Spermidine suppresses glial inflammation and parkinsonian abnormalities in ATP13A2 deficiency

Technical Summary: Spermidine suppresses glial inflammation and parkinsonian abnormalities in ATP13A2 deficiency

Problem Statement
Pathogenic loss-of-function variants in ATP13A2 (PARK9) cause Kufor-Rakeb syndrome (KRS), a severe juvenile-onset parkinsonism, and are implicated in early-onset Parkinson's disease (PD). ATP13A2 encodes an endolysosomal polyamine exporter responsible for transporting spermidine (Spd) and spermine (Spm) from the lysosomal lumen to the cytosol. While ATP13A2 dysfunction is known to cause lysosomal polyamine accumulation, impairing lysosomal degradative capacity and sensitizing cells to oxidative stress, the specific mechanisms linking this transporter defect to the early onset of neuroinflammation and motor deficits remain incompletely defined. Furthermore, it is unclear whether the resulting polyamine dysregulation is a primary driver of pathology or a secondary consequence, and whether therapeutic modulation of polyamine levels can rescue disease phenotypes.

Methodology
The study employed a multi-model approach combining in vivo mouse models, in vitro cellular assays, human postmortem tissue analysis, and Drosophila models:

• Mouse Models: Atp13a2 knockout (KO) and wild-type (WT) C57/BL6J mice were utilized. Phenotyping included motor tests (open field, pole test, limb clasping) and non-motor assessments (buried pellet, stool analysis) at various ages (PND14, 10 weeks, 6 months, 12 months).
• Polyamine Modulation: Mice were subjected to negative modulation (DFMO to inhibit synthesis; DenSpm to induce catabolism) and positive modulation (oral or intraperitoneal supplementation of Spd or Spm) starting from PND2 or in adulthood.
• Metabolomics: Targeted LC-MS was performed on plasma, brain, and liver tissues to quantify polyamine species (Ornithine, Putrescine, Spd, Spm, and acetylated derivatives).
• Cellular and Molecular Analysis:
  • Histology: Immunohistochemistry and immunofluorescence were used to assess gliosis (GFAP, Iba-1, CD68), lysosomal dysfunction (Lamp1, Galectin-3), and NF-κB signaling in mouse brains and human postmortem tissue.
  • Microglia Isolation: Magnetic cell sorting (MACS) and primary culture of microglia from Atp13a2 KO and WT mice were used to analyze gene expression (qPCR for Trem2, Lgals3, cytokines), morphology (aspect ratio), mitochondrial ROS (MitoSox), and cytokine secretion (TNF-α, IL-6, IL-10).
  • Mechanistic Probes: The polyamine transport inhibitor AMXT 1501 and siRNA-mediated knockdown of Atp13a3 were used to determine the uptake mechanism of exogenous Spd.
• Human Models: Postmortem brain tissue from a KRS patient and human iPSC-derived microglia (harboring an ATP13A2 c.1306 frameshift mutation) were analyzed for inflammatory markers and oxidative stress.
• Drosophila Models: Drosophila models of parkinsonism (anne/Atp13a2, Pink1, nutcracker/FBXO7, iPLA2-VIA) were treated with dietary Spd to assess motor function (SING assay) and dopaminergic neuron integrity.

Key Contributions and Results

1. Early Polyamine Deficiency Precedes Symptoms: In Atp13a2 KO mice, a transient but significant reduction in brain polyamines (Putrescine, Spd, Spm) was detected at PND14, preceding the onset of overt motor deficits (which appeared at 10 weeks) and gliosis. While polyamine levels eventually increased in the brain at 18 months, the early deficit was identified as a critical pathogenic window.
2. Polyamine Depletion Exacerbates Pathology: Pharmacological depletion of polyamines (via DFMO or DenSpm) in Atp13a2 KO mice significantly reduced survival and worsened motor and non-motor deficits compared to untreated KO mice, confirming that reduced polyamine availability drives disease severity.
3. Spermidine, but not Spermine, Rescues Phenotypes: Oral supplementation with Spd, initiated either early (PND2) or in adulthood (7 months), robustly rescued motor deficits (locomotion, coordination), non-motor deficits (anxiety-like behavior, olfaction), and survival in Atp13a2 KO mice. In contrast, Spm supplementation failed to rescue behavioral deficits and exacerbated toxicity in KO mice, likely due to lysosomal accumulation.
4. Mechanism: Glial Inflammation and Lysosomal/Mitochondrial Dysfunction:
   • Atp13a2 deficiency induced early microgliosis and astrogliosis, characterized by NF-κB activation, increased pro-inflammatory cytokines (TNF-α, IL-6), and a Disease-Associated Microglia (DAM) transcriptional profile.
   • Microglia in KO mice exhibited enlarged Lamp1-positive lysosomes and increased Galectin-3 accumulation, indicating lysosomal membrane damage.
   • Spd supplementation reversed these effects, normalizing lysosomal health, reducing mitochondrial ROS, and suppressing NF-κB signaling and cytokine release.
5. Role of Atp13a3 in Spd Uptake: In the absence of Atp13a2, microglia upregulated the related transporter Atp13a3. The anti-inflammatory and protective effects of exogenous Spd were abolished by the transport inhibitor AMXT 1501 and by Atp13a3 knockdown, demonstrating that Spd rescue depends on cellular uptake via Atp13a3.
6. Translational Relevance:
   • Postmortem analysis of a KRS patient confirmed increased microglial reactivity (CD68, Iba-1, GFAP) in the midbrain and striatum.
   • Human iPSC-derived ATP13A2-deficient microglia showed increased inflammation and oxidative stress, which was attenuated by Spd treatment.
   • In Drosophila models of parkinsonism (anne, Pink1, nutcracker), Spd treatment restored dopaminergic innervation and improved motor performance, suggesting broader applicability beyond ATP13A2 deficiency.

Significance
The paper identifies early brain polyamine deficiency as a reversible, mechanistic driver of neuroinflammation and parkinsonian dysfunction in ATP13A2 deficiency, rather than merely a downstream consequence. It redefines ATP13A2-associated pathology as a glia-mediated metabolic inflammatory disorder. The study establishes spermidine supplementation as a potential therapeutic strategy that acts by restoring polyamine homeostasis via the compensatory transporter Atp13a3, thereby counteracting lysosomal dysfunction, mitochondrial oxidative stress, and neuroinflammation. These findings suggest that polyamine modulation could be a viable therapeutic avenue not only for Kufor-Rakeb syndrome but potentially for a subset of other parkinsonian disorders characterized by similar glial and metabolic dysregulation.