Long-term perampanel treatment in A53T BAC-SNCA mice with PFF injection

Validation Score: 0.950 Price: $0.50 Lewy body disease A53T BAC-SNCA transgenic mice Status: proposed

What This Experiment Tests

Validation experiment designed to validate causal mechanisms targeting SNCA in A53T BAC-SNCA transgenic mice. Primary outcome: α-synuclein pathology spread and neurodegeneration

Description

This comprehensive experiment evaluated the disease-modifying potential of perampanel in A53T BAC-SNCA transgenic mice injected with PFFs. Mice received PFF injections followed by either perampanel or vehicle treatment for 2 and 9 months after PFF removal. The study assessed α-synuclein pathology spreading from first-order injection sites to second- and higher-order brain regions, as well as neurodegeneration. This experiment directly tests whether perampanel can prevent transsynaptic propagation of α-synuclein pathology and associated neuronal loss.

TARGET GENE

SNCA (Score: 0.605)

MODEL SYSTEM

A53T BAC-SNCA transgenic mice

ESTIMATED COST

TIMELINE

0 months

PATHWAY

protein aggregation, transsynaptic propagation, neurodegeneration

SOURCE

extracted_from_pmid_41508763

PRIMARY OUTCOME

α-synuclein pathology spread and neurodegeneration

Scoring Dimensions

📖 Wiki Pages

NCT04724941: Prodromal Alpha-Synuclein Screeningmechanism SNCA-A53T Alpha-Synuclein Neuronscell SNCA-Overexpressing Dopaminergic Neuronscell snca-proteinprotein SNCA A53Tdisease SNCA Gene Variants and Mutationsdisease SNCA→Alpha-synuclein→Aggregation→Lewy Bodies→Synucpathway SNCA — Alpha-Synucleingene SNCA — Alpha-Synuclein Gene Entity Pagegene SNCA→Alpha-synuclein→Parkinson's Disease Causal Chpathway A53T Alpha-Synuclein Transgenic Mouse Model of Parmodel Brain Regionsindex Neurodegenerationdisease

Protocol

Study Overview

Title: Long-term perampanel treatment in A53T BAC-SNCA mice with preformed α-synuclein fibril (PFF) injection Model: A53T BAC-SNCA transgenic mice (C57BL/6J background) Treatment: Perampanel (AMPA receptor antagonist) or vehicle, administered chronically Induction: Bilateral intrastriatal PFF or vehicle injection at 3 months of age Study Duration: 6 months post-injection (9 months of age at endpoint)

PHASE 1: Animal Ordering and Baseline Baseline (Week -4 to 0)

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Study Overview

PHASE 1: Animal Ordering and Baseline Baseline (Week -4 to 0)

1.1 Animal Ordering

Source: Jackson Laboratory (B6;C3-Tg(SNCA* A53T)23C notice/j)
Genotype: Backup PCR confirmation of human SNCA-A53T transgene
Sex: Male mice (reduces estrus cycle variability)
Age at arrival: 6-8 weeks
Number: n=120 total (see power analysis below)
Group 1: A53T + PFF + Perampanel (n=30)
Group 2: A53T + PFF + Vehicle (n=30)
Group 3: A53T + Vehicle + Perampanel (n=20)
Group 4: WT + PFF + Vehicle (n=20)
Group 5: A53T + PFF + Perampanel (n=20) — satellite cohort for pharmacokinetics

1.2 Baseline Behavioral Testing (Week -2 to 0)

Open field test (baseline locomotor activity)
Rotarod performance test (baseline motor coordination)
Wire hang test (baseline grip strength)
Body weight measurement

PHASE 2: Stereotaxic Surgery (Week 0)

2.1 Pre-operative Preparation

Anesthesia: 2% isoflurane in O₂ at 1 L/min
Analgesia: Carprofen 5 mg/kg SC, administered 30 min pre-operatively
Eye protection: Artificial tear ointment applied

