Copper dependent ERK1/2 phosphorylation is essential for the viability of neurons and not glia

Kaustav Chakraborty; Sumanta Kar; Bhawana Rai; Reshma Bhagat; Nabanita Naskar; Pankaj Seth; Arnab Gupta; Ashima Bhattacharjee

doi:10.1093/mtomcs/mfac005

. 2022 Jan 22;14(4):mfac005. doi: 10.1093/mtomcs/mfac005

Copper dependent ERK1/2 phosphorylation is essential for the viability of neurons and not glia

Kaustav Chakraborty ¹, Sumanta Kar ², Bhawana Rai ³, Reshma Bhagat ^4,², Nabanita Naskar ⁵, Pankaj Seth ⁶, Arnab Gupta ⁷, Ashima Bhattacharjee ^8,^✉

PMCID: PMC8975716 PMID: 35150272

Abstract

Intracellular copper [Cu(I)] has been hypothesized to play role in the differentiation of the neurons. This necessitates understanding the role of Cu(I) not only in the neurons but also in the glia considering their anatomical proximity, contribution towards ion homeostasis, and neurodegeneration. In this study, we did a systematic investigation of the changes in the cellular copper homeostasis during neuronal and glial differentiation and the pathways triggered by them. Our study demonstrates increased mRNA for the plasma membrane copper transporter CTR1 leading to increased Cu(I) during the neuronal (PC-12) differentiation. ATP7A is retained in the trans-Golgi network (TGN) despite high Cu(I) demonstrating its utilization towards the neuronal differentiation. Intracellular copper triggers pathways essential for neurite generation and ERK1/2 activation during the neuronal differentiation. ERK1/2 activation also accompanies the differentiation of the foetal brain derived neuronal progenitor cells. The study demonstrates that ERK1/2 phosphorylation is essential for the viability of the neurons. In contrast, differentiated C-6 (glia) cells contain low intracellular copper and significant downregulation of the ERK1/2 phosphorylation demonstrating that ERK1/2 activation does not regulate the viability of the glia. But ATP7A shows vesicular localization despite low copper in the glia. In addition to the TGN, ATP7A localizes into RAB11 positive recycling endosomes in the glial neurites. Our study demonstrates the role of copper dependent ERK1/2 phosphorylation in the neuronal viability. Whereas glial differentiation largely involves sequestration of Cu(I) into the endosomes potentially (i) for ready release and (ii) rendering cytosolic copper unavailable for pathways like the ERK1/2 activation.

Keywords: neuron, glia, copper, Wilson, ERK1/2, differentiation

Graphical Abstract

Introduction

The brain is the largest storehouse of copper, second to the liver. Copper level varies in different regions of the brain as well as at different stages of the development. The copper level in the subventricular zone of the rat brain increases in an age dependent manner.¹ However, copper level decreases with age in the human brain.² Variation in the level, at the developmental stages, reflects changes in its utilization and its importance. Besides copper distribution also varies in the different regions of the brain,³ thereby reflecting the heterogeneity in its utilization. However, the exact nature of utilization of Cu(I) towards triggering the pathways during the neuronal and glial differentiation is not well understood. The critical role of the maintenance of the copper level in the CNS is emphasized by the occurrence of CNS dysfunctions associated with the copper homeostasis disorders. Mutation in the copper transporting ATPase ATP7B leads to accumulation of the excess intracellular copper in the liver, kidney, and brain known as Wilson disease (WD).⁴ Apart from the hepatic symptoms, WD involves neuropsychiatric symptoms.⁵ Defects in ATP7A causes three X-linked recessive disorders—Menkes disease, occipital horn syndrome, and spinal muscular atrophy. Menkes disease, caused by systemic copper deficiency, involves atrophy of grey and white matter involving focal degeneration of all layers of the cerebral cortex extending to other regions of the CNS. The degeneration involves both the neurons and the astrocytes.⁶ The mo-br mice exhibit distinct neuronal and glial abnormalities suggesting role of intracellular copper in both cell types. The abnormal axonal extension and synaptogenesis in these mice demonstrates role of copper towards the neurite extension.⁷ Moreover, ATP7A expression has also been detected in the developing neurites during synaptogenesis suggesting involvement of copper mediated pathways in the neurodevelopment related to learning and memory.⁸ This emphasizes the necessity to investigate the role of the intracellular copper homeostasis in the neuronal and glial differentiation.

In this study, we have investigated the changes in the cellular copper homeostasis during the neuronal and glial differentiation using two cell-based model system. Neuronal differentiation accompanies increased mRNA for the plasma membrane copper importer CTR1 and increased intracellular copper. Utilization of this excess copper is evident from the retention of the primary copper ATPase-ATP7A in the trans-Golgi network (TGN). One of the specific pathways triggered by the intracellular copper is phosphorylation of ERK1/2 during the neuronal differentiation. Our study demonstrates that ERK1/2 phosphorylation is essential for the viability of the neurons. On the other hand, the differentiated glia has low intracellular copper and subsequent downregulation of the ERK1/2 phosphorylation. Despite low intracellular copper in the glia, ATP7A localizes into the RAB 11 positive vesicles along the neurite processes.

Results

Differentiation of the PC-12 cells (neuron) involves increased intracellular copper in contrast to the differentiation of the C-6 (glia) cells

PC-12 cells are plated on collagen. To induce differentiation, cells are treated with nerve growth factor (NGF) as described in the methods. The cells showed increase in the neurite length (Fig. 1A) and 5.06-fold higher expression of β-III tubulin (Fig. 2A). However, 0.86-fold lower expression of the glial fibrillary acidic protein (GFAP) demonstrates the purity of the neuronal lineage (Fig. 2A right). C-6 cells were plated and treated with dBcAMP and theophyllin for 48 h, whereby they demonstrated progressive neurite growth and branching of the neurites (Fig. 1A, B). 4.65-fold higher mRNA level for GFAP and 0.8-fold lower mRNA level for β-III tubulin confirm the glial differentiation (Figs. 1B and 2B). The GFAP expression was further confirmed by confocal microscopy (Fig. 1B). The PC-12-derived neurons have 3.18× increased intracellular copper compared to the undifferentiated ones (Fig. 2C). Increased intracellular copper accompanying the neuronal differentiation suggests its importance towards the process. However, the glia, differentiated in serum deprived condition contains 5.4× lower intracellular copper compared to the undifferentiated cells (Fig. 2D).

Fig. 1 — Differentiation of the PC-12 and C-6 cells into neuron and glia, respectively. (A) (upper panel) Bright field images (at 20× magnification) of undifferentiated and differentiated PC-12 cells. Increased neurite length is observed following treatment with NGF. (A) (lower panel) Bright field images (at 40× magnification) of undifferentiated and differentiated C-6 cells. Increased length and branching of neurites are noticed with the treatment of dBcAMP and theophylline. (B) Undifferentiated and differentiated C-6 cells immunostained for GFAP. Increased expression of GFAP along the neurites confirms glial differentiation. Image acquired at 63× optical zoom; scale bar, 5 μm.

Fig. 2 — Changes in the cellular copper homeostasis accompanying the neuronal and the glial differentiation. (A) Increased mRNA for β-III tubulin (left) and decreased mRNA for GFAP (right) confirm differentiation of the PC-12 cells into the neurons (number of experimental replicates––4). Differentiated PC-12 cells have 5.06 ± 1.9-fold higher expression of β-III tubulin and 0.86 ± 0.06-fold lower expression of GFAP compared to the undifferentiated cells (**P-value—0.0015 in both cases). (B) Increased mRNA for GFAP and decreased mRNA for β-III tubulin confirm differentiation of the C-6 cells into the glia (number of experimental replicates––4). Differentiated C-6 cells have 4.65 ± 0.74-fold higher expression of GFAP and 0.8 ± 0.09-fold lower expression of β-III tubulin compared to the undifferentiated cells (**P-value—0.0028, *P-value—0.0257). (C) Level of the intracellular copper in the undifferentiated (4.53 ± 0.11 ng/mg protein) and the differentiated (14.38 ± 0.18 ng/mg protein) PC-12 cells as determined by ICP-MS (number of experimental replicates—3, ****P-value ˂0.0001). (D) Level of intracellular copper in the undifferentiated (2.87 ± 0.48 ng/mg protein) and differentiated (0.53 ± 0.18 ng/mg protein) C-6 cells as determined by ICP-OES (number of experimental replicates—4, ***P-value—0.0004). (E) Level of the intracellular copper in the nuclear, mitochondrial, and post mitochondrial fractions in the undifferentiated and differentiated PC-12 cells as determined by ICP-OES (number of experimental replicates—2). Level of copper is O.26 ± 0.01 ng/mg protein (undifferentiated cells) and 0.24 ± 0.01 ng/mg protein (differentiated cells) in the nuclear fraction. Level of copper is 0.21 ± 0.07 ng/mg protein (undifferentiated cells) and 1.32 ± 0.02 ng/mg protein (differentiated cells) (**P-value—0.0018). Level of copper is 0.55 ± 0.27 ng/mg protein (undifferentiated cells) and 6.5 ± 0.23 ng/mg protein (differentiated cells) in the post mitochondrial fraction (**P-value—0.0016). (F) Relative expression of the CTR1 mRNA in the undifferentiated and the differentiated PC-12 cells (number of experimental replicates––3). The differentiated PC-12 cells have 3.3 ± 1.09-fold higher expression of the CTR1 mRNA compared to the undifferentiated cells (**P-value—0.0043).

The distribution of the intracellular copper to the major intracellular locations in the PC-12-derived neurons was investigated. Intracellular copper was measured and compared in the nucleus, mitochondria, and the post mitochondria fraction (containing the cytosol and other membrane encapsulated organelles including the Golgi). The PC-12-derived neurons did not show any changes in intracellular copper in the nuclear fraction compared to the undifferentiated ones (Fig. 2E). Significant copper accumulation (6.2× higher) was noticed in the mitochondria of the differentiated neurons (Fig. 2E). Highest accumulation of the intracellular copper was noticed in the post mitochondria fraction, which contains mainly the cytosol and the Golgi. Intracellular copper, in this fraction, is 12× higher in the differentiated neurons compared with the undifferentiated cells (Fig. 2E). This demonstrates that the bulk of the intracellular copper is utilized in the cytosol and the Golgi-based secretory pathway. Increased utilization of the intracellular copper in the TGN based secretory pathway has been previously demonstrated in the SH-SY5Y-derived neurons.⁹ To further confirm this, localization of the intracellular labile copper was investigated using the Copper Fluor-4 (CF-4). The CF-4 has been demonstrated to bind to intracellular copper.¹⁰ Copper, in the PC-12-derived neurons, is localized mostly in the soma (Fig. 3 lower panel). The neurites did not show appreciable CF-4 signal. Soma includes both the Golgi and cytosol. Therefore, the copper estimation and CF-4 experiments demonstrate localization and hence utilization of the copper mainly in the cytosol and the Golgi-based secretory pathway in the PC-12-derived neurons.

