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

PRIMARY OUTCOME

α-synuclein pathology spread and neurodegeneration

EXPECTED OUTCOMES

1. **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). 2. **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). 3. **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). 4. **Perampanel effect on pathology:** Perampanel treatment will reduce p-α-synuclein pathology by 50±15% in A53T/PFF mice compared to vehicle (p = 0.003, effect size Cohen's d = 0.95). 5. **Microglial activation:** Iba-1+ cell density in SNc will be 2.8±0.6-fold higher in vehicle vs. perampanel groups (p = 0.001), correlating with pathology burden (r = 0.72). 6. **Survival/body weight:** Vehicle-treated A53T/PFF mice will show 12±3% body weight loss from baseline by month 6, while perampanel-treated mice maintain 98±4% of baseline (p = 0.02). 7. **Pharmacokinetics:** Perampanel brain concentrations will reach 0.45±0.12 μM at steady state, consistent with target engagement of AMPA receptors (target: >0.3 μM). ---

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). - **Effect size threshold:** Cohen's d ≥ 0.6 for primary pathology outcome, confirming meaningful biological effect beyond statistical significance. - **Reproducibility:** Independent replication cohort (n=10/group) shows consistent direction of effect with p < 0.10 in confirmatory analysis. - **Dosing adequacy:** Plasma perampanel concentrations confirmed ≥0.3 μM (pharmacokinetic target) in all treated animals at terminal harvest, with <20% coefficient of variation. - **Safety profile:** Perampanel-treated mice show no significant difference in overall survival (≥90% survival in both groups), no treatment-related liver/kidney pathology, and body weight maintained ≥85% of baseline throughout study.

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) **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 ---

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