Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper

Article abstract of DOI:10.1021/jacs.0c05727

Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed, copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the same time copper misregulation is associated with inflammation and neurodegeneration. As such, chemical probes that can track dynamic changes in copper with spatial resolution, especially in loosely bound, labile forms, are valuable tools to identify and characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we designed copper-directed acyl imidazole dyes that operate through copper-mediated activation of acyl imidazole electrophiles for subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively, after exposure to inflammatory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper, an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic signaling and stress analytes in biology.

Full text of DOI:10.1021/jacs.0c05727

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Activity-Based Sensing with a Metal-Directed Acyl Imidazole
Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper
Sumin Lee,○ Clive Yik-Sham Chung,○ Pei Liu, Laura Craciun, Yuki Nishikawa, Kevin J. Bruemmer,
Itaru Hamachi, Kaoru Saijo, Evan W. Miller, and Christopher J. Chang*
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ABSTRACT: Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed,
copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the
same time copper misregulation is associated with inflammation and neurodegeneration. As such, chemical probes that can track
dynamic changes in copper with spatial resolution, especially in loosely bound, labile forms, are valuable tools to identify and
characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for
copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we
designed copper-directed acyl imidazole dyes that operate through copper-mediated activation of acyl imidazole electrophiles for
subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and
fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types
in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct
changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon
activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively,
after exposure to inflammatory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper,
an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic
signaling and stress analytes in biology.
resulting from aberrant increases in reactive oxygen species
(ROS) that can lead to oxidative damage to proteins, lipids,
and DNA/RNA.15,16 Such stress responses contribute to
diseases including cancer,17−19 neurodegenerative diseases
such as Alzheimer’s, Parkinson’s, and Huntington’s dis-
eases,20−24 and genetic disorders such as Menkes and Wilson’s
diseases.25−27 These correlations are intriguing, but the
INTRODUCTION
■
Copper is an essential element for human health.1 Indeed,
enzymes harness the potent redox activity of this metal
nutrient to perform functions spanning energy generation,
pigment synthesis, and epigenetic modifications.1−7 Owing to
the high metabolic and signaling needs of the brain, copper is
found in particularly high concentrations in this organ,8−12
where work from our laboratory showed that activation of
neurons causes a marked redistribution of cellular copper to
affect the excitability of cultured neurons,13 with subsequent
studies identifying that these labile copper pools regulate
spontaneous activity of circuits in retinal tissue12 and sleep
behavior in zebrafish models in vivo.14 However, misregulation
of copper homeostasis can lead to cellular malfunctions
Received: May 26, 2020
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Scheme 1. Synthesis of (a) CD649 and (b) CD433; (c) Schematic Cartoon Showing the Working Principle of CD Dyes for
Fluorescent Labeling of Proximal Proteins at Sites of Elevated Labile Copper
underlying causal contributions of copper homeostasis to
function and disease in the brain and central nervous system
remain insufficiently understood. To this end, a number of
chemical technologies have been developed to probe biological
copper fluxes by molecular imaging, including fluores-
cent,11−14,18,28−47 and magnetic resonance imaging
(MRI),48−56 and bioluminescent57 copper indicators. These
reporters can achieve high selectivity and signal-to-noise
responses for copper ion imaging from cellular to tissue to
whole animal settings. Application of these reagents, in
conjunction with complementary biochemical, cellular, and
animal studies, have given rise to the emerging concept of
transition metal signaling, where such elements can reversibly
modulate the activity of protein targets by fast and reversible
binding beyond the active site.4,17,58,59
In this context, most fluorescent copper probes are designed
to recognize labile copper ions based on thioether-rich
receptors or tris[(2-pyridyl)methyl]amine (TPA) receptors.40
These receptors have been exploited to trigger a fluorescent
turn-on or ratiometric response by reversible coordination of
copper ions or by activity-based sensing through irreversible
uncaging of a fluorophore40,60−63,18 or luciferin.57 Despite
advances in designing probes for studying copper biology, a