Fig. 3 — Localization of labile copper in the differentiated (PC-12) neurons under basal condition. Differentiated PC-12 cells show presence of copper in the entire soma, including the perinuclear region, as visualized by CF-4. Absence of detectable signal in the cells incubated with control CF-4 (upper panel) demonstrates the specificity of CF-4 for intracellular copper.

Increased intracellular copper parallels increased expression of CTR1 mRNA in the differentiated PC-12 (neuron) cells but not in differentiated C-6 (glia) cells

To further correlate how the intracellular copper, in the differentiated neurons, is distributed to the different intracellular locations, we investigated the mRNA expression of different chaperones and transporters designated to deliver copper to the different intracellular locations. 3.3-fold higher level of the mRNA for CTR1 was observed (Fig. 2F) in the differentiated PC-12 cells. This suggests that differentiation of PC-12 cells into the neurons involves an increased expression of the plasma membrane copper transporter CTR1 leading to the entry of copper into the cells. Moderate increase in Copper Chaperone for SOD1 (CCS) and mitochondrial chaperone COX17 was observed (Supplementary Fig. S1A) in the PC-12-derived neurons. COX17 delivers Cu(I) to SCO1, SCO2, and COX11 for incorporation into the Cu_A and Cu_B sites of cytochorome C oxidase on the inner membrane of the mitochondria. COX17 localizes in the cytosol and mitochondria of the yeast cells¹¹ and mainly in the mitochondria of the mammalian cells.¹² CCS abundance has been detected in the neurons of human and rodent brain.¹³ Moderately high expression (1.77-fold) of the mRNA for the mitochondrial copper importer, SLC25A3¹⁴ is also observed in the PC-12-derived neurons (Supplementary Fig. S1A). Higher mRNA level for CCS, COX17, and SLC25A3 might be related to the increased mitochondrial copper demand accompanying the differentiation. This is further supported by the observation of higher mitochondrial copper level in the PC-12-derived neurons (Fig. 2E). Although intracellular copper is high, expression of mRNA for the primary copper ATPase-ATP7A and its delivery chaperone ATOX1 did not change (Supplementary Fig. S1A). Neither undifferentiated nor differentiated (PC-12) neurons nor (C-6) glia expressed ATP7B (data not shown). Higher expression of the copper chaperone ATOX1 along with ATP7A has been previously demonstrated to accompany the differentiation of the SH-SY5Y cells along with the increase in the intracellular copper.⁹ But such an increase in the expression of ATOX1 and ATP7A is not observed in the PC-12-derived neurons, despite an increase in the intracellular copper in these neurons. This can be due to the fact that the neurons, in this case, originate from a different cell type. However, higher copper level has been observed in the post mitochondria fraction (Fig. 2E) containing the cytosol and Golgi compartments in the PC-12-derived neurons. Experiments with the CF-4 probe demonstrate localization of the copper in the soma region, which includes its localization in the perinuclear Golgi region (Fig. 3). This suggests that the bulk of the cytosolic copper, in the PC-12-derived neurons, might be transported into the TGN like the SH-SY5Y-derived neurons. But the results also suggest presence of high level of copper in the cytosol suggesting its utilization in this compartment as well.

In contrast, glial differentiation does not show any change in the mRNA expression for any chaperones and transporters including CTR1 (Supplementary Fig. S1B). Differentiation of C-6 cells into the glia is performed in copper deprived media (without serum), and the differentiated glia contain less intracellular copper (Fig. 2D). The unchanged level of the CTR1 mRNA, under low intracellular copper, is intriguing. It suggests that maintenance of the low intracellular copper is crucial for the glial differentiation.

ATP7A shows different localization in the differentiated PC-12 (neuron) and C-6 (glia) cells

ATP7A traffics to the vesicles under high intracellular copper. Since the PC-12-derived neurons have high intracellular copper, we wanted to investigate if ATP7A shows vesicular localization in the PC-12-derived neurons. Interestingly, ATP7A showed localization exclusively in the TGN (Fig. 4A) despite high intracellular copper. Quantitation of the colocalization of ATP7A with TGN marker, TGN38 showed significantly increased colocalization in the differentiated PC-12 cells compared to the undifferentiated ones (Fig. 4B). But whether ATP7A could perform copper dictated anterograde trafficking under very high intracellular copper needed to be tested in these cells. We utilized Copper Fluor (CF-4) to visualize intracellular labile copper in the differentiated PC-12 cells. CF-4 has been demonstrated to be able to detect labile copper in the living cells.¹⁰ Differentiated PC-12 cells, treated with 100 μM CuCl₂ for 2 h, demonstrated a higher intensity of CF-4 signal, suggesting higher probe incorporation compared to the basal condition (Fig. 5) demonstrating intracellular copper accumulation. Concurrently, ATP7A showed vesicular localization in addition to the TGN in the differentiated cells treated with 25–100 μM CuCl₂ (Fig. 6). This demonstrates that ATP7A, in the differentiated neurons, is capable of copper dictated anterograde trafficking from the TGN when treated with high doses of externally added copper. Further, this demonstrates that the high intracellular copper in these neurons is not excess and that there might be utilization of the copper by multiple physiological pathways. High doses of externally added copper for short time intervals do not permit its intracellular utilization. On the contrary, the physiological high copper in the differentiated neurons is utilized towards various pathways thereby retaining ATP7A in the neurons. This utilization can be multiple. Firstly, the neuronal copper can be utilized towards the differentiation, thereby retaining ATP7A in the TGN instead of the vesicles. Secondly, a bulk of the cytosolic copper can be transported into the Golgi-based secretory pathway as already demonstrated⁹ during the neuronal (SH-SY5Y) differentiation.

Fig. 4 — Localization of ATP7A in the undifferentiated and differentiated PC-12 (neuron) cells. (A) Localization of ATP7A with TGN38 in the PC-12 cells. ATP7A colocalizes primarily with TGN38 in the perinuclear (soma) area. (B) Quantitation of the colocalization of ATP7A with TGN38 in the undifferentiated and differentiated PC-12 cells. PCC has been measured and represented [n = 17 cells (undifferentiated), n = 17 cells (perinuclear for differentiated); ***P-value—0.0005].

Fig. 5 — Intracellular copper accumulation in the differentiated PC-12 cells in response to 100 μM CuCl₂ treatment. Intracellular labile copper has been visualized after incubating the cells with CF-4. Images have been acquired at 63× optical magnification with 2.99 (upper panel) and 1.8 (lower panel) digital zoom.

Fig. 6 — Trafficking of ATP7A from the TGN into vesicles in response to CuCl₂ treatment in the differentiated PC-12 cells.

In the differentiated C-6 glia, ATP7A shows colocalization with TGN38 and Golgin97 in the perinuclear location (Fig. 7A, C). In addition, ATP7A also localizes into non-Golgi vesicular structures (Fig. 7A–C) along the neurites. Such vesicular localization of ATP7A is intriguing as the differentiated glia has low intracellular copper. TGN has been demonstrated to interact with the recycling endosomal system in the neurons playing important role in the trafficking of the synapse related proteins.^9,15 Since the role of copper has been implicated in the maturation of the synaptic vesicles and their release,^16,17 we wanted to investigate if these ATP7A vesicles, in the glial neurites, could be TGN compartments. ATP7A, in the neurites, does not colocalize with TGN38 and Golgin97 (Fig. 7B, C).

To confirm this, we performed Stimulated Emission Depletion Microscopy (STED) to visualize these structures. At such a resolution, ATP7A in the vesicles is not seen to colocalize with TGN38 and Golgin97, suggesting that these are not extensions of the TGN (Supplementary Fig. S2A). This suggests that ATP7A exits the TGN and localizes into the vesicles in the glia, despite low intracellular copper. Further, we investigated if these vesicles are part of the canonical TGN exit pathway for ATP7B.

Lysosomal exocytosis has been demonstrated to be the mechanism of ATP7B mediated copper export.¹⁸ Although not demonstrated for ATP7A, we wanted to investigate if these vesicular structures play a role in glial copper export involving the lysosomal route. We observed that the vesicular ATP7A in the neurites does not colocalize with LAMP1, suggesting that these are not lysosomes (Supplementary Fig. S3).

ATP7B has been reported to sort from the TGN to the basolateral endosomes prior to trafficking to the apical domain of the polarized WIF-B cells.¹⁹ It has been further demonstrated that MyosinVb localizes ATP7B to the gamma tubulin rich domains prior to apical sorting at high copper.²⁰ Therefore, it is probable that post-TGN-exit, both ATP7A and ATP7B follow a partially overlapping basolateral trafficking itinerary. The trafficking itinerary of ATP7A has not been studied in the polarized system. Since glia are unique polarized cells, we wanted to investigate if ATP7A vesicles are in the gamma tubulin rich domains. But vesicular ATP7A, in the glia, does not colocalize with gamma tubulin (Supplementary Fig. S4) suggesting that the vesicles are not part of the Microtubule Organizing Centre (MTOC).

We further investigated if ATP7A, in these vesicles, localizes to the late endocytic pathway²¹ for ready release of copper. STED microscopy showed no colocalization of ATP7A with Rab7 (Supplementary Fig. S2B). Further, we investigated if the ATP7A containing vesicular structures in the glial processes represent RAB11²² positive recycling endosomes. We observed colocalization of ATP7A and RAB11 in the vesicles along the glial (neurite) processes (Fig. 8, Supplementary Fig. S5). Additionally, we also observed considerable colocalization of ATP7A with RAB11 in the perinuclear region (Fig. 8A, B, Supplementary Fig. S5) of both differentiated and undifferentiated glial cells. This suggests that, under basal conditions, ATP7A localizes to the recycling endosomes in addition to the TGN, in these cells. These RAB11 positive compartments extend to the neurites with differentiation and ATP7A resides into some of those compartments in the glia (Fig. 8A, B). Super-resolution microscopy (STED) shows ATP7A either in colocalization or in juxtaposition with RAB11 both in the perinuclear (Fig. 9A) and along the neurites in the glia (Fig. 9B).

