Neuroprotective copper bis(thiosemicarbazonato) complexes promote neurite elongation

Abstract

Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, Cu(II)(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that Cu(II)(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, Cu(II)(atsm), but at a higher concentration. Induction of neurite elongation by Cu(II)(gtsm) was restricted to neurites within the length range of 75-99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM Cu(II)(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that Cu(II)(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM Cu(II)(gtsm), suggesting a potential link between Cu(II)(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes.

Figure 1. The effect of Cu^II(gtsm) on MTT reduction, LDH release and Cu levels.

The effects of CuCl₂, (gtsm)H₂, Cu^II(atsm) and Cu^II(gtsm) (25 and 50 nM) on NGF-treated PC12 cells was assessed. (A) The treatments used did not affect MTT reduction with the exception of 25 and 50 nM Cu^II(gtsm) that inhibited MTT reduction slightly (n = 4/treatment). (B) LDH analysis of Cu^II(gtsm)-treated cell cultures indicates that concentrations of up to 100 nM can be used with no significant increase in LDH release (n = 4/treatment). (C) ICP-MS results showed that 1 hr exposure to 50 nM Cu^II(gtsm) increased cellular Cu content significantly and at 18 hr produced a significantly higher increase in Cu content than at 1 hr (n = 6/treatment). Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Cu^II(gtsm) effects on neurogeneration in NGF-treated PC12 cells.

The effect of Cu^II(gtsm) (25, 50 and 100 nM, 18 hr) on total neurite numbers and neurite elongation was examined. (A) Cu^II(gtsm) reduced the mean number of neurites per cell in a dose dependent manner (n = 3/treatment). (B) Cu^II(gtsm) induced an increase in neurite elongation (assessed as mean neurite length) (n = 3/treatment). Values are mean ± SEM. **p<0.01, ***p<0.001.

Figure 3. Cu^II(gtsm) effects on neurite elongation of NGF-treated PC12 cells.

(A) The effect of CuCl₂, (gtsm)H₂, Cu^II(atsm) and Cu^II(gtsm) (25 and 50 nM, 18 hr) on neurite elongation (assessed as % neurites 2x or more than cell body width). Cu^II(gtsm) induced a significant increase in neurite elongation (at both concentrations used) whereas the other treatments had no effect (n = 3/treatment). (B) The effect of 50 nM Cu^II(gtsm) on neurite elongation was examined further by grouping neurites according to length (measured in µm). Cu^II(gtsm) induced a significant increase in the number of neurites in the 75–99 µm range. Values are mean ± SEM. *p<0.05, **p<0.01.

Figure 4. The effect of Cu^II(atsm) on Cu levels and neurite elongation.

(A) ICP-MS results showed that 18 hr exposure to 50 nM Cu^II(atsm) increased cellular Cu content slightly while 500 nM had no effect on cellular Cu levels (n = 6/treatment). (B) The effect of Cu^II(atsm) (500 nM, 18 hr) on neurite elongation (assessed as % neurites 2x or more than cell body width). Cu^II(atsm) at 500 nM concentration induced a significant increase in neurite elongation similar to that induced by 50 nM Cu^II(gtsm) (n = 4/treatment). Values are mean ± SEM. *p<0.05, **p<0.01.

Figure 5. The effect of Cu^II(gtsm) on kinase activation in NGF-treated PC12 cells.

Activation of several cell signaling kinases was examined to determine which pathways might be involved in Cu^II(gtsm)-mediated neurite elongation. (A&B) Cu^II(gtsm) treatment decreased activation of ERK, JNK and p38 (n = 3/treatment). Values are mean ± SEM. *p<0.05

Figure 6. Timecourse of Cu^II(gtsm)-mediated ERK and JNK phosphorylation and the effect of specific kinase inhibition on neurite elongation in NGF-treated PC12 cells.

(A & B) Cu^II(gtsm) (18 hr, 50 nM) decreased ERK and JNK activation from the 8 hr timepoint. (C) Neither JNK, ERK nor GSK3 inhibition had an effect on neurite elongation. Values are mean ± SEM. *p<0.05, ***p<0.001.

Figure 7. The localised effect of Cu^II(gtsm) on JNK phosphorylation (pJNK) in NGF-treated PC12 cells.

Cu^II(gtsm)-mediated effect on pJNK was examined using immunofluorescence with rabbit anti-phospho-JNK primary antibody (Cat. # 4668, CST) and the percentage of longer neurites that have a pJNK node was quantified. (A) Images of pJNK immunofluorescence (B) The percentage of neurites that are two times or more than cell body width were increased by Cu^II(gtsm) and the percentage of these longer neurites that have a pJNK node was found to be unchanged (n = 4/treatment). Values are mean ± SEM. **p<0.01.

Figure 8. Effect of Cu^II(gtsm) on phosphatase activity and effect on calcineurin.

Following exposure to Cu^II(gtsm) (50 nM, 18 hr) cells were assayed to determine overall phosphatase activity and also specifically for calcineurin activity. Cells were also exposed to the specific calcineurin inhibitor FK506 at 5 and 10 nM concentrations (18 hr) with and without Cu^II(gtsm) (50 nM) and neurite elongation was assessed. (A) Cu^II(gtsm) inhibited total phosphatase activity by 28% (n = 3/treatment). (B) Cu^II(gtsm) inhibited calcineurin activity by 45% (n = /treatment). (C) Cu^II(gtsm) and 5 nM FK506 each enhanced neurite elongation but when combined, their effects were blocked. At 10 nM concentration the FK506 effect on neurite elongation was not as strong, and again, when combined with Cu^II(gtsm) the enhanced elongation was blocked (n = 4/treatment). Values are mean ± SEM. *p<0.05, **p<0.01.