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Communications BiologyCell BiologyPeer-reviewed journal article

LRRK2-mutant microglia and neuromelanin synergize to drive dopaminergic neurodegeneration in an iPSC-based Parkinson's disease model

Lucas Blasco-AgelliD, Meritxell Pons‐EspinaliD, Veronica TestaiD, Gerard RochiD, Jara Montero-MuñoziD, Irene Fernández‐CarasaiD, Valentina BaruffiiD, Marta González-SepúlvedaiD, Yvonne Richaud‐PatíniD, Senda Jiménez‐DelgadoiD, Thais CuadrosiD, Joana M. Cladera-SastreiD, Joan CompteiD, Zoe Manglano-ArtuñedoiD, Salvador VenturaiD, Manel JuaniD, Eduardo TolosaiD, Ángel RayaiD, Miquel VilaiD, Antonella ConsiglioiD

PDFDOIPreprint

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (30 Jul 2025) · CC BY 4.0

LRRK2 G2019S iPSC-derived microglia exposed to neuromelanin, but not α-synuclein fibrils, reduce healthy dopaminergic neuron numbers in co-culture; ivermectin prevents this, and post-mortem midbrain supports it.

Little was known about how LRRK2-mutant microglia respond to neuromelanin, a PD-relevant endogenous stimulus, or whether human iPSC-derived microglia respond like primary ones. This work provides an isogenic human neuron-microglia co-culture for testing immunomodulatory drugs.

Key results

  1. ShownLRRK2 G2019S microglia-like cells (hMG) secrete more IL-1β, IL-6 and TNFα after LPS stimulation, and separately show greater synaptosome phagocytosis, than control and gene-corrected isogenic hMG.

    Higher IL-1β, IL-6, TNFα after 24 h LPS (100ng/ml) in L2-PD vs CTL and L2-PD^corr hMG (one-way ANOVA, Fisher LSD); synaptosome uptake higher (Kruskal-Wallis, Dunn); N=3 independent experiments; p<0.05 and p<0.01 thresholds. · Functional validation of hMG; Figure legends

  2. ShownAfter neuromelanin (NM) exposure, L2-PD hMG take up more NM, move farther, and show higher pro-inflammatory mRNA, IL-6 release and reactive oxygen species than controls.

    16 h NM uptake higher in L2-PD vs CTL and corrected hMG; at 24 h higher IL-1β, IFNγ, IL-6, C3, TNFα mRNA and IL-6 release; DCF ROS fold change N=3 CTL, N=5 L2-PD, N=7 L2-PD^corr (Tukey ANOVA). · L2-PD hMG increases NM uptake and releases ROS; Figure legends

  3. SuggestedOnly NM-activated L2-PD hMG lowered the fraction of TH+ dopaminergic neurons in co-culture; hMG alone caused no loss, and α-synuclein fibrils with L2-PD hMG caused none either.

    NM 0.5 ug/mL for 8 h, 1 hMG per 2 neurons; one control neuron line (SP11) and L2-PD2 (SP13) hMG; N=3 experiments; fibrils 10 ug/mL for 8 h gave no loss. Effect magnitudes are not stated in the text. · L2-PD hMG induces dopaminergic neuronal…; 2D and 3D Neuron/Microglia co-culture systems; Figure legends

  4. ShownIvermectin prevented NM-induced dopaminergic neuron loss in L2-PD hMG co-cultures and reduced pro-inflammatory cytokine mRNA in L2-PD hMG monocultures.

    3 µM ivermectin for 6 h restored TH+ fraction (one-way ANOVA, Tukey); lower IL-1β, IL-6, TNFα and C3 mRNA in L2-PD1 (SP12) hMG; N=3 independent experiments. · Treatment with Ivermectin prevents L2-PD hMG…; 2D and 3D Neuron/Microglia co-culture systems; Figure legends

  5. ShownPost-mortem midbrain showed more IBA1+ microglia in both idiopathic and LRRK2 PD, more amoeboid cells in LRRK2 PD, and a larger rise of amoeboid cells near released NM in LRRK2 PD than in iPD or controls.