Fig. 8 — Localization of ATP7A in the RAB11 positive compartment in the perinuclear region (soma) and along the neurites in the differentiated C-6 (glia) cells. (A) Localization of ATP7A with RAB11 as viewed by confocal microscopy. ATP7A colocalizes with RAB11 both in the perinuclear region and the neurites. (B) Quantitation of the colocalization of ATP7A with RAB 11 in the undifferentiated and differentiated (both perinuclear soma and neurite) PC-12 cells. PCC has been measured and represented [n = 17 cells (undifferentiated), n = 17 cells (perinuclear for differentiated), and n = 27 (neurites)].

Fig. 9 — Colocalization of the ATP7A with RAB11 in the perinuclear (soma) and neurite in differentiated (C-6) glia as seen in STED microscopy. (A) (upper): ATP7A colocalizes with RAB11 in the perinuclear soma. (A) (lower): A Magnified area from the upper panel shows both ATP7A overlapping and juxtaposing with RAB11 demonstrating its presence in the RAB11 positive compartment. (B) (upper): ATP7A colocalizing with RAB11 positive compartments in the neurites. (B) (lower): Magnified area from the upper panel shows colocalization and juxtaposition of ATP7A with RAB11.

RAB11 and TGN38+Golgin97 positive compartments overlap considerably in the perinuclear region of the differentiated glia (Fig. 10C, D, Supplementary Fig. S6). In addition to localizing on the overlapping compartments, ATP7A also resides on exclusive RAB11 positive compartments in the perinuclear glia (Fig. 10C, D). Such overlapping of TGN38+Golgin97 and RAB11 compartments is not observed in the neurites (Fig. 11B, Supplementary Fig. S6), where ATP7A localizes only on RAB11 compartment (Fig. 11). Together, the results demonstrate that ATP7A resides on both TGN38+Golgin97 and RAB11 positive compartments, including the overlapping compartments in the perinuclear glia. With differentiation, TGN38+Golgin97 and RAB11 positive compartments extend into neurites in the glia, where the compartments gain exclusivity (no overlap of TGN38+Golgin97 and RAB11). Among them, ATP7A localizes only on the RAB11 positive compartment in the neurites. Increased localization into the recycling endosomes, with differentiation, suggests a role of these vesicles in either ready release or storage of the copper. However, ATP7A in the vesicles does not colocalize with TfR2 (Supplementary Fig. S7). Recycling endosomal markers, RAB11 and TfR, have been demonstrated to have distinct intracellular localization in the PC-12-derived neurons.²³ ATP7A, in the glial neurites, also does not colocalize with early endosomal markers EEA1 (Supplementary Fig. S8) and RAB5 (Supplementary Fig. S9).

Fig. 11 — ATP7A localizes into only the recycling endosomes (RAB11) in the neurites of the differentiated C-6 (glia) cells. (A) Localization of ATP7A, TGN38+Golgin97 and RAB11 in the neurites of the differentiated C-6 (glia). (B) (left) Merged (ATP7A, TGN38+Golgin97, RAB11) image demonstrating colocalization of ATP7A (green) with RAB11 (red) and not TGN38+Golgin97 (cyan) in the neurite. The nuclei have been indicated in blue. (B) (right) An enlarged region, in the neurite showing colocalization (yellow) of ATP7A (green) with RAB11 (red) and lack of any colocalization of ATP7A with TGN38+Golgin97 (cyan).

Vesicles in the neurite processes, in the differentiated (C-6) glia, contain copper

ATP7A is observed to localize to vesicles in the glial neurites, under basal condition. Some of these ATP7A containing vesicles colocalize with the RAB11 positive recycling endosomes. We wanted to investigate if these vesicles contain copper. Live C-6-derived glia were incubated with the molecular probe CF-4 and control Copper Fluor-4 (control CF-4)¹⁰. Control CF-4, having a similar dye scaffold, does not detect copper and shows little or no signal in the differentiated C-6 glia (Fig. 12 upper panel). However, CF-4 signal has been detected in both the soma (Fig. 12 middle panel) and the neurites (Fig. 12 middle and lower panel, Supplementary video 1). This demonstrates that the vesicles in the glial neurites contain labile copper. Therefore, the ATP7A containing vesicles in the glial neurites might also contain copper. To further confirm this, the copper dictated retrieval of these ATP7A containing vesicles to the TGN was examined.

Fig. 12 — Localization of labile copper in the differentiated (C-6) glia. Differentiated C-6 cells show presence of copper both in the soma and neurite (middle panel) and in the neurites (lower panel) as visualized by CF-4. Absence of detectable signal in the cells incubated with control CF-4 (upper panel) demonstrates the specificity of CF-4 for intracellular copper.

Localization of the ATP7A into the RAB 11 positive vesicles in the differentiated C-6 (glia) cells can be retrieved back to the TGN upon copper chelation

Anterograde trafficking of ATP7A from the cytosolic vesicles to TGN has been demonstrated at low or basal intracellular copper. ATP7A localizes into the RAB11 positive recycling endosomes in the differentiated glia. We wanted to investigate if this vesicular localization of ATP7A, along the glial processes, can be reversed to TGN upon further lowering of the intracellular copper level. To investigate this, C-6 cells were differentiated and treated for 2 h with 30 μM copper chelator ammounium tetrathiomolybdate (TTM) to chelate intracellular copper. Copper chelation led to the disappearance of the ATP7A from the vesicular structures and localized it entirely into the TGN (Fig. 13). This suggests that the vesicular ATP7A can be retrieved back to the TGN upon further lowering of the intracellular copper level. Further, we investigated whether this vesicular ATP7A is crucial for the glial differentiation. C-6 cells were plated, grown, and differentiated in the presence of 30 μM TTM. Also, under this condition, TTM treatment did not affect the differentiation as is evident from the neurite generation (Fig. 14A) and unaffected level of GFAP mRNA (Fig. 14B). These cells also showed complete depletion of the ATP7A containing vesicles in the glial processes with colocalization of ATP7A entirely with TGN38+Golgin97 (Fig. 14C). This confirms that ATP7A containing vesicles, in the glial neurites, contain labile copper. Further, it suggests that the localization of ATP7A into the RAB11 positive vesicles, in the glial neurites, does not play a role towards the differentiation of the glial cells.

Fig. 13 — Retrieval of ATP7A from the RAB 11 positive vesicles (neurites) to the TGN (perinuclear) upon copper chelation. C-6 cells were differentiated as described, and then treated with 30 μM TTM for 2 h. Disappearance of the ATP7A containing vesicles along the neurites and retention of ATP7A in the TGN38 and Golgin97 positive TGN is observed in response to the copper chelation.

Fig. 14 — Chelation of the intracellular copper does not affect the differentiation of the C-6 cells into glia. C-6 cells are plated and differentiated in the presence of 30 μM Ammonium Tetrathiomolybdate (TTM). (A) Bright field image of the C-6 cells differentiated in presence and absence of TTM. (B) Relative mRNA level of GFAP in the glial cells cultured and differentiated in presence and absence of TTM (no of experimental replicates—3). C-6 cells cultured and differentiated in the presence of TTM showed similar normalized expression (4.97 ± 0.9 fold) of GFAP as the C-6 cells differentiated under basal condition (5.2 ± 0.9 fold) (P-value—0.67). (C) ATP7A localization in the glial cells differentiated in presence and absence of TTM. Copper chelation leads to the absence of ATP7A in vesicles along the neurites.

Induction of the copper dependent ERK1/2 phosphorylation during the neuronal and not the glial differentiation

Retention of ATP7A into the TGN despite high intracellular copper suggests a role of the copper towards the differentiation of the PC-12 cells into neurons. Activation of ERK1/2, through phosphorylation, has been observed during the differentiation of the embryonic stem (ES) cells into neurons.²⁴ ERK1/2 activation is dependent on two Cu(I) ions binding to its upstream activator MEK1/2.²⁵ We wanted to investigate the status of the ERK1/2 phosphorylation during the differentiation of neuronal and glial cells. PC-12-derived neurons have 2.75× increased ERK1/2 phosphorylation than the undifferentiated cells (Fig. 15A). We wanted to investigate if ERK1/2 phosphorylation accompanies neuronal differentiation and is not just associated with the differentiation of the PC-12 cells. To confirm this, we investigated ERK1/2 phosphorylation during the differentiation of the human foetal brain derived neuronal progenitor cells. These neurons also showed increased ERK1/2 phosphorylation (2×) like the PC-12-derived neurons (Fig. 15B). This suggests that a part of this increased intracellular copper in the neurons is utilized towards activating pathways essential for the differentiation, and ERK1/2 activation is one of them. Interestingly, we observed 3.3× downregulation of ERK1/2 phosphorylation in the differentiated C-6 cells (glia) compared with the undifferentiated cells (Fig. 15C). The glia, having low intracellular copper, have downregulated ERK1/2 phosphorylation. One of the potential implications of the vesicular localization of ATP7A, in the glia, might be to render the copper unavailable for the pathways like ERK1/2 phosphorylation. The differentiated PC-12 and the C-6 cells demonstrate a nice correlation of the intracellular copper level and ERK1/2 phosphorylation.

Fig. 15 — ERK1/2 phosphorylation in the differentiated neurons and glia. (A) Increased ERK1/2 phosphorylation in the differentiated PC-12 (neuron) cells. 92.63 ± 2% of ERK1/2 is phosphorylated in the differentiated PC-12 cells in contrast to 33.69 ± 2.03% in the undifferentiated cells (number of experimental replicates—3, ****P-value ˂ 0.0001). (B) Increased ERK1/2 phosphorylation in the neurons differentiated from the human foetal brain derived Neuronal Progenitor Cells (hNPC). Proportion of the ERK1/2 phosphorylation is 2 ± 0.5 in the neurons in contrast to 0.94 ± 0.1 in the hNPC (number of experimental replicates—3, *P-value–0.0346). (C) Decreased ERK1/2 phosphorylation in the differentiated C-6 (glia) cells. ERK1/2 phosphorylation is decreased to 20 ± 12.13% in the differentiated C-6 (glia) in contrast to 66 ± 20.4% in the undifferentiated cells (number of experimental replicates—5, **P-value–0.0034).