2.2 PFF Preparation

α-Synuclein preformed fibrils (PFF) from recombinant human α-synuclein (rPeptide P-100-1)
Dissolved in PBS at 5 mg/mL, fibrillized by shaking at 37°C, 1000 rpm for 7 days
Sonicated to generate ~50 nm fragments (Active Motif #02802)
Concentration: 5 μg/μL (2.5 μg per side)

2.3 Stereotaxic Coordinates (Bilateral Intrastriatal)

AP: +0.5 mm from bregma
ML: ±2.0 mm from midline
DV: -3.0 mm from skull surface
Volume: 0.5 μL per side, injected at 0.1 μL/min
Needle: 33-gauge, 5 μL Hamilton syringe with pump

2.4 Post-operative Care

Carprofen 5 mg/kg SC q24h for 3 days post-surgery
Recovery monitoring: weight, hydration, wound integrity
Inclusion criteria: Post-operative recovery within 48h, no neurological deficits

PHASE 3: Drug Administration (Week 0 to Week 24)

3.1 Perampanel Preparation

Perampanel (Adamas Pharmaceuticals / Esai Inc.)
Formulation: 0.5% methylcellulose in water
Fresh preparation weekly, stored protected from light at 4°C

3.2 Dosing Regimen

Treatment group: Perampanel 3 mg/kg/day
Control groups: 0.5% methylcellulose vehicle (equal volume)
Route: Oral gavage, once daily at 9:00 AM
Volume: 10 mL/kg
Rationale: 3 mg/kg achieves brain concentrations ~0.5 μM (human equivalent of 8 mg/day clinical dose)

3.3 Compliance Monitoring

Body weight measured weekly
Clinical observations: grooming, posture, activity level
Food/water consumption recorded

PHASE 4: Longitudinal Behavioral Assessment (Months 1, 3, 6 post-surgery)

4.1 Motor Function Tests (Monthly)

4.1.1 Rotarod Test

Apparatus: Columbus Instruments Rotamex-5
Protocol: 4 rpm accelerating to 40 rpm over 300 sec, max trial 600 sec
Trials: 3 trials/day, 30 min ITI, 3 consecutive test days
Outcome: Latency to fall (sec)

4.1.2 Open Field Test

Apparatus: 40×40 cm arena, infra-red tracking (MED-OFA-510)
Duration: 30 min
Parameters: Total distance (cm), vertical rearing, center time (sec)
Lighting: 300 lux

4.1.3 Wire Hang Test

Protocol: Inverted wire mesh (40×40 cm, 1 mm grid)
Latency to fall: Max 300 sec
3 trials, 30 min ITI

4.1.4 Grid Walk Test (Footfault)

Apparatus: Wire mesh floor (12×12 mm grid), 50 cm elevated
Duration: 5 min
Outcome: Footfaults/total steps (%)

4.2 Non-Motor Assessment (Months 3, 6)

4.2.1 Nest Building Test

Individual housing with compressed cotton nestlets (5×5 cm)
24h scoring: 1-5 scale (Shirley et al., 2008)
Photographed at 0h, 2h, 6h, 24h

4.2.2 Olfactory Test

Habituation/dishabituation: Social odor discrimination
3 consecutive presentations of same odor, then novel odor
Sniffing duration measured

PHASE 5: Tissue Collection and Processing (Month 6)

5.1 Terminal Procedure

Terminal body weight recorded
Cardiac perfusion: 0.9% saline + 0.1% heparin under isoflurane anesthesia
Brain extraction: Whole brain, hemispheres separated

5.2 Tissue Dissection

Left hemisphere: Flash frozen in liquid N₂, stored at -80°C
Striatum (for biochemistry)
Substantia nigra (for biochemistry)
Cortex (for biochemistry)
Hippocampus (for biochemistry)
Right hemisphere: Fixed in 4% PFA/PBS for 48h, then cryoprotected in 30% sucrose/PBS
Serial 40 μm sagittal sections on microtome

5.3 Blood Collection (Terminal)

Trunk blood, collected in EDTA tubes
Plasma isolated by centrifugation (2000×g, 15 min, 4°C)
Stored at -80°C for pharmacokinetics