    Figure 4 cohort N=30 control, N=9 iPD, N=6 L2-PD; ANOVA with Tukey for counts, Kruskal-Wallis with Dunn for the near-minus-far NM difference; iPD amoeboid counts were intermediate. · Microglial activation is increased in…; Human post-mortem brain tissue; Figure legends

Methods, data and reproducibility

Isogenic iPSC-derived microglia-like cells (LRRK2 G2019S, gene-corrected, healthy donor) were challenged with LPS, NM extracts or α-synuclein fibrils, and co-cultured with control iPSC-derived dopaminergic neurons. Findings were compared with IBA1 staining of post-mortem midbrain.

  • iPSC lines: one healthy donor, two LRRK2 G2019S PD patients, two gene-corrected isogenic lines, differentiated to microglia-like cells · human · n = one CTL, two L2-PD, two L2-PD^corr lines
  • Control iPSC-derived ventral midbrain dopaminergic neurons (SP11) on astrocyte feeder; TH+/MAP2+ efficiency about 47% · human
  • NM extracts purified from tyrosinase-expressing, differentiated SH-SY5Y (TR5TY6) cells · human
  • Post-mortem midbrain sections (substantia nigra) from LRRK2 G2019S PD, idiopathic PD and age-matched controls, IBA1 immunostaining · human · n = Fig 4: 30 control, 9 iPD, 6 L2-PD; Methods: 6 L2-PD, 32 controls
  • Source data and key resources table (Zenodo repository) · 10.5281/zenodo.15681445
Methods & tools
Luminex MILLIPLEX cytokine multiplex assay, qRT-PCR (2^-ΔΔCt, β-Actin normalization), pHrodo-labelled synaptosome phagocytosis assay, DCFH2-DA ROS assay, Time-lapse imaging with ImageJ motility tracking, Sholl analysis (Simple Neurite Tracer), IMARIS, Olympus V200 Desktop 3.3 AI-based microglia morphology algorithm, Kaluza flow cytometry software, GraphPad Prism, Ivermectin, Lipopolysaccharide stimulation
Reagents & environment
GraphPad Prism 7.0a (statistics section) and 8.0.1 (data availability section); Olympus V200 Desktop 3.3; Chemotaxis and Migration Tool version 2.0; ImageJ, IMARIS, Kaluza.
Reproducibility gaps
No code was generated. Source data are in Supplementary Data 1 and a Zenodo key resource table (DOI only). Further information is available from the corresponding author; post-mortem tissue came from a biobank.
Limitations (4)
  • Patient-derived dopaminergic neurons were not included, so the study does not test whether neuron susceptibility itself changes under pathological conditions. · stated by the authors
  • The full mechanisms of NM-driven neuroinflammation remain unresolved. Ivermectin also has a direct neuroprotective effect, so its rescue may not act purely via microglia; future work is planned to avoid direct contact with neurons. · stated by the authors
  • Neuron-loss co-cultures used one control neuron line (SP11) and one L2-PD hMG line (SP13) in N=3 experiments, and the text gives no effect magnitudes, so generalization across patients is untested. · noted in this summary
  • The post-mortem cohort is unbalanced (6 L2-PD vs 30 controls in the Figure 4 legend), and the Methods report 32 controls, so group counts are inconsistent and power for L2-PD comparisons is low. · noted in this summary

Read this if you study microglia in Parkinson's disease, LRRK2 biology, or want an isogenic iPSC neuron-microglia co-culture for testing immunomodulatory drugs against neuromelanin toxicity.

Citations
16
FWCI
8.79
Field percentile
top 1%

Abstract

Parkinsons disease (PD) is a progressive, incurable neurodegenerative disorder characterized by the loss of neuromelanin (NM)-containing dopamine neurons (DAn) in the substantia nigra of the midbrain. Non-neuronal cells are increasingly recognized as contributors to PD. We generated human microglia-like cells (hMG) from induced pluripotent stem cells (iPSC) derived from patients with LRRK2 PD-causing mutations, gene-corrected isogenic controls, and healthy donors. While neither genotype induced neurodegeneration in healthy DAn, LRRK2 hMG become hyperreactive to LPS stimulation, exhibiting increased cytokine expression, reactive oxygen species, and phagocytosis. When exposed to NM-containing particles, but not -synuclein fibrils, LRRK2 hMG trigger DAn degeneration, in a process that is prevented by pre-treatment with the immunomodulatory drug ivermectin. Finally, post-mortem analysis of midbrain tissue of LRRK2-PD patients show increased microglia activation around NM-containing neurons, confirming our in vitro findings. Overall, our work highlights NM-activated microglias role in PD progression, and provides a model for testing therapeutic targets.

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