Intracellular copper chelation reduces neurite formation and ERK1/2 phosphorylation in the differentiated PC-12 (neuron) cells

Since the differentiation of neurons involves ERK1/2 phosphorylation,²⁴ and ERK1/2 phosphorylation is copper dependent,²⁶ we wanted to investigate if the increased intracellular copper in the differentiated PC-12 cells is utilized towards ERK1/2 phosphorylation. We investigated the effect of chelation of intracellular copper on ERK1/2 phosphorylation and differentiation of the PC-12-derived neurons. Chelation of the intracellular copper during the differentiation by treatment with TTM does not affect the differentiation as is evident from the unchanged neurite length (Supplementary Fig. S10A, B). Also, ERK1/2 phosphorylation does not decrease under such conditions (Supplementary Fig. S10D). We hypothesized that the treatment with 10 μM TTM at the onset of differentiation could not chelate the bioavailable intracellular copper. Therefore, we treated the PC-12 cells with a higher dose (30 μM) of TTM during the differentiation. But under this condition, there was also no effect on the differentiation of the PC-12 cells (Supplementary Fig. S10A lower panel, right and S10B). Estimation of the intracellular copper level demonstrated that the treatment with 10 μM TTM during differentiation could only reduce 29% of the intracellular copper (Supplementary Fig. S10C), in the presence of the high expression of CTR1. This suggests that chelation of intracellular copper at the onset of differentiation is unable to chelate bioavailable Cu(I) and remove Cu(I) from the MEK1/2 thereby failing to affect ERK1/2 phosphorylation.

To efficiently reduce copper mediated ERK1/2 activation, we cultured PC-12 cells for 48 h in 10 μM TTM and differentiated these cells in the presence of the copper chelator. Estimation of intracellular copper, under this condition, demonstrated a decrease in 77% of the intracellular copper thereby suggesting depletion of the bioavailable Cu(I) (Supplementary Fig. S10C). Under this condition, we observed that copper chelation decreased neurite length at the onset of differentiation (T1––48 h after the first treatment with NGF) (Fig. 16A, B). This data demonstrate that intracellular copper triggers pathways crucial for the generation of the neurites. 3.9× decrease in the mRNA for β-III tubulin (Fig. 16C) in these cells confirms that the chelation of the intracellular copper affects the differentiation of the PC-12 cells. Neurite outgrowth is observed in the T1phase (Fig. 16B) of the differentiation. However, we observed no change in the ERK1/2 phosphorylation in the T1 phase (Fig. 16D, lower panel). But, we observed a 4× (approximately) increase in the ERK1/2 phosphorylation in the T2 (Fig. 16D, lower panel). This suggests that ERK1/2 phosphorylation might not play a role in the generation of the neurites. Chelation of intracellular copper led to 2× decrease in the ERK1/2 phosphorylation in the T2 (Fig. 16D) confirming that the intracellular copper, in the neurons, is also utilized towards phosphorylating ERK1/2, in addition to generation of the neurites. Therefore, the neuronal copper is not solely utilized towards the ERK1/2 phosphorylation. Our data indicate that in addition to ERK1/2 activation, intracellular copper triggers pathways crucial for the neurite outgrowth. Besides a bulk of the cytosolic copper is transported into the secretory pathway as demonstrated.⁹ Based on the observed increase in the ERK1/2 phosphorylation in the T2 phase of differentiation, we hypothesized its role towards the neuronal viability.

U0126 mediated inhibition of the ERK1/2 phosphorylation decreases the viability of the PC-12-derived neurons

ERK1/2 phosphorylation (T2) is not increased at a phase during which generation of the neurites (T1) is observed in the PC-12 cells (Fig. 16B, D). Therefore, ERK1/2 is not likely to affect the neurite outgrowth. Since ERK1/2 is not activated at the initial phase of differentiation, we hypothesized that it plays a role in the neuronal viability. We investigated the effect of the inhibition of ERK1/2 phosphorylation by the MEK1/2 inhibitor, U0126, on the PC-12-derived neurons. PC-12 cells (undifferentiated) were treated with 2–8 μM MEK1/2 inhibitor U0126. The treatment did not affect the viability of the undifferentiated cells (Supplementary Fig. S11A). A significant concentration dependent inhibition of the ERK1/2 phosphorylation was observed in the PC-12-derived neurons when differentiated in the presence of U0126 (Supplementary Fig. S11B). ERK1/2 phosphorylation is decreased (2.27×) to 47.7% in the cells differentiated in the presence of 2 μM U0126 (Supplementary Fig. S11B). Under this condition, the viability of the differentiated cells is reduced to 42.9% at T2 (Fig. 17C) and 32.6% at T3 (Fig. 17A–C) as demonstrated by the neutral red incorporation assay. Similarly, ERK1/2 phosphorylation is decreased (5.16×) to 21% in the PC-12 cells differentiated in the presence of 6 μM U0126 (Supplementary Fig. S11B). Under this condition, the viability of the differentiated cells is reduced to 15.5 and 24.02% at T2 (Fig. 17C) and T3 (Fig. 17A–C), respectively. A decrease in the β-III tubulin expression (Fig. 17D) confirms the selective loss of the neuronal cells in response to the treatment.

Fig. 17 — Inhibition of the ERK1/2 phosphorylation by U0126 affects the viability of the differentiated PC-12 (neurons) cells. (A) and (B) Bright field images of the PC-12 cells differentiated in the presence or absence of the MEK1/2 inhibitor U0126. Panel (A) represents T1 and panel (B) represents T3 stage. (C) Percentage viability of the PC-12 cells differentiated in the presence or absence of the U0126 (number of experimental replicates—3). (D) Relative mRNA expression of β-III tubulin in the PC-12-derived neurons under similar conditions as mentioned (T2 phase—**P-value—0.0042, *P-value—0.0137) [T3 phase—**P-value—0.0069 (vehicle and 2 μM U0126) and 0.0021 (vehicle and 6 μM U0126)] (number of experimental replicates—3). (E) Rescue of ERK1/2 phosphorylation inhibited by U0126. PC-12 cells are plated and differentiated to T1 phase. Differentiation is continued (in T2) in the presence of U0126 inhibitor for 24 h and rescued by removal of the U0126 (U0126 + basal) and continuing the differentiation for another 48 h. Western immunoblot (upper) showing the total and phosphorylated ERK1/2 and percentage (lower) of phosphorylated ERK1/2, calculated from the densitometry in response to U0126 and U0126 + basal (number of experimental replicates—3, ***P-value—0.0001). (F) Rescue of the viability of the differentiated PC-12 cells treated with U0126 (as before). U0126 treatment for 24 h (in T2) reduce cell viability to 52.5 ± 10.85%. Cell viability is rescued to 98.7 ± 2.07% after withdrawing U0126 and continuing the differentiation for 48 h. Number of experimental replicates—3, **P-value—0.0048 (vehicle and U0126), **P-value—0.0029 (U0126 and U0126 + basal).

As already demonstrated, the maximum increase in ERK1/2 phosphorylation is observed during the T2 phase of the PC-12 differentiation (Fig. 16D). Therefore, ERK1/2 phosphorylation was inhibited by the treatment with U0126 at the onset of the T2 phase for 24 h. This leads to the decrease in ERK1/2 phosphorylation to 24.7% (Fig. 17E). Under this condition, the viability of the differentiated PC-12 cells is reduced to 52.5% (Fig. 17F). However, decrease in the ERK1/2 phosphorylation is rescued to 73.3% upon withdrawal of the U0126 after 24 h and continuing the differentiation for the next 48 h till the beginning of the T3 phase (Fig. 17E). Under this condition, the viability of the differentiated PC-12-derived neurons could be rescued to 98.7% (Fig. 17F).

Expression of the constitutively active ERK1 and ERK2 mutants do not affect the differentiation of the neurons and glia but can rescue the viability of the neurons

To further confirm that ERK1/2 phosphorylation affects the viability rather than neurite generation or β-III tubulin expression, the effect of the constitutively phosphorylated ERK1 and ERK2 mutants was investigated on the differentiation of the PC-12 neurons. PC-12 cells stably expressing either ERK1 harbouring the R84S mutation or ERK2 harbouring the R65S mutation was generated. The mutations have been previously demonstrated to render the proteins phosphorylated constitutively at the activatory TEY and other motifs.²⁷ Expression of HA and His tag confirms expression of the ERK1 (R84S) and ERK2 (R65S) mutants (Supplementary Fig. S12A–C). MEK1/2 mediated phosphorylation of ERK1/2 was inhibited by U0126 in both PC-12 and C-6 cells. Under such conditions, an uninhibited phosphorylation of either ERK1 or ERK2 in both cell lines (Supplementary Fig. S12D, E) confirms constitutive phosphorylation of ERK1 (R84S) and ERK2 (R65S) in both cell lines. The ability of the PC-12 cells, expressing the constitutively active mutants, to differentiate into neurons is also confirmed (Supplementary Fig. S12F). Expression of ERK1 (R84S) or ERK2 (R65S) could not rescue differentiation in the PC-12 cells cultured and differentiated in the presence of TTM. PC-12 cells stably expressing ERK1 (R84S) or ERK2 (R65S) showed decreased neurite outgrowth (Fig. 18A, B) similar to the untransfected cells. This is further accompanied by the significant decrease in the expression of β-III tubulin in the PC-12 cells expressing ERK1 (R84S) or ERK2 (R65S), cultured and differentiated in the presence of TTM (Fig. 18C). The results provide further confirmation that ERK1/2 activation does not regulate neurite outgrowth or β-III tubulin expression in the PC-12-derived neurons. Further, the hypothesis that ERK1/2 activation is essential for the viability of the neurons was tested. PC-12 cells are differentiated in the presence of the MEK1/2 inhibitor U0126. Inhibition of MEK1/2 mediated phosphorylation of ERK1/2 by 6 μM U0126 reduced the viability of the untransfected PC-12-derived neurons to 55.04% (Fig. 18D) as demonstrated before. However, neurons expressing the constitutively active mutants ERK1 (R84S) or ERK2 (R65S) demonstrated 129% and 139.61% viability, respectively in presence of U0126. This demonstrates that expression of the constitutively active ERK1 or ERK2 mutants could rescue the U0126 (MEK1/2 inhibitor) mediated effect on the viability of the neurons. This confirms that ERK1/2 activation regulates the viability of the PC-12-derived neurons but does not affect the neurite generation and β-III tubulin expression.

