A collection of familial PD knock-in models, related to Supplemental figure 1.

(a) Scheme of the knock-in strategy where the first common exon in all Drosophila transcripts (left) was replaced by an attP-flanked mini-white gene using CRISPR/Cas9 mediated homologous recombination, creating a null mutant, and then replaced by wild-type or pathogenic mutant human or fly CDS using PhiC31 mediated integration (right). The chromosomal positions of the genes are indicated. All knock-ins are in the same isogenic genetic background (cantonized w1118). (b) Phenotypic analysis of PD mutants (1) Median survival under starvation. n>50 animals per genotype. (2) Mitochondrial membrane potential measured by ratiometric TMRE fluorescence at neuromuscular junction (NMJ) boutons in 3rd instar larvae. n≥4 animals per genotype and 10 boutons from ≥3 NMJs per animal. (3) Depolarisation amplitude quantified from electroretinograms (ERGs), recorded after 7 days of light exposure. n≥20 animals per genotype. (4) Quantification of startle-induced-negative geotaxis (SING) at 5±1 d after eclosion (young) and 25±1 d after eclosion (old). 95% of pink1 mutants died < 25 d and were tested at 15 d. Values are normalized to control (see methods). Variance of control measurements are in grey. Bars are mean ± s.e.m; *, p<0.05 ANOVA/Dunnett’s test.

Single-cell RNA sequencing reveals that olfactory projection neurons are impaired across young PD fly models, related to Supplemental figure 2.

(a) tSNE of 226k cells characterized by single-cell RNA sequencing of entire young (5 day old) fly brains (controls and PD knock-in mutants). Colors indicate the cell types; 186 of which were identified (See methods). Key cell types are encircled, including dopaminergic neurons (DAN, blue), Mushroom body Kenyon cells (orange) and olfactory projection neurons (OPN, green). (b-f) tSNE of the cells from 5 selective PD knock-in mutants (5 day old). Black cells are those with a significant transcriptomic change. Key cell types labeled in (a) are indicated. Note that OPN are consistently affected across mutants, while DAN are not at this early stage.

Common DEGs in cholinergic neuron-rich brain regions of human PD patients and PD fly models, related to Supplemental figure 3.

(a-a”) Schematic of the sunburst plot indicating GO terms for each sector (a) and the mapping of the DEGs in nucleus basalis of Meynert (NMB), 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”). (b) Gene Ontology 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.

Synaptic defects in OPN of PD mutants.

(a) Schematic of a head-fixed awake fly for live Ca2+-imaging through a window in the head capsule. (b) Confocal image of GCaMP3-fluorescence expressed in OPN using GH146-Gal4, indicating the locations of the cell bodies, antennal lobe and the calyx (Scale bar: 50 µm). (c-d) Confocal image of the stimulus-induced change of fluorescence (peak amplitude) in the synaptic region of the calyx of a control and a hLRRK2G2019S knock-in animal (c) and quantification of fluorescence change (± SEM) over time (d); Arrowhead: time of stimulus application (10 mM Nicotine, See methods). (e, f) Images of GFP fluorescence marking the synaptic area of OPN in the calyx of control (e) and hLRRK2G2019Sknock-ins (f); Scale bar is 20 µm. (g) Quantification of GCaMP3 peak amplitude at OPN synapses in the calyx following stimulation (10 mM Nicotine) in controls, wild-type CDS knock-ins, and in the PD knock-in mutants where the wild-type CDS is not (-) or is (+) expressed in OPN (OPN>wt CDS). (h) Quantification of the GFP fluorescence area of OPN synapses in the calyx (based on GCaMP3 signal) in controls, wild-type CDS knock-ins, and in the PD knock-in mutants where the wild-type CDS is not (-) or is (+) expressed in OPN (OPN>wt CDS). For (g, h): n≥5 animals per genotype. *, p<0.05 in ANOVA/Dunnett.

OPN dysfunction causes hyposmia in PD mutants.

(a, b) Olfactory performance of PD knock-in flies and controls when given the choice between a blend of motor oil and banana odors (a) or a blend of beer and wine odors (b). (c, d) Olfactory performance of PD knock-in flies and LRRKKO when the relevant wildtype CDS is not (-) or is (+) expressed selectively in OPN (or T1 cholinergic interneurons, T1>hR39B, as a negative control). For (a-d): Bars are mean ± s.e.m. n≥5 assays, ≥200 flies per genotype. *, p<0.05 in ANOVA/Dunnett.