kolf2 ipscs  (STEMCELL Technologies Inc)

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet: ( a ) Schematic representation of gene editing strategy to knock-in the G2019S mutation in the LRRK2 locus. ( b ) Sanger sequencing of a single gene edited clone showing successful homozygous editing of the indicated nucleotide. ( c ) KOLF2-1J wild-type (LRRK2 WT ) control iPSCs and KOLF2-1J LRRK2 G2019S/G2019S (LRRK2 G2019S ) iPSCs show normal expression of pluripotency markers OCT4 (yellow), SOX2 (purple), NANOG (purple), and TRA-1–81 (green). Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. ( d ) KOLF2-1J wild-type (LRRK2 WT ) control and KOLF2-1J LRRK2 G2019S/G2019S (LRRK2 G2019S ) mutant ventral midbrain neural progenitor cells show normal expression of FOXA2 (magenta), LMX1A (green), and OTX2 (green), confirming that the neural progenitor cells are ventral midbrain-specific and capable of differentiating into DAN. Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. ( e ) Quantification of the frequency of TH+/MAP2+DAN. Statistical significance calculated with an ordinary t-test: ns, not significant. Error bars represent mean ± SEM. Data were collected in three independent vmDAN differentiations. ( f ) Differentiation protocol used.

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques: Knock-In, Mutagenesis, Sequencing, Control, Expressing

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet: ( a–a” ) Schematic of the sunburst plot indicating Gene Ontology (GO) terms for each sector ( a ) and the mapping of the DEGs in nucleus basalis of Meynert (NBM), nucleus accumbens, and putamen brain samples idiopathic PD patients (with LRRK2 risk mutations) and controls ( a’ ) and mapping of the DEGs found commonly in fly and human samples ( a” ). Inner rings represent the different GO categories (indicated in a), with their subcategories in the outer rings, rings (in a’–a”) are color-coded according to enrichment Q-value. ( b ) GO analysis of DEG in cholinergic neurons of young PD fly models and NBM neurons of PD patients. Redundant terms were removed. Color: adjusted p-value. ( c ) Schematic of the DEGs found commonly in fly PD models (blue) and human PD samples (black) manually sorted according to their previously described synaptic functions. contains the summarized results of the DEG analysis of fly brains and postmortem human brain samples and the SynGO analysis.

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques:

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet: ( a , left) Synaptic area of dopaminergic neuron (DAN) innervating the mushroom body in aged PD models (Aux R927G , synj R258Q , or LRRK2 G2019S ) and with or without GH146-Gal4-driven expression of the wild-type PD gene ( aux or synj ) or EndoA S75D , respectively. Bars: mean ± SEM. n≥5, *p<0.05 in ANOVA, Dunnett’s test. ( a , right) Startle-induced negative geotaxis (SING) of the PD models with or without GH146-Gal4-driven expression of wild-type gene or endoA S75D . Points: mean ± SEM. n≥5, *p<0.05 in two-way ANOVA. Gray zone: variance of controls. ( b ) Odor choice performance, stimulus-induced changes in synaptic Ca 2+ signal and olfactory projection neuron (OPN) synapse area of young controls and hLRRK2 G2019S flies with or without chronic nicotine (Nic) feeding (up to 1 day before testing). Bars: mean ± SEM. n≥5 assays, *p<0.05 in ANOVA, Dunnett’s test. ( c ) SING, stimulus-induced changes in synaptic Ca 2+ and DAN synapse area of aged controls and hLRRK G2019S flies with or without chronic application of nicotine. Bars: mean ± SEM. n≥5 assays, *p<0.05 in ANOVA, Dunnett’s test. ( d ) Confocal images of differentiated (60 days) wild-type and LRRK2 G2019S ventral midbrain DAN labeled with the ventral midbrain marker FOXA2, dopaminergic marker TH, and neuronal marker MAP2. Scale bar: 20 µm. ( e ) Scheme of the treatment protocol and spontaneous Ca 2+ activity ( e’ ) and amplitude ( e’’ ) of human induced DAN, 2 days after 20 days of no treatment (Ctrl), nicotine (Nic) treatment or nicotine+mecamylamine (Nic+Meca) treatment. Bars: mean ± SEM. n≥60 DAN from three independent differentiations, *p<0.05 in ANOVA, Dunnett’s test.

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques: Expressing, Labeling, Marker, Activity Assay

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques: Knock-Out, Knock-In, Control, Mutagenesis, Transfection, Construct, Plasmid Preparation, Expressing, Imaging, Recombinant, Sequencing, Multiplex Assay, Software