Lysosomal storage disorders (LSDs) are a group of more than 50 inherited diseases, each caused by a defect in the cell’s garbage-disposal system. The lysosome, the organelle responsible for breaking down fats, sugars, and proteins, fails to clear certain molecules, which accumulate to toxic levels. Most LSDs affect the brain, but how each gene defect leads to neuronal dysfunction has been difficult to study: the mutations are individually rare, and generating models has required time-consuming genetic engineering for each one.
A team led by J. Wade Harper at Harvard Medical School has removed that bottleneck. Building a library of 23 human embryonic stem cell lines, each with a homozygous knockout of a different LSD gene, the researchers differentiated them into both cortical-like and dopaminergic-like neurons and profiled their proteomes, approximately 10,000 proteins per line, to map the downstream consequences of each defect. The work was published July 1 in PNAS.
“This is a powerful community resource,” said Felix Kraus, a co-first author of the study. “For the first time, we can directly compare the molecular consequences of many different lysosomal defects side by side in the same experimental system.”
A systematic approach
The 23 genes targeted in the study include the most common sphingolipidoses — GBA1 (Gaucher’s disease), ASAH1 (Farber disease), HEXA (Tay-Sachs), SMPD1 (Niemann-Pick types A and B) — as well as 12 genes responsible for neuronal ceroid lipofuscinoses (the Batten disease family), and others including NPC1, NPC2, and MCOLN1 (mucolipidosis type IV). All were created on the same H9 embryonic stem cell background, with an inducible genetic switch for rapid conversion into neurons.
The team used two differentiation protocols: one that produces cortical-like neurons (iN cells) and one that produces midbrain dopaminergic-like neurons (iDA cells). The latter are particularly relevant because mutations in GBA1, the gene that causes Gaucher’s disease, are the strongest known genetic risk factor for Parkinson’s disease, which selectively kills dopaminergic neurons.
Using high-resolution mass spectrometry, the researchers quantified approximately 10,000 proteins per cell line at multiple time points (days 30, 50, and 70 of differentiation), and developed computational methods to identify which protein complexes were disrupted.
Cell-type-specific vulnerabilities
The most striking finding is that the same gene knockout produces fundamentally different molecular effects in cortical versus dopaminergic neurons. For example, GBA1 deficiency caused severe mitochondrial OXPHOS (oxidative phosphorylation) defects in dopaminergic neurons: proteins of mitochondrial Complex I were substantially downregulated, and the effect was coordinated across the entire respiratory chain. In cortical neurons, the same mutation produced only minor mitochondrial changes.
“This is cell-type specificity at the molecular level,” Harper said. “It explains why different LSDs have different neurological presentations, even when the underlying biochemistry seems similar.”
The ASAH1 knockout showed a particularly dramatic cell-type difference. In dopaminergic neurons, loss of ASAH1 caused negative correlations across synaptic proteins — essentially, the synapse was falling apart. In cortical neurons, the same proteins were positively correlated. Functional validation using calcium imaging confirmed that ASAH1-deficient dopaminergic neurons fired significantly less than controls, while cortical neurons were only mildly affected. Electron microscopy revealed disorganized presynaptic structures with fewer vesicles of uneven size.
A convergent pathway
Across the 23 knockouts, mitochondrial and synaptic proteins were the most consistently affected compartments. This convergence suggests that diverse lysosomal defects — despite their different molecular substrates — may damage neurons through a common pathway: lysosomal dysfunction leads to mitochondrial impairment, which in turn disrupts synaptic function and energy metabolism.
The study also identified a specific protein complex-inference pipeline that can detect which multi-protein assemblies are destabilized in each mutant. Among the disrupted complexes were mitochondrial Complex I (in GBA1-deficient neurons), the BLOC-1 complex (in MCOLN1-deficient neurons), and glutamate receptor complexes (in GBA1 and MCOLN1 mutants).
Several caveats apply. The cells are induced neurons — they recapitulate key features of cortical and dopaminergic neurons but lack the full maturity, network complexity, and glial support of brain tissue. The 23 genes represent only a subset of the 50-plus known LSDs; mucopolysaccharidoses and glycoproteinoses are not yet included. And the knockouts are complete gene deletions, whereas most human patients carry partial loss-of-function mutations.
Nevertheless, the toolkit and its accompanying proteomic data — deposited in the MASSIVE repository (accession MSV000099237) — are publicly available, and the approach can be extended to additional genes and cell types. The study was funded by the Aligning Science Across Parkinson’s initiative, the National Institutes of Health, and the Warren Alpert Foundation.
Source: Kraus, F., He, Y., Jiang, Y. et al. “Lysosomal storage disorder toolkit for decoding proteome landscapes in cortical-like and dopaminergic-like induced neurons.” PNAS 123(27), e2609132123 (2026). DOI: 10.1073/pnas.2609132123