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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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Richard Wilson

LAB HEAD: Dr Richard Wilson

PROVIDER: PXD032191 | Pride | 2022-12-07

REPOSITORIES: Pride

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Project description:Background Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties. In our previous study, we found that TGF-β1 played a critical role in inhibiting apoptosis and increasing neuronal activity in murine cortical neurons following trauma. However, the precise mechanisms underlying the neuroprotective actions of TGF-β1 on the cortex require further investigation. Methods Thus, in this study, we were aimed to investigate the regulatory function of TGF-β1 on neuronal autophagy and apoptosis using an in vitro primary cortical neuron trauma-injury model. Results To establish the landscape of differentially expressed genes (DEGs) with or without TGF-β1 (10ng/ml) treatment for 24 hours, we performed RNA-sequencing. We observed significant enrichment of DEGs related to autophagy, apoptosis, and the lysosome pathway in trauma-injured cortical neurons. Additionally, transmission electron microscopy (TEM) confirmed the presence of autophagosomes as well as autophagolysosomes. Western blot analysis revealed upregulation of autophagy-related protein light chain 3 (LC3)-Ⅱ/LC3-Ⅰ, sequestosome 1 (SQSTM1)/p62, along with apoptosis-related protein Cleaved-caspase3 in trauma-injured primary cortical neurons. Furthermore, mechanically injured neurons showed an upregulation of lysosomal marker protein lysosomal marker protein (LAMP1) and lysosomal enzyme mature cathepsin D (mCTSD), but a decrease in the activity of CTSD enzyme. These results indicated that apoptosis was up-regulated in mechanically injured neurons at 24 hours, accompanied by lysosomal dysfunction and impaired autophagic flux. Notably, TGF-β1 significantly reversed these changes. Conclusions Therefore, our findings suggested that TGF-β1 exerted neuroprotective effects on mechanically injured neurons by reducing lysosomal dysfunction, decreasing the accumulation of autophagosomes and autophagolysosomes, and enhancing autophagic flux.

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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

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