| In Vitro | In vitro activity: Latrepirdine increases succinate dehydrogenase activity (MTT-assay), mitochondrial membrane potential (DeltaPsim), and cellular ATP levels in primary mouse cortical neurons and human neuroblastoma cells (SH-SY5Y). Latrepirdine enhances mitochondrial function both in the absence and presence of stress and Dimebon-treated cells are partially protected to maintain cell viability. Latrepirdine leads to enhanced mTOR- and Atg5-dependent autophagy in cultured mammalian cells. latrepirdine stimulates MTOR- and ATG5-dependent autophagy, leading to the reduction of intracellular levels of APP metabolites, including Aβ in cultured cells. Latrepirdine stimulates the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Latrepirdine increases intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane in primary neuron.
Cell Assay: N2a cells, stable human cervical carcinoma (HeLa) cells expressing EGFP-LC3, and mouse embryonic fibroblasts (MEFs) derived from wildtype mice or ATG5-/- mice are maintained in “growth medium” (high glucose Dulbecco's modified Eagle's medium supplemented with 10% FBS and 100 units/mL Penicillin/Streptomycin) at 37°C, 5% CO2. N2a cells stably transfected with APPK670N, M671L are maintained in growth medium supplemented with 0.2 mg/mL G418. Cells are washed 1× with ice cold PBS (pH 7.4) then incubated with either Latrepirdine (5 nM, 500 nM or 50 μM) or vehicle (growth medium). Following 3-, 6-, or 24-hour of treatment, cells are washed 1x with ice cold PBS, and collected in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM Pepstatin, 1 mM PMSF, 1% Triton X-100, EDTA-free mini-complete protease inhibitor cocktail tablet) then centrifuged (14,000 RPM) for 15 minutes at 4°C. For time-course experiments, cells are washed 2× with ice-cold PBS (pH 7.4) and incubated for the indicated time in serum-free DMEM containing 50 μg/mL CHX or 50 μg/mL Cycloheximide (CHX)+50 μg/mL Chloroquine (CQ). Baseline (T0) samples are collected immediately prior to treatment |
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| In Vivo | Latrepirdine treatment of TgCRND8 transgenic mice is associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. Latrepirdine administration results in increased levels of the biomarkers thought to correlate with autophagy activation in the brains of TgCRND8 (APP K670M, N671L, V717F) or wild-type mice, and that treatment is associated with abrogation of behavioral deficit, reduction in Aβ neuropathology, and prevention of autophagic failure among TgCRND8 mice. |
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