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Fonseca-Ornelas L, Stricker JM, Soriano-Cruz S, Weykopf B, Dettmer U, Muratore CR, Scherzer CR, Selkoe DJ. Parkinson-causing mutations in LRRK2 impair the physiological tetramerization of endogenous α-synuclein in human neurons. NPJ Parkinsons Dis. 2022 Sep 16;8(1):118. PubMed.
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National Institute on Aging
This report pairs of IPSC neurons where LRRK2 mutation status associates with a shift in the tetramer-monomer ratio in neurons and an increase in pS129-synuclein. This latter observation is particularly interesting as the assay for measuring pS129-synuclein in neurons is robust and could be used across laboratories to validate the main results of the published work in multiple additional IPSC clones and presumably in knock-in mouse neurons. It may be of interest to note here that we have been working to make isogenic LRRK2 lines available as part of larger project to model multiple single variant causes of neurodegeneration (Human iPS Cells, The Jackson Lab).
Considering open questions that come from this report, it would be critical to further understand the mechanism(s) by which LRRK2 genotype affects synuclein metabolism. The application of two structurally distinct kinase inhibitors reversed all effects, including those on synuclein but also on Rab10 phosphorylation. However, it is curious that the authors used very extended and very high levels of inhibitors. Typically, measures of LRRK2 activity including pS1292 LRRK2 and Rab12 or Rab29 are responsive to inhibitors within one hour, including in vivo (Kluss et al., 2021). Additionally, pRAb10 is not very responsive to the LRRK2 G2019S mutation in some tissues (Iannotta et al., 2020; Fan et al., 2021).
These considerations may indicate that neuronal LRRK2, as evaluated here, may have different regulation than does LRRK2 in other contexts. Possibly relevant is that in non-neuronal cells, we, and several other labs, see a strong relationship between LRRK2 activity and lysosomal function (e.g., Bonet-Ponce et al., 2020) but work in neurons suggests a role of LRRK2 in autophagolysosome transport (Boecker et al., 2021). Thus, it would be important to understand whether synuclein is responsive to LRRK2 genotype in neurons because it is either not being degraded, which seems unlikely given that total synuclein levels are unchanged, or whether the distribution of protein between soma and synapse is altered.
The other unanswered question from this paper is whether an altered monomer-tetramer ratio, as evaluated by cross-linking then western blots, is responsible for any LRRK2 mutation effects. I don’t think we know at this stage whether loss of tetramers is damaging to neurons, although the association with dominant mutations of both LRRK2 and SNCA would imply so. It is notable that only a small amount of cross-linked material is recruited into the potential tetramers (Figure 2a in the current paper, for example). It would be interesting to repeat these experiments in vivo using LRRK2 knock-in (and knockout) alleles that have been endogenously introduced into the mouse genome. Unfortunately, at least in baseline conditions, those animals don’t have obvious TH cell loss so one would have to then challenge the animals further to be able to test relationships between tetrameric and phosphorylated synuclein, LRRK2 genotype, and dopamine cell loss. However, such experiments would then form the basis of reversal of both LRRK2 activation and synuclein tetramer formation; this could be dissected out, to some degree, using the SCD inhibitor reported by this group to reverse synuclein tetramer formation irrespective of LRRK2 genotype, presumably without affecting LRRK2 activity per se.
References:
Kluss JH, Mazza MC, Li Y, Manzoni C, Lewis PA, Cookson MR, Mamais A. Preclinical modeling of chronic inhibition of the Parkinson's disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo. Mol Neurodegener. 2021 Mar 19;16(1):17. PubMed.
Iannotta L, Biosa A, Kluss JH, Tombesi G, Kaganovich A, Cogo S, Plotegher N, Civiero L, Lobbestael E, Baekelandt V, Cookson MR, Greggio E. Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues. Cells. 2020 Oct 22;9(11) PubMed.
Fan Y, Nirujogi RS, Garrido A, Ruiz-Martínez J, Bergareche-Yarza A, Mondragón-Rezola E, Vinagre-Aragón A, Croitoru I, Gorostidi Pagola A, Paternain Markinez L, Alcalay R, Hickman RA, Düring J, Gomes S, Pratuseviciute N, Padmanabhan S, Valldeoriola F, Pérez Sisqués L, Malagelada C, Ximelis T, Molina Porcel L, Martí MJ, Tolosa E, Alessi DR, Sammler EM. R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils. Acta Neuropathol. 2021 Sep;142(3):475-494. Epub 2021 Jun 14 PubMed.
