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Lipofuscin accumulation in aging and CLN1 is associated with deficient de-S-acylation, lysomitochondrial dysfunction, and lipid dyshomeostasis

ABSTRACT: Lipofuscin is an autofluorescent material that accrues in brain tissues with age and in Neuronal Ceroid Lipofuscinosis (NCL), a neurodegenerative disease with pediatric onset. The distribution, composition, and organellar origin of lipofuscin have remained unclear despite its widespread presence in aged tissues and involvement in neurodegeneration. Here, we elucidate lipofuscin composition in mouse and human brain and assemble a reference neuroanatomical atlas of lipofuscin accumulation with age and NCL (Type 1; CLN1) progression across 425 fine brain regions. We identify a primary role of the lysosomal-mitochondrial axis in the formation of lipofuscin pathology via multimodal mass spectrometry, ultrastructural analyses,and assays of cellular and enzymatic metabolism. We find the protein and lipid composition of age and CLN1 lipofuscin to be remarkably similar. Dissection of implicated molecular pathways reveals protein Sacylation and unsaturated lipid homeostasis as central processes involved in lipofuscin deposition during aging and CLN1. Notably, >95% of lipofuscin resident proteins can be S-acylated and many are substrates of the enzyme PPT1, validating a seminal hypothesis that CLN1 lipofuscin contains these lipid-modified proteins. Further, we discover deficient de-S-acylation is correlated with lipofuscin load in healthy aging, as the specific de-S-acylation enzyme activity of PPT1 is found to decline with advancing age. Finally, we identify lipid metabolite biomarkers of lipofuscin, including long-chain polyunsaturated fatty acids, bis(monoacylglycerol)phosphate (BMP), and oxidized phosphatidylethanolamine (OxPE) lipid species. Overall, we provide a comprehensive redefinition of lipofuscin neuropathology and a resource for studying aging, lysosomal storage disorders, and neurodegeneration.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

DISEASE(S): Neuronal Ceroid Lipofuscinosis 3

SUBMITTER: TuKiet Lam

LAB HEAD: TuKiet Lam

PROVIDER: PXD054766 | Pride | 2026-05-06

REPOSITORIES: Pride

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Source:

			Action	DRS
	HFX25-4744_Tieze_BDBB1_Lipo.raw	Raw
	HFX25-4749_Tieze_BDBB1_Total.raw	Raw
	HFX25-4754_Tieze_BDBB2_Lipo.raw	Raw
	HFX25-4759_Tieze_BDBB2_Total.raw	Raw
	HFX25-4764_Tieze_BDBB13_Lipo.raw	Raw

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Project description:Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1 result in early onset neurodegeneration, therefore, we hypothesized that the levels of proteins extracted from purified brain membranes of newborn mice will differ. One of the main effects of palmitoylation is changing the hydrophobic index of proteins and thus affecting these proteins interactions with different membranal domains. Assuming that PPT1 substrates are over palmitoylated in Ppt1-/- brains it is possible that its localization to specific membranal domain is different than in the wild-type brains. We isolated Triton X-100 soluble membranes from three wild-type and three Ppt1-/- brains and identified the proteins by PalmPISC. Many of the identified proteins were found to be palmitoylated. A second feature, which was noted is that many of the differential proteins (62 out of 88, 70%) can be detected in the cilia proteome (http://v3.ciliaproteome.org/cgi-bin/index.php). This observation led to test whether ciliary proteins differ between wild-type and Ppt1-/-. Cilia were prepared from newborn dissociated brain cell cultures and analyzed by MS

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Lipofuscin accumulation in aging and CLN1 is associated with deficient de-S-acylation, lysomitochondrial dysfunction, and lipid dyshomeostasis

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