The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in human neurons

Abstract

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, α-synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation.

Fig 1.. Rab8a KO and Rab10 KO human neurons have altered lysosomal morphology and function.

Representative confocal images of Rab8a KO, Rab10 KO and isogenic WT control neurons stained with LAMP1, MAP2 and DAPI (A). Lysosomal parameters including lysosomal count, average lysosomal area and lysosomal pH were assessed by high-content imaging of LysoTracker Red or LysoSensor staining (B-I). Western blot analysis of levels of glycosylated LAMP1, (J-M). All lysosomal analyses were collated from 3–4 independent experiments for each of three differentiations with 10–20 wells per genotype, per experiment, on 21-day old iNs. (*, p < 0.05; **, p < 0.001; ***, p < 0.0001; ****, p < 0.0001; one-way ANOVA Tukey’s post-hoc).

Fig 2.. Rab8a KO and Rab10 KO cells exhibit alterations in Golgi distribution

Rab8a KO, Rab10 KO and isogenic WT control iPSCs were fixed and stained for Cis-Golgi (GM130), Trans-Golgi (TGN46) and LAMP1 (A, D). Z-stack confocal images were 3D reconstructed in Imaris (Bitplane) and the mean distance between CGN stacks (B), distance of CGN to nucleus (C), TGN volume (E), TGN distance to nucleus (F), colocalization between TGN46 and LAMP1 (G) and number of lysosomes per cell were analyzed (H). (N>50 cells over three independent experiments; **, p < 0.001; ***, p < 0.0001; ****, p < 0.0001; one-way ANOVA Tukey’s post-hoc).

Fig 3.. Rab8a KO human neurons accumulate insoluble α-synuclein and secrete α-synuclein in media.

Rab8a KO, Rab10 KO and WT neurons were collected at D21 and extracted in NP-40 containing lysis buffer (soluble fraction), followed by SDS resuspension of the pellet (insoluble). Densitometry analysis represents α-synuclein levels normalized to actin (B, C, E, F). (G, H) Media was collected at D21 and analyzed by dot-blot for total and oligomeric α-synuclein. Protein yields of neuronal lysates were used to normalize for variability in cell density and analyze equivalent amounts of conditioned media. (*, p < 0.05; ***, p < 0.0001; ****, p < 0.0001; one-way ANOVA Tukey’s post-hoc).

Fig 4.. Rab10 KO increases phospho-Tau species in human neurons

Human cortical neurons were collected on D21 and phosphorylated (AT8; pTau: pS205 & pT205 Tau) and total Tau levels were assessed in Rab8a KO (A) and Rab10 KO (B) cells compared to isogenic controls (n=3 differentiations, one-way ANOVA Tukey’s post-hoc). (C) The ratio of each pTau band (54kDa, 62kDa & 70kDa) out of the total pTau signal was plotted. (D) Densitometry analysis represents total Tau levels normalized to actin. (n=3 differentiations, one-way ANOVA Tukey’s post-hoc).