Bonet-Ponce L, Beilina A, Williamson CD, Lindberg E, Kluss JH, Saez-Atienzar S, Landeck N, Kumaran R, Mamais A, Bleck CK, Li Y, Cookson MR. LRRK2 mediates tubulation and vesicle sorting from lysosomes. Sci Adv. 2020 Nov;6(46) Print 2020 Nov PubMed.
Boecker CA, Goldsmith J, Dou D, Cajka GG, Holzbaur EL. Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes. Curr Biol. 2021 May 24;31(10):2140-2154.e6. Epub 2021 Mar 24 PubMed.
View all comments by Mark CooksonThese results add to the collective body of data that LRRK2 kinase activity appears to (albeit subtly) push the normal biology of α-synuclein toward states associated with pathological conformations.
Next steps will be determining how LRRK2 directs α-synuclein away from tetrameric states, whether acting directly, or indirectly through Rab phosphorylation or mitochondrial interactions, for example.
The new tools we have, including LRRK2 inhibitors that work at low nanomolar levels, and pT73-Rab10 antibodies that are quite effective for measuring LRRK2 kinase activity, build more confidence in the conclusions that are made.
View all comments by Andrew WestJohns Hopkins University
This fascinating finding builds on the authors’ previous works highlighting an increase in the ratio of α-synuclein monomers and tetramers as a molecular mechanism driving PD. They show in this paper that LRRK2 mutations negatively affect the ratio of monomer to tetramer and increase phosphorylation at pSer129, effects that are rescued by an SCD inhibitor and a LRRK2 inhibitor. These results add to the collective body of data that genetic modifiers could affect the formation of tetramers of α-synuclein, and that changes in lipid homeostasis are an important mechanism in the formation and PD pathogenesis.
This is impressive work. That said, I think we need more information regarding:
1) the use of isogenic LRRK2 lines,
2) understanding the direct causal relationship between LRRK2 mutation and tetramer formation,
3) how LRRK2 mutation induces changes in lipid homeostasis,
4) SCD inhibition, how the accumulation of polysaturated lipids by LRRK2 mutation plays a role in the inhibition of tetramer formation-lipidomics data derived from cells with LRRK2 mutation would be informative for the answer,
5) whether the amount of specific lipids is important, or whether changes in the lipid composition of specific organelles or presynaptic terminal membrane are important.
Also, it would be important to understand whether α-synuclein 129 phosphorylation could inhibit tetramer formation.
View all comments by Hanseok KoINSERM
Fonseca-Ornelas and colleagues in Dennis Selkoe’s lab have used human induced pluripotent cells to study the relationships between LRRK2 and α-synuclein tetramers, which their lab has proposed in the past decade to be a “healthy” form of α-synuclein because it is resistant to pathological aggregation. The authors detect the tetramers using a cross-linking assay whereby they appear as different bands on western blots ranging from 60 to 100 kDa (compared to the 14 kDa monomer) and hence may be loosely described as lower-order complexes of α-syn. While previous work has reported that LRRK2 activity may be required for α-synuclein-mediated pathological events, such as cell death, α-synuclein phosphorylation, or cell-to-cell transmission of the protein, this is the first report that explores links between LRRK2 and these tetrameric species.
Assuming that such lower-order complexes are more desirable than α-syn monomers, the finding that the lower-order complex/monomer ratios are reduced in induced neuronal cells harboring pathological LRRK2 mutations suggests a potential pathway leading to adverse, long-term effects in these patients. Also, the observation that the lower-order complex/monomer ratio increased after treatment with LRRK2 kinase inhibitors further confirms that LRRK2 may be an important upstream regulator of α-syn toxicity.
Interestingly, the lower-order complex/monomer ratio also increased upon changes in cellular lipid composition, with conditions preventing the generation of monounsaturated fatty acids being favorable. This suggests that lipids are important in defining the equilibrium between α-synuclein monomer and its protective lower-order complexes. It remains to be verified if this is a direct effect of the lipids on α-synuclein that overrides the effects of LRRK2 mutants, or if the lipid composition in cells affects the functions of the LRRK2 mutants.
Should the described phenomenon be confirmed in larger cohorts of patients, this lower-order complex/monomer ratio may be interesting to use in stratifying patients for clinical trials or as a phenotype in therapeutic screening efforts.
View all comments by Jean-Marc TaymansMake a Comment
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