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  • Plasmid-reprogramming of human fibroblasts

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    This protocol is part of a collection of protocols for the paper, "Glucocerebrosidase, a Parkinson's disease-associated protein, is imported into mitochondria and regulates complex I assembly and function"

  • HeLa S3 ATG3 KO

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    HeLa cell line bearing knock out of ATG3.

  • Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

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    The authors used proteomics to develop a quantitative snapshot of the proteins involved in lysophagy. Among the proteins identified, they found that TAX1BP1 and TBK1 are both required for lysophagy.

  • Gavage – food supplementation for stalled weight gain in mice

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    Feeding protocol for Ndufs2fl/fl mice,

  • Cell culture, transfection, and imaging

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    This protocol describes general procedures for culturing HeLa cells, transient transfection, and imaging using an Andor Dragonfly spinning disk confocal system.

  • L1 retrotransposons drive human neuronal transcriptome complexity and functional diversification

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    The genetic mechanisms underlying the expansion in size and complexity of the human brain remains poorly understood. L1 retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution is largely unknown. Using multi-omic profiling we here demonstrate that L1-promoters are dynamically active in the developing and adult human brain. L1s generate hundreds of developmentally regulated and cell-type specific transcripts, many which are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived lncRNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPRi-silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

  • Lentivirus plasmid: pLV[Exp]-U6>KANSL1_P1_Seq1-hPGK>mApple

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    Plasmid: Plasmid vector encoding a sgRNA sequence which targets the KANSL1 promoter from CRISPRi, under a U6 promoter and a mApple fluorescent reporter. Generated by Vectorbuilder in the pLV backbone.

  • LRRK2 kinase activity regulates GCase level and enzymatic activity differently depending on cell type in Parkinson’s disease

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    Leucine-rich repeat kinase 2 (LRRK2) is a kinase involved in different cellular functions, including autophagy, endolysosomal pathways, and immune function. Mutations in LRRK2 cause autosomal-dominant forms of Parkinson’s disease (PD). Heterozygous mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), are the most common genetic risk factors for PD. Moreover, GCase function is altered in idiopathic PD and in other genetic forms of the disease. Recent work suggests that LRRK2 kinase activity can regulate GCase function. However, both a positive and a negative correlation have been described. To gain insights into the impact of LRRK2 on GCase, we performed a comprehensive analysis of GCase levels and activity in complementary LRRK2 models, including (i) LRRK2 G2019S knock in (GSKI) mice, (ii) peripheral blood mononuclear cell (PBMCs), plasma, and fibroblasts from PD patients carrying LRRK2 G2019S mutation, (iii) patient iPSCs-derived neurons; (iv) endogenous and overexpressed cell models. In some of these models we found a positive correlation between the activities of LRRK2 and GCase, which was further confirmed in cell lines with genetic and pharmacological manipulation of LRRK2 kinase activity. GCase protein level is reduced in GSKI brain tissues and in G2019S iPSCs-derived neurons, but increased in fibroblasts and PBMCs from patients, suggesting cell-type-specific effects. Overall, our study indicates that LRRK2 kinase activity affects both the levels and the catalytic activity of GCase in a cell-type-specific manner, with important implications in the context of therapeutic application of LRRK2 inhibitors in GBA1-linked and idiopathic PD.

  • pGST2-OPTN (S177D, S473D)

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    Plasmid: For the purification of phosphorylation mimic protein.

  • HEK293::TMEM192-3xHA YIPF4-/-

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    HEK293 cell line with TMEM192 gene C-terminally tagged with 3xHA epitope using CRISPR/Cas9. YIPF4 knockout also done. Derived from fetal kidney, human origin, adenovirus transformed.

  • HEK293 ::TMEM192-HA YIPF4-/-; YIPF3-/-

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    HEK293 ::TMEM192-HA YIPF4-/-; YIPF3-/- YIPF3/YIPF4-/- DKO HEK293; HEK293 YIPF3-/-;YIPF4-/- PubMed=37757899; Characteristics: Using CRISPR/Cas9 TMEM192 was C-terminally tagged on both alleles with a 3xHA epitope (from parent cell line). Knockout cell: Method=CRISPR/Cas9; HGNC; 21023; YIPF3. Knockout cell: Method=CRISPR/Cas9; HGNC; 28145; YIPF4. Transformant: NCBI_TaxID; 28285; Adenovirus 5. Derived from site: In situ; Fetal kidney; UBERON=UBERON_0002113. NCBI_TaxID=9606; ! Homo sapiens (Human) RRID:CVCL_D1J3 ! HEK293::TMEM192-3xHA YIPF4-/- Female Fetus Transformed cell line

  • Isotope tracing in health and disease

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    Here, the authors review recent work utilizing metabolic tracing to study health and disease, and highlight its application to interrogate subcellular, intercellular, and in vivo metabolism.

  • Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons

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    The authors describe the procedure and equipment used for live imaging of axonal cargoes.

  • Differentiation of human medium spiny neurons (MSNs) from induced pluripotent stem cells (iPSCs)

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    This protocol generates human medium spiny neurons (MSNs) from induced human pluripotent stem cells (iPSCs).

  • pCAG-GST-ATG13 (402-450)

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    Plasmid: Mammalian expression of GST tagged ATG13 (402-450).

  • His-ATG3-K208D

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    Plasmid for ATG3 mutant K208D overexpression in E.Coli.

  • PTEN-induced kinase 1 (PINK1) and Parkin: Unlocking a mitochondrial quality control pathway linked to Parkinson’s disease

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    This review focuses on understanding the PINK1/Parkin-mediated mitochondrial quality control pathway through the lens of abherrant immune activation as a driver of dopaminerigic neuron loss following the loss of PINK and Parkin.

  • The lipid flippase ATP10B enables cellular lipid uptake under stress conditions

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    ATP10B mutations are linked to Parkinson's and Lewy body disease. ATP10B acts as a lipid transporter in late endo-/lysosomes, enhancing phosphatidylcholine uptake in cells under stress conditions like rotenone treatment.

  • Nuclei Isolation and Sorting from Frozen Human Temporal Cortex V2

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    Protocol focuses on isolating and sorting nuclei from frozen human temporal cortex.

  • GI Transit Assay

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    This test is used to check for possible signs of constipation and alteration in gut transit.

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