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Proteomics of CLN3-deficient murine microglia identifies a disease associated phenotype with lysosomal alterations

ABSTRACT: Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3∆ex7/8 mice, a genetically accurate disease model. Loss of CLN3 function in microglia leads to lysosomal storage material accumulation and abnormal morphology of subcellular organelles. We also discovered pathological proteomic signatures consistent with defects in lysosomal function and indicative of abnormal lipid metabolism. CLN3-deficient microglia were unable to efficiently turnover myelin and metabolize the associated lipids, showing defects in lipid droplet formation and cholesterol accumulation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Microglial Cell

DISEASE(S): Neuronal Ceroid Lipofuscinosis 3

SUBMITTER: Stephan Mueller

LAB HEAD: Stefan F. Lichtenthaler

PROVIDER: PXD048550 | Pride | 2024-10-23

REPOSITORIES: Pride

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			Action	DRS
	20200513_150032_CLN3_Microglia_.sne	Other
	CLN3_Microglia_ConditionSetup.xls	Xls
	CLN3_reports.zip	Other
	E3005_WT.raw	Raw
	E3006_CNL3.raw	Raw

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Lysosomal alterations and altered electrophysiological activity in atypical juvenile neuronal ceroid lipofuscinoses (CLN3 1 kb deletion, E295K) patient-derived cortical neurons

Project description:Juvenile neuronal ceroid lipofuscinoses (JNCL) is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with JNCL carry at least one allele with 1 kb deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based JNCL models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we have corrected a 1 kb deletion mutation in human induced pluripotent stem cells (iPSC) of a compound heterozygous patient (CLN3 Δ1 kb and E295K), to generate an isogenic control to be used for disease modeling along with the unedited CLN3 patient iPSCs. iPSC-derived neurons carrying this particular CLN3 mutation, CLN3 neurons, had lower levels of spike, burst and network burst activity for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis on day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis of LAMP1 expression revealed a new finding where LAMP1 was hyperglycosylated in CLN3 neurons on DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

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Project description:The lysosome has many cellular roles, including degrading and recycling macromolecules and signaling to the mTORC1 growth regulator. Lysosomal dysfunction occurs in various human diseases, including common neurodegenerative diseases as well as monogenic lysosomal storage disorders (LSDs). For most LSDs the causal genes have been identified, but in many cases the function of the implicated gene is unknown. Here, we develop the LysoTag mouse line for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We apply it to the study of CLN3, a lysosomal transmembrane protein of unclear function whose loss causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and stable isotope tracing experiments show that CLN3 is required for their lysosomal egress. Loss of CLN3 also alters upstream glycerophospholipid catabolism in the lysosome. Our results suggest that CLN3 is a lysosomal effluxer of GPDs and reveal Batten disease as the first, to our knowledge, neurodegenerative LSD with a primary defect in glycerophospholipid metabolism.

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