Fig. 18 — Expression of the constitutively active ERK1 (R84S) and ERK2 (R65S) cannot rescue differentiation but can rescue survival of the PC-12-derived neurons. (A) Bright field images of PC-12 cells, either untransfected (UT) or stably expressing constitutively active ERK1 (R84S) or ERK2 (R65S) mutants, cultured and differentiated in the presence of 10 μM TTM. (B) Estimation of the neurite length in the above cells. (C) Relative expression of mRNA for β-III tubulin in the above cells. Untransfected cells cultured and differentiated under basal condition show 2.57 ± 0.14-fold higher expression of β-III tubulin compared to the undifferentiated cells. Whereas, untransfected PC-12 cells cultured and differentiated in the presence of TTM has 0.96 ± 0.12-fold lower expression of β-III tubulin compared to the undifferentiated ones. PC-12 cells expressing the constitutively active ERK1 (R84S) or ERK2 (R65S) mutants, cultured and differentiated in the presence of TTM express 1.18 ± 0.45-fold and 0.772 ± 0.03-fold lower level of β-III tubulin mRNA respectively compared to the undifferentiated cells. ****P-value is < 0.0001 (number of experimental replicates—2). (D) Stable expression of the constitutively active mutants of ERK1 and ERK2 can rescue the viability of the PC-12-derived neurons. Untransfected (UT) PC-12 cells as well as those expressing the constitutively active ERK1 and ERK2 mutants are differentiated in presence or absence of 6 μM U0126. U0126 treatment reduced viability of the UT cells to 55.04 ± 1.9%. But cell viability in the ERK1 (R84S) and ERK2 (R65S) expressing neurons is 129 ± 3.9% and 139.61 ± 0.31%, respectively (number of experimental replicates—3). ****P-value is < 0.0001.

C-6 cells stably expressing the constitutively phosphorylated ERK1 or ERK2 can differentiate similarly to the untransfected cells when cultured and differentiated in the presence of TTM (Fig. 19A). This is further confirmed by the unchanged level of GFAP in these cells (Fig. 19B). This demonstrates that the presence of ERK1/2 phosphorylation does not affect the differentiation of the C-6-derived glia.

Fig. 19 — Expression of the constitutively active ERK1 (R84S) and ERK2 (R65S) does not affect the differentiation of the C-6-derived glial cells. (A) Bright field images of the untransfected C-6 cells and C-6 cells stably expressing the constitutively active ERK1 or ERK2, cultured and differentiated in the presence of 30uM TTM. (B) Relative expression of mRNA for GFAP in the above cells. Untransfected (UT) C-6 cells cultured and differentiated in the presence of TTM show 3.17 ± 0.42-fold higher expression of GFAP compared to the undifferentiated cells. C-6 cells stably expressing the constitutively active ERK1 or ERK2 also express 2.78 ± 0.18-fold and 3.17 ± 0.22-fold higher mRNA for GFAP (respectively) compared to the undifferentiated cells (number of experimental—2). P-value—0.0605 (Untransfected and ERK1 mutants) and 0.992 (untransfected and ERK2 mutants).

Discussion

This is the first comparative study of the changes in the cellular copper homeostasis during the neuronal and glial differentiation. Although the existing cell-based model might not exactly mimic the in vivo situation, yet they provide a glimpse of the role of cellular copper homeostasis during the neuronal and glial differentiation. We have observed the differential role of intracellular copper in triggering the pathways in the neurons and the glia during differentiation. Differentiation of the PC-12 cells involves increased intracellular copper, whereas differentiated glia have low intracellular copper (Fig. 20). The requirement of copper for the differentiating neurons is evident from the increased level of CTR1 mRNA accompanying the differentiation and decreased neurite generation in response to copper chelation. However, the ATP7A transcript remains unchanged with the differentiation of the PC-12 cells into neurons. The expression of ATP7A has been observed to increase with the differentiation of the SH-SY5Y cells.⁹ Interestingly, ATP7A is retained into the TGN despite high intracellular copper in the PC-12-derived neurons (Fig. 20). This localization suggests utilization of the high intracellular copper towards the differentiation. The intracellular copper has multiple utilizations. The present study demonstrates that the copper is utilized towards triggering pathways crucial for the neurite generation and ERK1/2 activation (Fig. 20). The cytosolic copper, in the neurons, is also transported to the Golgi-based secretory pathway.⁹ Perinuclear localization of copper in the soma of the PC-12-derived neurons supports copper utilization into the TGN. Cytosolic utilization along with transport into the TGN is further supported by the presence of high copper in the post mitochondria fraction of the differentiated PC-12 neurons. A part of the intracellular bioavailable copper is also transported into the mitochondria (present study), as demonstrated by high mitochondrial copper along with the increased mRNA for COX17 and SLC25A3 in the PC-12-derived neurons.

We report, for the first time, copper dependent ERK1/2 activation during the differentiation of the PC-12 and human foetal brain derived neuronal progenitor cells. Therefore, ERK1/2 activation is one of the cytosolic pathways triggered by the intracellular copper during the differentiation of the neurons. Copper dependence of ERK1/2 phosphorylation has already been demonstrated in melanoma cells^25,26 and further confirmed by our experiments in the neurons. Inhibition of the ERK1/2 activation by U0126 affects the viability of the neurons, highlights the importance of this copper dependent pathway towards the neuronal differentiation. Experiments with the constitutively active ERK1 and ERK2 mutants have confirmed the role of ERK1/2 activation towards the neuronal viability. Moreover, it demonstrates that ERK1/2 activation does not regulate the generation of neurites and expression of β-III tubulin during the neuronal differentiation. ERK1/2 phosphorylation has been observed to be associated with the differentiation of the ES cells into neurons.²⁴ The present study demonstrates the role of the process towards the neuronal viability. Further, we demonstrate the opposite status of the copper level and ERK1/2 phosphorylation during the neuronal and glial differentiation (Fig. 20). ERK1/2 phosphorylation has been observed to be involved in the flavanone mediated survival of the cortical neurons from peroxide induced neurodegeneration.²⁸ This involves phosphorylation of BAD leading ultimately to the inhibition of caspase-9 and caspase-3. Activated ERK1/2 has also been demonstrated to activate pathways leading to inhibition of BIM expression. ERK1/2 can also directly bind to the BIM_EL splice variant and target it towards ubiquitination and proteasomal degradation.²⁹ ERK1/2 mediated viability of the PC-12 neurons might involve one or more of these pathways. Nevertheless, this requires further investigation. In addition to ERK1/2 phosphorylation, intracellular copper regulates pathways that affect the neurite generation during neuronal differentiation. This is demonstrated by the decreased neurite generation and β-III tubulin expression in the PC-12 cells cultured and differentiated in the presence of TTM. Further studies are warranted to investigate those copper regulated pathways.

The glia, having low intracellular copper, have ATP7A localizing into both TGN (perinuclear) and RAB11 (neurites) positive vesicles (Fig. 20). The vesicular localization of ATP7A into the RAB 11 positive compartments is reported for the first time. Experiments with CF-4 suggest that the vesicles contain copper. The presence of such vesicles at low intracellular copper is intriguing. ATP7A expression has been reported in the developing axons in the olfactory neurons.³⁰ Extensions of the Golgi compartments and TGN38 have been detected in the endosomes¹⁵ in the neurons. The mannose-6-phosphate receptor recycles between TGN and late endosomes in the neuronal processes.³¹ But ATP7A containing vesicles, identified in the glia, do not overlap with any of the classical TGN markers. Also, RAB11 positive recycling endosomes have been implicated in neurite extension in the PC-12-derived neurons, dorsal root ganglia, and hippocampal neurons.³² But we did not observe such vesicles in the neurons. It is observed only in the C-6-derived glia. Chelation of the intracellular copper in the glia, leading to the disappearance of these compartments, does not affect the glial differentiation. Therefore, the primary function of these vesicles might be to store copper either for supply to the neurons or to bypass the cytosolic copper, thereby making it unavailable for the MAP kinases (Fig. 20). However, expression of the constitutively phosphorylated ERK1 or ERK2 mutants does not affect the differentiation of the C-6 glia. Keeping in mind the already implicated role of the glia in maintaining ion homeostasis,³³ we hypothesize that the glia might be the key regulator of copper homeostasis in the brain. The ability of the glial cells to sequester copper into RAB11 positive vesicles underlies its ability to reportedly³⁴ withstand copper-induced toxicity. Cellular copper homeostasis has earlier been suggested to play a role in neuronal differentiation. However, intracellular copper has been hypothesized to be diverted towards the secretory pathway aiding into the differentiation of the SH-SY5Y cells.⁹ Our study demonstrates a direct route of copper utilization during neuronal differentiation, in addition to incorporation into the cuproenzymes.

Experimental procedures

Cell culture, differentiation

The PC-12 cells are differentiated as reported³⁵ with minor modifications. Cells maintained in DMEM (DMEM-high glucose, GIBCO) containing 10% horse serum (heat inactivated, GIBCO), 5% foetal bovine serum (heat inactivated, HiMedia), 1% Pen-Strep (HiMedia), 1% amphotericin-B (GIBCO) at 37°C, and 5% CO₂. The C-6 glioma cells are maintained in DMEM containing 10% FBS, 1% Pen-Strep, 1% amphotericin-B at 37°C, and 5% CO₂. For differentiation of PC-12, 2.5 × 10⁵ cells are plated on each well of a six-well plate coated with collagen and cultured overnight. The cells are rinsed twice with phosphate-buffered saline. Differentiation is initiated by adding 100 ng/ml NGF (Sigma-Aldrich) in PC-12 priming media containing DMEM and 0.5–1% horse serum. Three subsequent treatments are performed, first two at an interval of 48 h and the last one at 24 h after the second treatment. C-6 cells are differentiated as mentioned³⁶ with minor modifications. For differentiation of glia, C-6 cells (1 × 10⁵ cells) are plated on each well of a six-well plate and cultured for 48 h (approximately) to 50–60% confluency. The cells are then rinsed with phosphate-buffered saline and cultured in DMEM medium without foetal bovine serum for 1 h. Differentiation is initiated by adding 500 μM dBcAMP (N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt, Sigma-Aldrich) and 1 mM theophylline (MP Biomedicals) in culture medium without serum. Bright field images of undifferentiated and differentiated cells are acquired using the DM IL LED Fluo (Leica Microsystems) and the Carl Zeiss™ PrimoVert™ Inverted Microscope (ZEISS).

