrial biogenesis or in the copy number of the mitochondrial genome within individual organelles.

The researchers interpreted these findings cautiously, emphasizing that the study was designed to be exploratory rather than definitive. They noted that mtDNA copy number is known to vary widely among healthy individuals, and that the normal range for this parameter is broad enough that modest decreases may fall within the spectrum of healthy variation. They also acknowledged that the study used a single patient-derived cell line per disease subtype, which means that the observed changes could reflect donor-specific variability rather than disease-specific biology. Without replication across multiple patients per subtype, the findings cannot be definitively attributed to the disease itself.

Nevertheless, the pattern of decreased mtDNA content in specific subtypes — particularly types I, II, IIIA, and VI — is consistent with earlier reports of mitochondrial dysfunction in these diseases. Previous studies have documented reduced activity of the electron transport chain, the series of protein complexes that generates cellular energy, in fibroblasts and other cell types from patients with several mucopolysaccharidosis subtypes. Other work has reported increased production of reactive oxygen species, the damaging molecules generated as byproducts of mitochondrial energy production, and alterations in mitochondrial dynamics, the processes by which mitochondria fuse, divide, and move within the cell. The new study’s genetic findings add another layer to this accumulating evidence, suggesting that the mitochondrial genome itself may be altered in some disease subtypes.

The dissociation between structural normality and decreased mtDNA content also raises important questions about how mitochondria change in mucopolysaccharidoses. One possibility is that the diseases alter mitochondrial biogenesis — the process by which cells build new mitochondria — without changing the overall organization of the mitochondrial network. Another possibility is that the diseases reduce the copy number of the mitochondrial genome within individual organelles, a change that would be invisible to structural imaging but detectable by genetic analysis. Yet another possibility is that the observed decreases reflect a compensatory response, in which cells reduce mitochondrial content as a way of limiting the production of damaging reactive oxygen species. Distinguishing among these possibilities will require further study.

The researchers also emphasized the limitations of their cell model. Fibroblasts are readily accessible and easy to grow in the laboratory, making them a convenient model for studying rare diseases, but they may not fully recapitulate the biology of the tissues most affected by mucopolysaccharidoses, particularly the brain, skeleton, and heart. Skin fibroblasts from patients with these diseases do show some disease-related changes, including accumulation of glycosaminoglycans and alterations in gene expression, but the extent to which mitochondrial changes observed in fibroblasts reflect changes in other tissues remains uncertain. Future studies using neurons, cartilage cells, or heart muscle cells — or induced pluripotent stem cell-derived models of these tissues — may provide a more complete picture of mitochondrial involvement in the disease.

Despite these limitations, the study represents one of the broadest systematic analyses of mitochondria across the mucopolysaccharidosis spectrum, and its findings add important nuance to the ongoing debate about mitochondrial involvement in these rare diseases. The structural normality observed under the microscope suggests that mitochondria in these patient cells are not grossly disorganized, while the decreased mtDNA content in specific subtypes suggests that more subtle changes may be occurring at the level of the mitochondrial genome. The dissociation between these two levels of analysis highlights the importance of combining structural and genetic approaches in future studies of mitochondrial biology in rare diseases.

The findings also underscore the value of exploratory research in rare diseases. Mucopolysaccharidoses affect a small number of patients worldwide, and the rarity of these conditions makes large-scale clinical studies difficult to conduct. Laboratory-based analyses of patient-derived cells, such as the one conducted by the Polish team, offer a practical way to generate hypotheses about disease biology that can later be tested in more targeted studies. The new study’s findings — structural normality combined with decreased mtDNA content in specific subtypes — provide exactly this kind of hypothesis-generating evidence, pointing the way toward future research that may clarify the role of mitochondria in the pathology of these devastating conditions.

As the field moves forward, the challenge will be to integrate these exploratory findings with the accumulating evidence of mitochondrial dysfunction in mucopolysaccharidoses and to determine whether the observed changes are causes or consequences of the disease process. The new study’s cautious, hypothesis-generating approach reflects the reality of research in rare diseases, where definitive answers are hard to come by and progress often depends on the patient accumulation of suggestive evidence. For now, the Polish team’s findings add an important piece to the puzzle, suggesting that mitochondria in mucopolysaccharidoses may be more subtly altered than previously thought — and that the mitochondrial genome, rather than the organelle’s structure, may hold the key to understanding their involvement in these rare conditions.

Subject of Research: Mitochondrial morphology and mtDNA content in fibroblasts from patients with different types of mucopolysaccharidosis

Article Title: Mitochondrial morphology and mtDNA content in fibroblasts from patients with different types of mucopolysaccharidosis

Article References: Walczak, K., Gaffke, L., Pierzynowska, K., Sowa-Rogozińska, N., & Piotrowska, E. (2026). Mitochondrial morphology and mtDNA content in fibroblasts from patients with different types of mucopolysaccharidosis. Acta Biochimica Polonica, 73, Article 16345. https://doi.org/10.3389/abp.2026.16345

Image Credits: AI Generated

DOI: 10.3389/abp.2026.16345

Keywords: Mitochondrial, morphology, mtDNA, content, fibroblasts, patients, different, types, mucopolysaccharidosis, scientific research