Pre-clinical screening for re-purposed and novel compounds that reduce synucleinopathy, and investigation of LRRK2 activity and VPS35 function within human cell culture models of Parkinson’s disease
Human pluripotent stem cell (PSC) models of Parkinson’s disease (PD) are a promising tool for drug screening and for investigating disease mechanism. Mutations within LRRK2 are the most common cause of familial PD, where most mutations in LRRK2 increase its kinase activity. More recently a D620N mutation within VPS35, a component of the retromer complex, has been identified as an autosomal dominant cause of PD and was also shown to increase LRRK2 kinase activity. The Alessi lab have identified a subset of small Rab GTPases as substrates of LRRK2 and demonstrated that PD-associated hyperactive LRRK2 variants, G2019S-LRRK2 and R1441C/G-LRRK2, increase phosphorylation of these Rab substrates. Moreover, the D620N VPS35 mutation increased LRRK2-dependent Rab phosphorylation to a greater extent than G2019S LRRK2 or R1441C/G LRRK2 mutations. To investigate LRRK2 and VPS35 function in the context of cellular models of Parkinson’s, human pluripotent stem cells (PSCs) were differentiated into midbrain dopaminergic (mDA) neurons, macrophages, and microglia-like cells. We determined expression of VPS35 is high among all cell types, whereas LRRK2 is significantly higher within immune cells compared to mDA neurons. LRRK2 activity could be readily assessed within macrophages and microglia-like cells by quantification of pT73 Rab10. To investigate the effect of the D620N VPS35 mutation, CRISPR/Cas9 engineering was used to create D620N-VPS35 knock-in human embryonic stem cell (hESC) lines. We observed the D620N VPS35 mutation significantly increased LRRK2 activity within macrophages, but not mDA neurons. However, these cell lines were later found to also have an additional E617D mutation, which may, or may not have contributed to the observed results. hPSC-derived cellular models of PD can also be used to screen for compounds which reduce ⍺Synuclein pathology. To model Lewy-like synucleinopathy we seeded mDA neurons with ⍺Synuclein pre-formed fibrils (PFFs) and quantified the amount of phospho-Serine-129 (pS129) ⍺Synuclein formed in neurons. This screen involved compounds that are reported to target ⍺Synuclein directly either through reduction of endogenous ⍺Synuclein (Salbutamol, Clenbuterol, Riluzole), or targeting oligomeric forms of ⍺Synuclein (Anle138b). In addition, we investigated compounds which alter cellular processes implicated within ⍺Synuclein pathology, including a LRRK2 inhibitor (MLi-2), a PARP-1 inhibitor (Niraparib) and an Aldehyde dehydrogenase 2 (ALDH2) activator (Alda-1). Previous work has suggested Salbutamol, Clenbuterol, and Riluzole treatment reduced SNCA transcript levels, however we observed no effect in PSC-derived mDA neurons. Unexpectedly, we further observed a striking increase in PFF-induced pS129-⍺Synuclein pathology following regular treatment with high concentrations of Anle138b, Clenbuterol, Niraparib, and Riluzole. Conversely, we find the inhibition of LRRK2 activity with, MLi-2 had no effect on pS129-⍺Synuclein pathology within WT mDA neurons. Toxic aldehydes such as 4HNE and the dopamine metabolite DOPAL have been implicated in the acceleration of ⍺Synuclein pathology and subsequent neurodegeneration. ALDH2 is highly expressed in mDA neurons and is known to convert the DOPAL aldehyde into non-toxic DOPAC. Using a metabolomics approach, we report treatment with the small molecule ALDH2 activator, Alda-1, increased the metabolism of dopamine into DOPAC within mDA neurons. Furthermore, Alda-1 was able to partially rescue neuron death mediated by high concentrations of 4HNE, and it was the only compound to significantly reduce PFF-induced pS129-⍺Synuclein pathology in mDA neurons. Overall, this report provides further evidence of the potential function of LRRK2 and VPS35 within immune cells and mDA neurons. In addition, we highlight PSC derived neurons can be applied to drug screening model for synucleinopathy.