Experiment ID: PUMPS-PD-001
Hypothesis: Proteasome-Ubiquitin System Dysfunction Hypothesis in Parkinson's Disease
Primary Objective: Validate that UPS dysfunction is a primary driver of alpha-synuclein aggregation and dopaminergic neurodegeneration in PD
Study Type: Multi-phase translational study (preclinical + clinical)
Objective: Measure baseline proteasome activity in PD patient-derived cells
Models:
- iPSC-derived dopaminergic neurons from PD patients (LRRK2 G2019S,GBA, idiopathic)
- Age-matched healthy controls
- Isogenic controls with gene corrections
Endpoints:
- 20S proteasome chymotrypsin-like activity (fluorescent substrate)
- 20S proteasome trypsin-like activity
- 20S proteasome caspase-like activity
- 26S proteasome ATP-dependent activity
- Ubiquitin conjugate accumulation (Western blot)
- Alpha-synuclein turnover rates (pulse-chase)
Sample Size: n=10 PD iPSC lines, n=10 controls
Objective: Determine if UPS impairment specifically affects alpha-synuclein degradation
Methods:
- Proteasome inhibition (MG132, bortezomib) dose-response
- Alpha-synuclein half-life measurement with cycloheximide chase
- Ubiquitination status of alpha-synuclein
- Interaction with autophagy compensation
Objective: Validate UPS dysfunction as upstream driver in vivo
Models:
- A53T alpha-synuclein transgenic mice
- UCHL1 mutant mice (UCHL1^S18Y knock-in)
- Proteasome hypomorphic mice (PSMA3^D7/D7)
- Combination models
Interventions:
- Proteasome activators (natural compounds: EGCG, ursolic acid)
- Deubiquitinase modulators (UDCA, gene therapy)
- Proteasome subunit overexpression
Endpoints:
- Behavioral assessment (rotarod, cylinder test, gait analysis)
- Proteasome activity in substantia nigra
- Alpha-synuclein aggregation (IHC, Western blot)
- Dopaminergic neuron survival (TH+ neuron counts)
- Motor performance correlation
Objective: Confirm feed-forward loops between UPS dysfunction and alpha-synuclein
Measurements:
- Proteasome subunit composition changes
- Ubiquitin cascade alterations
- Autophagy compensation markers
- Mitochondrial function correlation
Objective: Validate peripheral proteasome activity as PD biomarker
Cohorts:
- Early PD (n=100)
- Moderate PD (n=100)
- Advanced PD (n=100)
- Healthy controls (n=100)
- Other neurodegenerative disease controls (n=50 each: AD, ALS)
Sample Types:
- Peripheral blood mononuclear cells (PBMCs)
- CSF proteasome activity
- Skin fibroblasts
- Exosomes
Endpoints:
- PBMC proteasome activity correlation with disease severity
- CSF proteasome activity and ubiquitin levels
- Predictive value for progression
- Specificity for PD vs. other diseases
Objective: Demonstrate target engagement of proteasome-enhancing interventions
Intervention: Dietary supplementation with proteasome-enhancing compounds
Endpoints:
- Peripheral proteasome activity change
- Biomarker correlation with motor outcomes
- Safety profile
| Endpoint |
Method |
Expected Result |
| Proteasome activity |
Fluorometric assay |
Reduced in PD vs. controls |
| Alpha-synuclein half-life |
Pulse-chase |
Prolonged in PD neurons |
| Ubiquitin conjugates |
Western blot |
Increased in PD |
| Dopaminergic neuron survival |
TH+ IHC |
Correlation with proteasome activity |
- Autophagy compensation markers (LC3, p62)
- Mitochondrial function (OCR, membrane potential)
- Neuroinflammation markers (IL-6, TNF-α in CSF)
- Motor function correlation (UPDRS, MoCA)
- Primary: t-test/Wilcoxon for proteasome activity (PD vs. controls)
- Correlation: Spearman correlation with UPDRS scores
- Survival analysis: Cox regression for progression
- Power: 80% power to detect 30% reduction at α=0.05
- Monitor for protein overload toxicity with proteasome activators
- Assess hepatic and renal function
- Document any unexpected behavioral changes
| Category |
Cost (USD) |
| Personnel |
$300,000 |
| iPSC lines |
$50,000 |
| Reagents |
$80,000 |
| Animal work |
$150,000 |
| Clinical sample collection |
$100,000 |
| Total |
$680,000 |
- Month 1-6: In vitro experiments
- Month 6-12: Preclinical animal studies
- Month 12-18: Clinical biomarker validation
- Month 18-24: Therapeutic pilot study
- Month 24: Final analysis and publication
- Demonstrate >30% reduction in proteasome activity in PD dopaminergic neurons
- Show proteasome activity correlates with alpha-synuclein burden
- Establish peripheral biomarker with >70% specificity for PD
- Identify lead proteasome-enhancing compound for further development