Human foetal neural stem cell culture and differentiation

Human foetal brain tissue is isolated from 10 to 15 week old aborted foetuses according to guidelines of the Institutional Human Ethics and Stem Cell and Research Committee of the National Brain Research Centre in compliance with the approvals of the Indian Council of Medical Research. The aborted tissue is used for research purpose only after obtaining the informed consent of the mother. Foetuses selected for the culture were healthy and had no sign of aneuploidy. The tissue is processed for neural stem cell (NSC) culture as described previously.^37,38 In brief, NSCs are isolated from the telencephalon of the foetal brain and are cultured onto the Poly-D-Lysine (Sigma-Aldrich) coated culture wares in neurobasal medium (Invitrogen). Media is added with the following: Neural Survival Factor-1 (Lonza), N2 supplement (Invitrogen), Bovine Serum Albumin (BSA) (Sigma-Aldrich), L-glutamine (Sigma), fibroblast growth factor (25 ng/ml) (bFGF) (Peprotech), epidermal growth factor (20 ng/ml) (EGF) (Peprotech), penicillin, and streptomycin solution (Invitrogen) and gentamycin (Sigma).

NSCs are passaged at least 9× before using them for experiments. NSCs were characterized for the expression of stemness markers- SOX2 and NESTIN by immunocytochemistry and PCR. More than 99% of the cells showed immunoreactivity towards stemness markers: NESTIN and SOX2. Also, cells are analysed for lineage-specific markers: GFAP (astrocytic marker) and MAP2 (neuronal marker), more than 95% cells are negative for GFAP and MAP2 when maintained in the above-mentioned stem cell medium. For differentiation of NSCs into neurons, EGF, and bFGF are replaced with brain-derived neurotrophic factor (10 ng/ml) (BDNF) (Peprotech) and platelet-derived growth factor (10 ng/ml) (PDGF-AB) (Peprotech) for 21days. Post differentiation cells are assessed for expression of MAP2 and Tuj-1; more than 95% cells are positive for these neural markers.

Measurement of intracellular copper by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Indictively Coupled Plasma-Optical Emission Spectrometry (ICP-OES)

PC-12 cell pellets (undifferentiated and differentiated) are washed with ice cold phosphate buffered saline (PBS) and solubilized in RIPA Lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, and protease inhibitor cocktail), and centrifuged at 600Xg in 4°C to remove the cell debri and unlysed cells. Lysate is digested in 50% extrapure nitric acid (Finar) at 55°C for an hour. Digested sample is diluted with water to a final concentration of 3% nitric acid. Metal estimation analysis is performed using a Thermo Fisher Scientific X-Series 2 ICP-MS. Data were quantified using a SRM2711a—Montana II soil moderately elevated trace element concentration standard (NIST—National Institute of Standards and Technologies). Copper concentration is normalized to total protein and represented.

For estimation of copper in the intracellular fractions, nucleus, mitochondria, and post mitochondria fraction is isolated as described.³⁹ In brief, PC-12 cells (control and differentiated) are suspended in lysis buffer (PBS containing 1 mM EDTA, 1 Mm EDTA, protease inhibitor cocktail, and 0.1 volume of 2.5M sucrose) and kept on ice for 1 h. After 1 h, cells are homogenized with a Dounce homogenizer (220 strokes). The homogenate is centrifuged at 600Xg for 10 min. The pellet (nucleus) is washed twice with PSE buffer (PBS containing 1 mM EDTA, 300 mM sucrose, 0.1% NP40, and a complete protease inhibitor cocktail) and resuspended in PSE buffer containing 0.2% triton X-100. The supernatant is centrifuged at 14,000 g for 20 min. The pellet (mitochondria) is re-suspended in PSE buffer containing 0.2% tritonX-100. The supernatant (in lysis buffer) is kept as a post mitochondrial fraction. The organelle fractions were confirmed by western immunoblot detection using organelle markers (Supplementary Fig. S13). The organelle markers used are Histone 2B (D2H6, Rabbit monoclonal Antibody, Cell Signaling Technology, nuclear marker), anti-VDAC1 (Abcam, mitochondrial marker), and β-actin (Santa Cruz Biotechnology, Inc, post mitochondrial/cytosolic fraction). The purity of a respective organelle fraction is checked by cross probing with the markers for the other two fractions (Supplementary Fig. S13). There is no band obtained in cross probing. For estimation of copper in the intracellular fractions, the lysates for each fraction are digested with HNO₃ as described before and filtered through a 0.45-μm syringe filter. Copper is estimated by ICP-OES by iCAP 7000 Series ICP-OES (Thermo Scientific).

For estimation of the total copper in the undifferentiated and differentiated PC-12, in response to the TTM treatment, samples (cell lysates) are similarly prepared as mentioned. The digested samples are filtered through a 0.45-μm syringe filter. Copper is estimated by iCAP 7000 Series ICP-OES (Thermo Scientific). The copper concentration is normalized to the total protein and represented. Each result was replicated at least for 3×.

Undifferentiated and differentiated C-6 cells are washed twice with PBS (pH: 7.5), trypisinized, centrifuged, and collected as pellets. The pellets are digested with 2% suprapur HNO3 (Merck) at a concentration of 3 ml/0.25 mg of the cell pellet for an hour at room temperature. The cell pellets are further digested at 100°C for an hour in a microwave (microwave digestion conditions: power = 800 W, temperature = 100°C, and hold time = 1 h). The digested sample is filtered through a 0.45-μm syringe filter. Elemental copper standard is prepared by digesting copper foil (Alfa Aeasar) with suprapur HNO3 under similar condition. Metal estimation has been done using ICP-OES using the iCAP DUO 6500 (Thermo Scientific). The copper concentration is normalized to total protein and represented. Each result was replicated at least for 5×.

Measurement of mRNA level by real time PCR

Undifferentiated and differentiated cells are cultured in a 60 mm dish. Cells are harvested and washed with PBS. RNA was extracted with the Nucleospin RNA isolation kit (Macherey-Nagel) according to the manufacturer's instruction manual. The cDNA is prepared by reverse transcription using the iScript™ Select cDNA Synthesis Kit (Biorad Laboratories and TaKaRa) using 1 μg of extracted mRNA in a final volume of 20 μl by using anchored oligo(dT) and random hexamer primer following the manufacturer's instructions with minor modifications. Real time PCR is performed using 2 μl of prepared cDNA in a 10 μl reaction volume with iTaqUniverSYBR Green SMX 1000 (Biorad Laboratories) using the Bio-rad CFX96 touch real time PCR instrument. The primers are used to amplify the respective rat cDNA (Supplementary table1). RPL29 cDNA is used as the reference gene. For PC-12 cells, the PCR included an initial denaturation at 95°C for 1 min followed by denaturation at 95°C for 30 s, annealing and extension at 60°C for 60 s (32 cycles). For C-6 cells, initial denuration is carried out for 30 s followed by denaturation for 5 s and annealing and extension at 60°C for 30 s (40 cycles). Relative mRNA abundance is estimated by the 2^–∆∆CT method. Each experiment is repeated at least thrice.

Immunofluorescence and image acquisition to investigate localization of ATP7A in the neurons (PC-12) and glia (C-6)

Undifferentiated and differentiated cells are cultured directly (C-6) or on collagen (PC-12) coated coverslips. Cells are first washed twice in 1X PBS. Cells are fixed with 4% paraformaldehyde in phoshate buffer saline for 3 min on ice, followed by chilled methanol and blocked with 3% (w/v) BSA. Coverslips with cells are inclubated in primary antibodies in 1% BSA [Albumin Bovine (pH 6–7) fraction V, 98%, Sicco Research Laboratories Pvt. Ltd] containing 0.1% Tween-20 overnight. Rabbit polyclonal anti-ATP7A antibody (Abcam, 1:300 dilution), mouse monoclonal TGN38 antibody (Novus Biologicals, 1:400 dilution), mouse monoclonal Golgin97antibody (Thermo Fisher Scientific, 1:400 dilution), mouse monoclonal LAMP1 (Developmental Studies Hybridoma Bank, University of Iowa1:400 dilution), mouse monoclonal Gama Tubulin (Invitrogen, 1:400 dilution), mouse monoclonal RAB 7 (Novus Biologicals, 1:50 dilution), goat polyclonal RAB11 (Santa Cruz Biotechnology Inc., 1:50 dilution), mouse monoclonal anti-RAB5 (Abcam., 1:100 dilution), mouse monoclonal anti-EEA (Abcam,1:100), and mouse monoclonal anti-GFAP (Sigma-Aldrich, 1:200) are used to detect the respective proteins. Goat anti-rabbit Alexa Flour 488 (Abcam), donkey anti-mouse Alexa Flour 568 (Thermo Fisher), donkey anti-goat Alexa Flour 568 (Thermo Fisher), and donkey anti-mouse Alexa Flour 633 (Thermo Fisher). Secondary antibodies are used at 1:2000 dilution for detection. Cells are mounted with Fluoroshield mounting medium (Sigma) (containing DAPI) and imaged with a confocal microscope (SP8 Lightening confocal microscope, Leica Microsystems). Image is acquired using an oil immersion 63X objective and deconvoluted using Leica application suiteX Lightning software (Leica Microsystems). The fluorescence intensity line profile plot is done using Image-J (MBF).

For STED microscopy, cells are first washed twice in 1X PBS for 3 min. Cells are fixed with 2% paraformaldehyde in PBS for 20 min at room temperature, followed by the treatment with 50 mM ammonium chloride for 20 min. Cells are blocked with 1% (w/v) BSA permiabilized with 0.075% saponin (Sigma-Aldrich) in phosphate buffer containing 1% BSA. Cells are incubated in primary antibodies in 0.5% BSA in PBS overnight at 4°C. Cells are incubated in secondary antibodies in 0.5% BSA in PBS for 90 min. Donkey anti-mouse Alexa Fluor 633 at 1:1000 dilution is used as a secondary antibody for the detection of RAB-7. The rest of the secondary antibodies (1:1000) used is like those for confocal microscopy. Cells are mounted in Prolong (R) goldmounting medium (without DAPI) (Cell Signalling Technologies) and imaged with a confocal microscope (SP8 Lightning confocal microscope with STED attachment, Leica Microsystems).

