Figure 1.
Dose-dependent neurotoxicity of berberine in primary neurons.
Primary neurons (cerebellar granule neurons [CGN; DIV7] and hippocampal neurons [HCN; DIV7]) treated with BBR for 6 hours at concentrations between 0.01 and 10 µM indicate a clear dose-dependent loss of neuronal cell viability with IC50 of roughly 3 µM. (A) CGN were stained for visualization of neurite (β-tubulin III, TUJ1) and nuclear (Hoechst 33342) morphology at 20X magnification. (B) Neuronal cell viability assessed by visual scoring of CGN nuclear morphology after 6-hour treatment with BBR. (C) Neuronal cell viability assessed by visual scoring of HCN nuclei morphology after 6-hour treatment with BBR. (D) Neuronal viability of CGN, as assessed by CellTiter-Glo assay measuring cellular ATP content, for 6-hour treatments with the indicated BBR concentrations. (E) Neuronal viability of CGN, as assessed by CellTiter-Glo assay for 24-hour treatments with the indicated concentrations. For A, green is β-tubulin III, blue is Hoechst; the scale bar represents 100 µm. For panels B, D, E, n = 5. For C, n = 3. For B–E, * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 2.
Berberine alters mitochondrial function and morphology, and causes caspase-independent cell death.
BBR causes rapid reduction in cell viability independent of caspase-3 activation, and changes in mitochondrial function and morphology. (A) Neuronal cell viability assessed by visual scoring of nuclear morphology reveals the largest drop in viability to take place between 4 and 6 hours. Pan-caspase inhibitor z-VAD-FMK does not affect BBR toxicity after cotreatment for 6 hours. (B) Western blot (WB) of CGN cell lysates shows caspase-3 cleavage and phosphorylation of c-Jun in serum/potassium deprived-neurons (K5) but not in BBR-treated neurons. Cotreatment with z-VAD-FMK prevents caspase-3 cleavage, but c-Jun remains phosphorylated as it functions upstream of caspase-3 in the neuronal apoptosis pathway. (C, D) WB of CGN lysates show that treatment with 10 µM BBR for 0.5–6 hours (C) or with 0.01–10 µM BBR for 6 hours (D) does not induce cleavage of caspase-3 or phosphorylation of c-Jun. (E) CGN were stained with TOM-20 (top row) or transduced with GFP-OMP25 lentivirus (bottom row) to visualize all mitochondria and neuronal mitochondria, respectively. Glial cells, marked with ‡, display larger nuclei, and lack of TUJ1-staining (top row). Mitochondria in untreated neurons display an elongated shape, and the proportion of rounded mitochondria (white arrows) is increased by BBR treatment. (F) BBR lowers mitochondrial membrane potential in CGN as evaluated by the increased JC-10 monomer/aggregate ratio (515/590 nm). (G) BBR at 3 and 10 µM causes a sharp increase in oxidative stress after 2 hours of treatment as assessed by the CM-H2DCFDA assay. (H) BBR treatment lowers the rate of resazurin reduction, but does not have an additive effect when coupled with maximal complex I inhibition with 10 μM rotenone. Orange *** indicate significant difference between control and BBR, blue *** indicate significant difference between control and rotenone, and red *** between BBR and BBR + rotenone-treated CGN. For E: (top row) blue is Hoechst, green is TOM-20 and red is β-tubulin III and (bottom row) blue is β-tubulin III, green is GFP-OMP25 and red is AIF; the scale bar represents 20 µm. For panels A, F, H, n = 4. For panel G, n = 5. For A, F, G and H, * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 3.
Cyclosporine A partially blocks berberine toxicity.
Pretreatment with CsA partially protects CGN against BBR toxicity. (A) Representative images of CGN pretreated 1 hour with and without 1 µM CsA or FK506 before the addition of 10 µM BBR. (B) Quantification of CGN cell viabilities shown in A. (C) LDH release assay from the conditioned media for the described treatments shows partial protection by CsA, but not for FK506. For A, green is β-tubulin III, blue is Hoechst; the scale bar represents 100 µm. For panel B, n = 4 and for C, n = 3. For B and C, * = p<0.05, ** = p<0.01.
Figure 4.
NMDAR antagonists memantine and MK-801 block BBR-induced cell death.
NMDA receptor antagonists memantine and MK-801 block toxicity caused by 10 µM BBR in CGN and HCN cultures. (A) Representative IF images of CGN (upper row) and HCN (lower row) pretreated with memantine and MK-801 before the addition of 10 µM BBR for 6 hours. (B) Quantification of CGN viabilities shown in A. (C) Quantification of neuronal cell viability of IF images of HCN. For A, green is β-tubulin III, blue is Hoechst; the scale bars represent 100 µm. For panels B and C, n = 3. For B and C, * = p<0.05, *** = p<0.001.
Figure 5.
Berberine sensitizes neurons to glutamate toxicity.
Pretreating CGN 18 hours with 300 nM BBR exacerbates glutamate excitotoxicity. (A) Representative IF images for CGN pretreated with 300 nM BBR for 18 hours before the addition of 20 or 50 µM glutamate for 6 hours. (B) Quantification of CGN cell viabilities shown in A. (C) LDH release assay from the conditioned media for the described treatments shows exacerbation of toxicity by 300 nM BBR with glutamate co-treatments. For A, green is β-tubulin III, blue is Hoechst; the scale bar represents 100 µm. For panels B, n = 3 and panel C, n = 4. For B and C, * = p<0.05, *** = p<0.001.
Figure 6.
Berberine sensitizes neurons to rotenone-induced injury.
Pretreatment with 30 nM BBR is sufficient to sensitize CGN to rotenone toxicity. (A) Representative images of CGN pretreated for 18 hours before the addition of 0.1 or 1 µM rotenone for 6 hours. (B) Quantification of CGN cell viabilities shown in A. (C) LDH release assay shows exacerbation of toxicity by BBR pretreatment. For A, green is β-tubulin III, blue is Hoechst; the scale bar represents 100 µm. For panels B and C, n = 3. For B and C, * = p<0.05, *** = p<0.001.