Biology subjects

Middleton, C. A.

Publications and source records attributed to Middleton, C. A..

3 recordsLinked to original sources

bioRxiv2020

Multiple pathways of LRRK2-G2019S / Rab10 interaction in dopaminergic neurons

BackgroundInherited mutations in the LRRK2 protein are the most common known cause of Parkinsons, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. Which Rabs interact with LRRK2 in dopaminergic neurons to facilitate normal and pathological physiological responses remains to be determined. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. We now ask if Rab10 is required for LRRK2-induced physiological responses in DA neurons. MethodsLRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, vision, circadian activity pattern and courtship memory determined in aged flies. ResultsRab10 loss-of-function rescued bradykinesia of the Proboscis Extension Response (PER) and visual defects of aged flies expressing LRRK2-G2019S in DA neurons. Rab10 knock-down however, did not rescue the marked sleep phenotype which results from dopaminergic expression of LRRK2-G2019S. Courtship memory is not affected by LRRK2 expression, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. ConclusionsWe conclude that, in Drosophila dopaminergic neurons, Rab10 is involved differentially in LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinsons. Graphical AbstractRab10 depletion ameliorates the proboscis extension bradykinesia and loss of synaptic signalling in the retina induced by LRRK2-G2019S expression (magenta arrows / orange crosses). Rab10 manipulation does not affect the sleep phenotype from LRRK2-G2019S (magenta arrow). Reduction of Rab10 facilitates conditioned courtship memory, but LRRK2 has no effect (yellow arrow). All manipulations of Rab10 and G2019S in dopaminergic neurons, shown in the outline of the brain (filled cells have high levels of Rab10). We conclude that Rab10 and LRRK2 interact in some, but not all dopaminergic neurons. This may underlie differences in the susceptibility of different human striatal dopaminergic cells to Parkinsons and explain why different symptoms initiate particular ages. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/316992v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1e9a923org.highwire.dtl.DTLVardef@15f7135org.highwire.dtl.DTLVardef@101525borg.highwire.dtl.DTLVardef@1c5da02_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

In vivo visual screen for dopaminergic Rab {leftrightarrow} LRRK2-G2019S interactions in Drosophila discriminates Rab10 from Rab3

LRRK2 mutations cause Parkinsons, but the molecular link from increased kinase activity to pathological neurodegeneration remains undetermined. Previous in vitro assays indicate that LRRK2 substrates include at least 8 Rab GTPases. We have now examined this hypothesis in vivo in a functional, electroretinogram screen, expressing each Rab with/without LRRK2-G2019S in selected Drosophila dopaminergic neurons. Our screen discriminated Rab10 from Rab3. The strongest Rab/LRRK2-G2019S interaction is with Rab10; the weakest with Rab3. Rab10 is expressed in a different set of dopaminergic neurons from Rab3. Thus, anatomical and physiological patterns of Rab10 are related. We conclude that Rab10 is a valid substrate of LRRK2 in dopaminergic neurons in vivo. We propose that variations in Rab expression contribute to differences in the rate of neurodegeneration recorded in different dopaminergic nuclei in Parkinsons.

neuroscience↗

bioRxiv2019

Neurodegeneration caused by LRRK2-G2019S requires Rab10 in select dopaminergic neurons

Inherited mutations in the LRRK2 protein are the commonest known cause of Parkinsons, but the molecular link from increased kinase activity to pathological neurodegeneration remains to be determined. In vitro (biochemical and cell culture) assays led to the hypothesis that several Rab GTPases might be LRRK2 substrates. Here we show that Rab10 potently modifies LRRK2-G2019S mediated electrophysiological responses in an in vivo screen, in which each Rab was overexpressed in Drosophila dopaminergic neurons. We therefore tested the effect of Rab10 loss of function on three LRRK2-G2019S phenotypes (vision, movement and sleep) that rely on dopaminergic circuits in both flies and mammals. The knock-out of Rab10 in vivo fully rescues the reduced responses induced by dopaminergic LRRK2-G2019S in visual and motor (reaching, proboscis extension) assays, but the sleep phenotype is unaffected. We show that Rab10 is expressed in dopaminergic (tyrosine hydroxylase positive) neurons controlling vision and proboscis movement, but undetectable in those controlling sleep, indicating that anatomical and physiological patterns of Rab10 are related. Our results support the idea that LRRK2 phosphorylates separate targets in distinct neurons and confirm that one degenerative pathway starts with Rab10. Although Rab3 is another putative substrate of LRRK2, it shows no synergy with G2019S and localises to a different subset of dopaminergic neurons from Rab10. We propose that variations in Rab expression may contribute to differences in the rate of neurodegeneration seen in different dopaminergic nuclei in Parkinsons.\n\nSignificance StatementA key question in Parkinsons is why dopamine neurons die particularly fast in some parts of the substantia nigra. We focused on the commonest Parkinsons-related mutation, LRRK2-G2019S. In vitro assays suggested that neurodegeneration may start by LRRK2-G2019S increasing phosphorylation of Rab10. We found Rab10 in fly dopamine neurons in visual and motor pathways, but not in the sleep system. Rab10 knock-out rescues G2019S-induced visual and movement degeneration, leaving sleep dysfunction unaffected. Thus, LRRK2 activates at least two pathways, one Rab10-dependent, leading to neurodegeneration in vivo. Rab3 is found in a different subset of dopaminergic neurons and shows no synergy with LRRK2-G2019S. We propose that variations in Rab expression contribute to differences in neurodegeneration seen in Parkinsons.

neuroscience↗