Quatitation of colocalization has been done by measuring the Pearson's correlation coefficient (PCC). Briefly, the images were analysed in ImageJ Fiji software and PCC quantification has been quantified by using the coloc tool.

Visualization of transferrin receptor 2 (TfR2) in the glia (C-6)

C-6 cells were plated on coverslips and cultured to 70% confluency. Cells were transfected with 700 ng of TFR2 (mcherry-TFR 20 plasmid, 55144, Addgene) using Lipofectamine 3000 (Invitrogen) following the manufacturer's protocol. Transfected cells were differentiated, fixed by 4% PFA, permeabilized with methanol, and prepared for confocal microscopy as described before.

Visualization of the intracellular copper by control CF-4 and CF-4 in the differentiated PC-12 and C-6 cells

PC-12 and C-6 cells are plated on a 30 mm glass bottomed confocal dish (SPL Life Sciences) and differentiated as described. Live cells were treated either with 0.8 μM CF-4 or control CF-4, ctrl CF-4 in DMEM without phenol red (Gibco) and incubated for 10 min at 37°C and 5% CO₂. After incubation, cells are washed twice with DMEM without phenol red to remove the excess probe and kept in DMEM without phenol red medium for an additional 20 min at 37°C and 5% CO₂. Imaging is performed on live cells at 37°C in DMEM without phenol red containing 20 mM HEPES (Gibco) and 1 mM Trolox (Sigma-Aldrich) at confocal microscope (Leica SP8) with 63X oil immersion lens. The copper bound CF-4 and the control CF-4 in the live cell were excited at 488 nm and emission was detected between 545 and 590 nm. For PC-12, images were taken in lightning mode under live conditions and represented. For C-6 cells, images were captured in a time lapse at an interval of 5 s. Videos were analysed at Leica Application Suite X (LAS X) and image J software and snapshot taken and represented. For experiments done under high intracellular copper conditions, cells (post differentiation) are treated with or without 100 μM copper for 2 h and washed twice with PBS and incubated with either CF-4 or control CF-4 as described.

Chelation of intracellular copper in the glia (C-6) and neuron (PC-12) by treatment with TTM

For investigating if ATP7A vesicles in glia (C-6) contain copper, undifferentiated and differentiated cells are plated on coverslips and cultured as described. Cells are treated with 30 μM ammounium tetrathiomolybdate (Sigma) for 2 h. Cells were fixed, permeabilized, blocked, and prepared for immunostaining as described in the previous section.

For investigating the effects of copper chelation on differentiation, glia (C-6) cells are plated on medium containing 30 μM TTM and cultured for 48 h. Differentiation is initiated (as described) in the presence or absence of 30 μM TTM. Cells are harvested and prepared for immunocytochemistry as described in the previous section.

For investigating the effect of copper chelation on ERK1/2 phosphorylation and PC-12 differentiation, cells were plated on six-well plates. Differentiation is performed (as described) in presence of 10 and 30 μM TTM. Alternatively, PC-12 cells are plated on six-well plates and cultured for 36 h (30–40% confluency) in the presence of 10 μM TTM. Differentiation was performed (as described) in the presence or absence of 10 μM TTM. Cells are harvested 48 h after the second treatment with NGF.

Cell lysis and western immunoblot blot for the detection of total and phospho-ERK1/2 in neuronal (PC-12), glial (C-6 cells), and human foetal brain derived neurons

Undifferentiated and differentiated cells are cultured on six-well plates. Cell pellets are resuspended with 100 μl of RIPA lysis buffer with a protease inhibitor cocktail (GCC Biotech Pvt. Ltd) and incubated on ice for 1 h. Cells are homogenized with the Dounce homogenizer with 220 strokes of the tight pestle. The homogenate is centrifuged at 600 × g for 10 min and supernatant is collected. Total protein in the lysate is estimated by the Bradford (Sigma-Aldrich) assay. 60 μg (PC-12 and C-6) and 40–50 μg (human foetal brain derived NPC and neurons) of lysate is mixed with gel loading dye (2% SDS, 2.5% ß mercapto-ethanol, 7.5% glycerol, 2M urea, and 0.005% bromophennol blue), boiled for 10 min at 95°C, kept at room temperature and resolved in 12% denaturing polyacrylamide gel. Protein is transferred to a nitrocellulose membrane (BioRad) at 180 mA current for 90 min. The membrane is blocked with 3% BSA in Tris buffered saline (TBS) at room temperature for 2 h and incubated overnight at 4°C with the primary antibody in 1% BSA and 0.1% Tween-20 in TBS. p44/42 MAPK (Erk1/2) (Cell Signalling Technologies) and Phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signalling Technologies) (dilution of 1:3300) are used as primary antibodies for detecting total and phopho-ERK1/2, respectively. Membrane is washed thrice with TBS (pH7.5) and thrice with TBS containing 0.1% Tween-20 (TBS-T). The membrane is incubated in goat anti-rabbit HRP conjugated secondary antibody (Sigma-Aldrich and Santa Cruz Biotechnology) for 2 h at room temperature. The membrane is washed thrice in TBS-T and thrice in TBS and chemiluminescence (clarity max, Biorad) is detected using the ChemiDoc™ Imaging System (170-01401) (Bio-Rad) instrument.

Neurite length estimation

In order to measure the length of the neurites of PC-12 cells (undifferentiated and differentiated in the presence or absence of TTM), bright field images of the cells are acquired and subsequently analysed with the help of Image-J. In order to perform the analysis, the resolution property (both in pixels and cm) of the image is acquired. The resolution of the width/height of the image is obtained in pixels and centimetres. The size of each pixel in centimetres is obtained using this information. The image is then opened in ImageJ. The scale of the software is calibrated beforehand in accordance with the previously mentioned, calculation. This is done by providing the pixel to centimetre value in the ‘set scale’ menu bar under ‘Analyze’ option in Image-J. Input of pixel aspect ratio property of the image is also required. Following this, the length of the neurite lengths is measured with the help of the ‘line selection tool’ in Image-J. The process of length measurement is repeated for at least in 50–100 cells. The average value is then downscaled to its appropriate and accurate unit (μM) by adjusting the magnifications.

Inhibition of the ERK1/2 phosphorylation by U0126 in PC-12 cells

For estimating the toxicity of U0126 on the PC-12 cells, 1.5 × 10³ undifferentiated cells are plated on each well of 96-well plate. Cells are treated with varying concentrations (0–8 μM) of U0126 for 48 h in complete medium.

For investigating the effect of ERK1/2 phosphorylation on PC-12 differentiation, 5 × 10⁴ cells are plated on a 6 cm dish and kept overnight. Cells are treated with or without U0126 (Cell Signalling Technology) in PC-12 priming media (DMEM containing only 0.5% of horse serum) for 2 h. After 2 h, differentiation is initiated by 100 ng/ml of NGF in the presence or absence of U0126. Cells are differentiated, as mentioned earlier in the presence or absence of U0126. ERK1/2 phosphorylation is investigated by western immunoblot as described before.

For estimation of neuronal viability, PC-12 cells are plated at 1.25 × 10⁴ cells in each well of a 12-well plate and grown for 18 h. Cells are differentiated in the presence or absence of 6 μM U0126.

Estimation of cell viability by neutral red dye incorporation assay

PC-12 cells are cultured in the presence or absence of U0126 as mentioned. Culture medium is aspirated as specific time points, and cells are washed twice with PBS (pH:7.4) and added with 40 μg/ml neutral red in complete medium (DMEM containing 10% horse serum and 5% FBS) and incubated for 2 h at 37°C and 5% CO₂. Cells are gently washed thrice with PBS followed by the addition of a distaining solution (50% ethanol and 1% acetic acid) and absorbance is recorded immediately at 540 nm either in a microplate reader (Varioskan LUX, Thermo Fisher) or a spectrophotometer (Eppendorf Biospectrophotometer, basic). Cell viability is expressed as a percentage to the viability of the untreated differentiated cells.

Generation of PC-12 and C-6 cells stably expressing the constitutively active mutants for ERK1 and ERK2

The lethal dose of G148 (Sigma-Aldrich) is determined for both PC-12 and C-6 cells. Briefly, PC-12 and C-6 cells are plated on 24-well plates in complete medium and cultured for 48 h. Cells are treated with an increasing concentration of G148 [in 20 mM HEPES (Gibco)] in the complete medium. The medium (containing G148) is replenished at a 5-day interval for each cell line. After 14 days, the minimum concentration of G148 at which there was complete death of the cells was determined. The lethal dose of G148 for PC-12 and C-6 was 0.9 and 2.2 mg/ml, respectively.

Constitutively active mutants of ERK1 bearing the R84S and ERK2 bearing the R65S mutations are selected for the study. These mutants have been demonstrated to be autophosphorylated at the activatory TEY motif and other residues.²⁷ PC-12 and C-6 cells are transfected either with His-Erk1(R84S) in pCEFL or HA-Erk2(R65S) in pCEFL using lipofectamine 3000 (Invitrogen) according to the manufacture’s protocol with minor modifications. The medium is supplemented with complete medium for the respective cell line 8 h post transfection and cultured for 48 h. After 48 h, the medium is supplemented with the respective lethal concentration of G418 as mentioned above and kept for 14 days. The media is replenished after every 5 days for each of the cell lines. After 14 days, surviving colonies of cells are isolated and subcultured. The stable expression is initially confirmed by investigating the reporter (HA or His) expression for both the constructs in both the cell lines (Supplementary Fig. S12). The antibodies used for the detection of the reporters are rabbit anti-HIS tag antibody (Sigma-Aldrich, dilution of 1:2000) and HA tag Rabbit monoclonal antibody (Cell Signaling Technology, dilution of 1:3000) and detected using goat anti-rabbit HRP conjugated secondary antibody (Sigma-Aldrich and Santa Cruz Biotechnology) and Alexa Fluor 488 (1:2000 dilution for His tag). Further, PC-12 and C-6 cells are treated with 6 and 12 μM U0126, respectively for 2–3 h to inhibit MEK1/2 and thereby MEK1/2 mediated phosphorylation of ERK1/2. Under such conditions, the phosphorylation of the constitutively active ERK1 and ERK2 mutants was confirmed using the method already described.

