Additional file 1 of Advanced patient-specific microglia cell models for pre-clinical studies in Alzheimer’s disease

Additional file 1. Supplementary figures

Intracellular Distribution of Fluorescent Copper and Zinc Bis(thiosemicarbazonato) Complexes Measured with Fluorescence Lifetime Spectroscopy

The intracellular distribution of fluorescently labeled copper and zinc bis­(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis­(thiosemicarbazonato) complex known as Cu^II(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu^II and Zn^II complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper­(II)­bis­(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper­(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper­(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu^II(atsm) in models of amyotrophic lateral sclerosis and Parkinson’s disease

Neuroprotective Copper Bis(thiosemicarbazonato) Complexes Promote Neurite Elongation

<div><p>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.</p></div

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

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

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

<p>(<b>A</b>) 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>B</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.</p

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

<p>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. (<b>A</b>) Images of pJNK immunofluorescence (<b>B</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.</p

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

<p>The effect of Cu^II(gtsm) (25, 50 and 100 nM, 18 hr) on total neurite numbers and neurite elongation was examined. (<b>A</b>) Cu^II(gtsm) reduced the mean number of neurites per cell in a dose dependent manner (n = 3/treatment). (<b>B</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.</p

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

<p>The effects of CuCl₂, (gtsm)H₂, Cu^II(atsm) and Cu^II(gtsm) (25 and 50 nM) on NGF-treated PC12 cells was assessed. (<b>A</b>) 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>B</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). (<b>C</b>) 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.</p

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

<p>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. (<b>A</b>) Cu^II(gtsm) inhibited total phosphatase activity by 28% (n = 3/treatment). (<b>B</b>) Cu^II(gtsm) inhibited calcineurin activity by 45% (n = /treatment). (<b>C</b>) 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.</p

Inhibition of TDP-43 Accumulation by <em>Bis</em>(thiosemicarbazonato)-Copper Complexes

<div><p>Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable <em>bis</em>(thiosemicarbazonato)copper^II complexes (Cu^II(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper^II (Cu^II(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper^II (Cu^II(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu^II(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu^II(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu^II(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu^II(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu^II(atsm) reduced accumulation of C-terminal (219–414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu^II(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.</p> </div