PHASE 6: Endpoint Analysis

6.1 Immunohistochemistry

6.1.1 α-Synuclein Pathology Staining

Primary antibody: Anti-phospho-α-synuclein (Ser129) (Abcam #ab59264, 1:500)
Secondary: Alexa Fluor 488 goat anti-rabbit (Thermo #A-11034, 1:500)
Counterstain: DAPI (1:1000)
Analysis: Stereological unbiased counting (Stereo Investigator, MBF Bioscience)
Regions: Striatum, substantia nigra pars compacta, cortex, hippocampus, amygdala

6.1.2 TH (Tyrosine Hydroxylase) Staining

Primary: Anti-TH (Pel-Freez #P40101, 1:1000)
Secondary: DAB visualization
Outcome: Optical density and stereological neuron count in SNc

6.1.3 Iba-1 (Microgliosis)

Primary: Anti-Iba-1 (Wako #019-19741, 1:500)
Analysis: Cell count and morphology (ramified vs. amoeboid)

6.1.4 GFAP (Astrogliosis)

Primary: Anti-GFAP (Cell Signaling #12389, 1:500)
Analysis: Fluorescence intensity

6.2 Biochemical Analysis

6.2.1 Sarkosyl Fractionation

Sequential extraction: TBS-soluble, TBS-insoluble/sarkosyl-soluble, sarkosyl-insoluble
α-Synuclein detection in each fraction by Western blot

6.2.2 ELISA Quantification

Total α-synuclein: BioLegend #844504
Phospho-α-synuclein (Ser129): Abcam #ab190805
Mouse α-synuclein (endogenous): R&D Systems #DY5928

6.2.3 Western Blot

12% SDS-PAGE, 20 μg protein/lane
Primary: Anti-α-synuclein (BD #610787, 1:1000)
Detection: ECL chemiluminescence (Bio-Rad #1705061)
Quantification: Image Lab software, normalized to β-actin (Cell Signaling #3700)

6.3 Pharmacokinetics

LC-MS/MS analysis of perampanel plasma concentrations
Brain penetration assessment (brain/plasma ratio)

6.4 Statistical Power

Primary endpoint: p-α-synuclein Ser129 burden in SNc
Expected effect size: 30% reduction (Cohen's d = 0.8)
α = 0.05, power = 0.80 → n = 26/group
Accounting for 15% attrition → n = 30/group

Expected Outcomes

Motor deficit onset: Vehicle-treated A53T/PFF mice will show significant rotarod impairment by 3 months post-injection, with latency to fall reduced to 45±12 sec compared to baseline of 180±15 sec (p < 0.001).

Pathology burden: Vehicle-treated A53T/PFF mice will exhibit p-α-synuclein (Ser129) pathology covering 35±8% of SNc area, with >10-fold increase over WT/PFF controls (p < 0.0001).

Neurodegeneration: TH+ neuron count in SNc will be reduced by 40±10% in vehicle-treated A53T/PFF mice versus perampanel-treated A53T/PFF mice (85±12% survival, p = 0.002).

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Success Criteria

Primary endpoint: Perampanel-treated A53T/PFF mice demonstrate ≥40% reduction in p-α-synuclein (Ser129) pathology in SNc compared to vehicle controls (p < 0.05, Mann-Whitney U test with Bonferroni correction).
Neuroprotection: TH+ neuron counts in SNc are preserved ≥70% in perampanel group versus 50% in vehicle group (p < 0.01, two-way ANOVA with Tukey post-hoc).
Motor preservation: Perampanel-treated mice maintain ≥60% baseline rotarod performance at 6 months versus ≤35% in vehicle group (p < 0.01, repeated measures ANOVA).

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Related Hypotheses (6)

Neuronal Subtype-Specific Alpha-Synuclein Expression Normalization0.571

Exosomal SNCA Propagation from Lysosome-Compromised Neurons Contains a Distinct Lysosomal Proteome S0.777

SNCA Aggregation Hijacks the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via 0.690

m6A Hypermethylation of SNCA mRNA Stabilises Alpha-Synuclein Transcript and Promotes Lewy Pathology0.662

Physiological SCFAs may reduce alpha-synuclein burden primarily through a gut-first or ENS-first mec0.650

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