Statistical analysis

Data were expressed as mean values +/− standard deviation. Students’ paired t-test is used to compare differences between two groups by GraphPad Prism 6 software (GraphPad Prism Inc, San Dieago, CA, USA). Statistically significant differences between them are indicated following the norms: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Material

mfac005_Supplemental_Files

Click here for additional data file.^{(3.1MB, zip)}

Acknowledgements

The authors thank Prof. Christopher J. Chang, University of California, Berkeley, CA, USA for sharing the control CF-4 and CF-4 probes. The authors thank Prof. David Engelberg, Hebrew University of Jerusalem, Israel for sharing the His-ERK1(R84S) and HA-ERK2(R65S) constructs. The authors thank Dr Somsubhra Nath (Saroj Gupta Cancer Centre and Research Institute, India), Dr Alok Ghosh (University of Calcutta, India), Prof. Jayasri Das Sharma, and Dr Rahul Das (Indian Institute of Science Education Research – Kolkata, India) for sharing the Histone 2B antibody, anti-VDAC1 antibody, HA tag antibody, and anti-His tag antibody, respectively.

Contributor Information

Kaustav Chakraborty, Amity Institute of Biotechnology, Amity University, Kolkata, India.

Sumanta Kar, Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India.

Bhawana Rai, Amity Institute of Biotechnology, Amity University, Kolkata, India.

Reshma Bhagat, Molecular and Cellular Neuroscience, Neurovirology Division, National Brain Research Centre, Manesar, India.

Nabanita Naskar, Chemical Sciences Division, Saha Institute of Nuclear Physics, Kolkata, India.

Pankaj Seth, Molecular and Cellular Neuroscience, Neurovirology Division, National Brain Research Centre, Manesar, India.

Arnab Gupta, Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India.

Ashima Bhattacharjee, Amity Institute of Biotechnology, Amity University, Kolkata, India.

Author contributions

A.B., A.G., and K.C. designed experiments. K.C., S.K., B.R., and R.B. performed experiments. K.C., S.K., B.R., and A.G. participated in data acquisition. K.C., A.B., and A.G. analysed the data. A.B., K.C., A.G., and R.B. wrote the manuscript. A.B., K.C., A.G., R.B., and P.S. critically evaluated the manuscript.

Funding

The study was supported by the Ramanujan Fellowship Project (SB/S2/RJN-106/2015) and Extramural Research Project (EMR/2016/003293) to A.B. and an Early Career Research Award (ECR/2015/000220) to A.G. from the Science and Engineering Research Board, Department of Science and Technology, Government of India. The work is further supported by the Wellcome Trust India Alliance Fellowship (IA/I/16/1/502369) and IISER-K intramural funding to A.G. K.C. is supported by the Indian Council of Medical Research–Senior Research Fellow (ICMR-SRF) (2020-7142/CMB-BMS).

Conflicts of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Data availability

The authors declare that all the required data have been presented in the manuscript. The datasets do not require submission to any publicly accessible repository. The datasets do not contain any software codes that need to be archived.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mfac005_Supplemental_Files

Click here for additional data file.^{(3.1MB, zip)}

Data Availability Statement

[bib1] 1. Fu S., Jiang W., Zheng W., Age-dependent increase of brain copper levels and expressions of copper regulatory proteins in the subventricular zone and choroid plexus, Front. Mol. Neurosci., 2015, 8, 22. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib3] 3. Lutsenko S., Bhattacharjee A., Hubbard A. L., Copper handling machinery of the brain, Metallomics, 2010, 2 (9), 596–608. [DOI] [PubMed] [Google Scholar]

[bib4] 4. Wilson S. A. K., Progressive lenticular degeneration: a familial nervous disease associated with cirrhosis of the liver, Brain 1912, 34 (4), 295–507. [DOI] [PubMed] [Google Scholar]

[bib5] 5. Lorincz M. T., Neurologic Wilson's disease, Ann. N. Y. Acad. Sci., 2010, 1184 (1), 173–187. [DOI] [PubMed] [Google Scholar]

[bib6] 6. Zlatic S., Comstra H. S., Gokhale A., Petris M. J., Faundez V., Molecular basis of neurodegeneration and neurodevelopmental defects in Menkes disease, Neurobiol. Dis., 2015, 81, 154–161. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib8] 8. El Meskini R., Crabtree K. L., Cline L. B., Mains R. E., Eipper B. A., Ronnett G. V., ATP7A (Menkes protein) functions in axonal targeting and synaptogenesis, Mol. Cell. Neurosci., 2007, 34 (3), 409–421. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9. Hatori Y., Yan Y., Schmidt K., Furukawa E., Hasan N. M., Yang N., Liu C. N., Sockanathan S., Lutsenko S., Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway, Nat. Commun., 2016, 7 (1), 10640. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10. Xiao T., Ackerman C. M., Carroll E. C., Jia S., Hoagland A., Chan J., Thai B., Liu C. S., Isacoff E. Y., Chang C. J., Copper regulates rest-activity cycles through the locus coeruleus-norepinephrine system, Nat. Chem. Biol., 2018, 14 (7), 655–663. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11. Beers J., Glerum D. M., Tzagoloff A., Purification, characterization, and localization of yeast Cox17p, a mitochondrial copper shuttle, J. Biol. Chem., 1997, 272 (52), 33191–33196. [DOI] [PubMed] [Google Scholar]

[bib12] 12. Suzuki C., Daigo Y., Kikuchi T., Katagiri T., Nakamura Y., Identification of COX17 as a therapeutic target for non-small cell lung cancer, Cancer Res., 2003, 63 (21), 7038–7041. [PubMed] [Google Scholar]

[bib13] 13. Rothstein J. D., Dykes-Hoberg M., Corson L. B., Becker M., Cleveland D. W., Price D. L., Culotta V. C., Wong P. C., The copper chaperone CCS is abundant in neurons and astrocytes in human and rodent brain, J. Neurochem., 1999, 72 (1), 422–429. [DOI] [PubMed] [Google Scholar]

[bib14] 14. Boulet A., Vest K. E., Maynard M. K., Gammon M. G., Russell A. C., Mathews A. T., Cole S. E., Zhu X., Phillips C. B., Kwong J. Q., Dodani S. C., Leary S. C., Cobine P. A., The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis, J. Biol. Chem., 2018, 293 (6), 1887–1896. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15. Sytnyk V., Leshchyns'ka I., Delling M., Dityateva G., Dityatev A., Schachner M., Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts, J. Cell Biol., 2002, 159 (4), 649–661. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16. Hartter D. E., Barnea A., Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67copper, Synapse, 1988, 2 (4), 412–415. [DOI] [PubMed] [Google Scholar]

[bib17] 17. Duncan C., Bica L., Crouch P. J., Caragounis A., Lidgerwood G. E., Parker S. J., Meyerowitz J., Volitakis I., Liddell J. R., Raghupathi R., Paterson B. M., Duffield M. D., Cappai R., Donnelly P. S., Grubman A., Camakaris J., Keating D. J., White A. R., Copper modulates the large dense core vesicle secretory pathway in PC12 cells, Metallomics, 2013, 5 (6), 700–714. [DOI] [PubMed] [Google Scholar]

[bib18] 18. Polishchuk E. V., Concilli M., Iacobacci S., Chesi G., Pastore N., Piccolo P., Paladino S., Baldantoni D., van I. S. C., Chan J., Chang C. J., Amoresano A., Pane F., Pucci P., Tarallo A., Parenti G., Brunetti-Pierri N., Settembre C., Ballabio A., Polishchuk R. S., Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis, Dev. Cell, 2014, 29 (6), 686–700. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19. Nyasae L. K., Schell M. J., Hubbard A. L., Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes, Traffic, 2014, 15 (12), 1344–1365. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Copper dependent ERK1/2 phosphorylation is essential for the viability of neurons and not glia

Kaustav Chakraborty

Sumanta Kar

Bhawana Rai

Reshma Bhagat

Nabanita Naskar

Pankaj Seth

Arnab Gupta

Ashima Bhattacharjee

Abstract

Graphical Abstract

Graphical Abstract.

Introduction

Results

Differentiation of the PC-12 cells (neuron) involves increased intracellular copper in contrast to the differentiation of the C-6 (glia) cells

Fig. 1.

Fig. 2.

Fig. 3.

Increased intracellular copper parallels increased expression of CTR1 mRNA in the differentiated PC-12 (neuron) cells but not in differentiated C-6 (glia) cells

ATP7A shows different localization in the differentiated PC-12 (neuron) and C-6 (glia) cells

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Fig. 11.

Vesicles in the neurite processes, in the differentiated (C-6) glia, contain copper

Fig. 12.

Localization of the ATP7A into the RAB 11 positive vesicles in the differentiated C-6 (glia) cells can be retrieved back to the TGN upon copper chelation

Fig. 13.

Fig. 14.

Induction of the copper dependent ERK1/2 phosphorylation during the neuronal and not the glial differentiation

Fig. 15.

Intracellular copper chelation reduces neurite formation and ERK1/2 phosphorylation in the differentiated PC-12 (neuron) cells

Fig. 16.

U0126 mediated inhibition of the ERK1/2 phosphorylation decreases the viability of the PC-12-derived neurons

Fig. 17.

Expression of the constitutively active ERK1 and ERK2 mutants do not affect the differentiation of the neurons and glia but can rescue the viability of the neurons

Fig. 18.

Fig. 19.

Discussion

Fig. 20.

Experimental procedures

Cell culture, differentiation

Human foetal neural stem cell culture and differentiation

Measurement of intracellular copper by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Indictively Coupled Plasma-Optical Emission Spectrometry (ICP-OES)

Measurement of mRNA level by real time PCR

Immunofluorescence and image acquisition to investigate localization of ATP7A in the neurons (PC-12) and glia (C-6)

Visualization of transferrin receptor 2 (TfR2) in the glia (C-6)

Visualization of the intracellular copper by control CF-4 and CF-4 in the differentiated PC-12 and C-6 cells

Chelation of intracellular copper in the glia (C-6) and neuron (PC-12) by treatment with TTM

Cell lysis and western immunoblot blot for the detection of total and phospho-ERK1/2 in neuronal (PC-12), glial (C-6 cells), and human foetal brain derived neurons

Neurite length estimation

Inhibition of the ERK1/2 phosphorylation by U0126 in PC-12 cells

Estimation of cell viability by neutral red dye incorporation assay

Generation of PC-12 and C-6 cells stably expressing the constitutively active mutants for ERK1 and ERK2

Statistical analysis

Supplementary Material

Acknowledgements

Contributor Information

Author contributions

Funding

Conflicts of interest

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

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