gap in the field is to improve control over the localization of
diffusible synthetic indicators to achieve higher spatial
resolution for copper and related analytes of interest. To
B
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Figure 1. Copper reactivity and selectivity of CD probes in aqueous buffer solutions. (a) LC chromatograms of the reaction between 5 μM CD649
and 10 μM Cu(I) in PBS containing 25 μM L-serine methyl ester and 2 mM GSH for 1 h, showing the complete conversion of CD649. (b) The
mass of intact CD649 probe was detected at a retention time of 8.0 min in the absence of Cu(I). (c) The copper-responsive reaction products of
CD649 with Cu(I)/2 mM GSH are detected at a retention time of 7.4 min. (d) LC chromatograms of 5 μM CD649 (top trace, black), 5 μM
CD649 incubated with 10 μM Cu(I) (red trace) for 30 min, and other biologically relevant metals at 10 μM (Na+, K+, Mg2+, Ca2+, Fe3+, Co2+, Ni2+,
Cu+, and Zn2+, gray traces) for 30 min in PBS in the presence of 25 μM L-serine methyl ester and 2 mM GSH. The LC data support that CD649
shows high selective reactivity toward copper ions. (e) In-gel fluorescence images and integrated fluorescence intensities of CD649 and lysozyme as
a model protein for activity-based sensing of copper via copper-induced bioconjugation. Lysozyme was preincubated with biologically relevant
metal ions for 5 min (s-block metal ions at 1 mM, d-block metal ions at 10 μM, BCS at 50 μM), followed by incubation with 1 μM CD649 at room
temperature for 2 h. In-gel fluorescence for SDS-PAGE was scanned by ChemiDoc MP and signal intensity was analyzed by ImageJ.
meet this need, we now present the design, synthesis, and
biological applications of a new type of activity-based sensing
(ABS) platform64,65 for fluorescent copper detection using acyl
imidazole bioconjugation chemistry. In ABS, the analyte is
detected by an analyte-triggered reaction rather than a simple
binding event, akin to activity-based protein profiling for
reading out protein activity rather than protein abundance. We
have prepared copper-directed acyl imidazole (CDAI or CD)
fluorescent probes that feature a thioether NS2 recep-
tor32,36,30,33 bearing a fused acyl imidazole linked to coumarin
(CD433) or Si-rhodamine (CD649) fluorophores, where
copper binding increases the electrophilicity of the acyl
imidazole unit for nucleophilic attack and covalent labeling
of proximal proteins with the fluorescent dye. This approach is
inspired by ligand-directed acyl imidazole (LDAI) reagents for
proteomics and activity-based protein profiling (ABPP).66−72
One of the advantages of utilizing this LDAI strategy for
sensing and imaging purposes is to overcome a major challenge
that small-molecule fluorescent probes can diffuse away from
their target upon binding. CD probes address this problem by
leveraging the direct coordination of Lewis acidic copper ions,
resulting in covalent bond formation between fluorescent
reporters and proximal proteins in the cell to preserve spatial
information of localized copper hotspots by minimizing
diffusion of the dye away from the site of the ABS reaction.
A second advantage is, unlike existing activity-based sensing
systems derived from TPA that require both Cu(I) and O2 to
trigger a signal change, the CD probes can be activated directly
by copper ions alone. The red-emissive CD probe CD649 was
utilized to image increases and decreases in labile copper pools
in live cells with exogenous copper supplementation or
depletion, respectively, as well as monitor differences in labile
copper status in genetic modified MEF cells with knockout of
the ATP7A copper-exporter P-type ATPase compared to the
wild-type congeners.
Moreover, to decipher fundamental copper biology in the
central nervous system, CD649 was applied to characterize
labile copper pools in the three major types of cells in the
brain: neurons, astrocytes, and microglia. In primary cultured
hippocampal neurons, CD649 revealed the translocation of
labile copper from somatic cell bodies to the dendrites during
neuronal depolarization. In parallel, CD649 identified global
increases in labile copper in microglia and decreases in
astrocytes during the inflammatory response. Taken together,
this work provides a unique approach to direct activity-based
sensing of copper that can retain spatial information with a
modular design concept that applied to other biological
analytes of interest. In addition, the foundational information
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Figure 2. Fluorescence imaging of labile copper pools in live HEK293T cells and MEF cells with wild-type or altered expression levels of the
copper transporter protein ATP7A using CD649. (a) Confocal fluorescence microscopic images of HEK 293T cells treated with 1 μM CD649
alone, CD649 and 2 μM Cu(gtsm) for Cu supplementation, or CD649 and 200 μM BCS for Cu deficiency. The cells were incubated with 2 μM
Cu(gtsm) for 2 h or 200 μM BCS in DMEM/10% FBS medium overnight and washed with DMEM/10% FBS and PBS, followed by incubation
with 1 μM CD649 in DPBS solution and then imaged after 15 min. (b) Chemical structures of Cu(gtsm) and BCS. (c) Normalized cellular
fluorescence intensities of the HEK 293T cells as determined using ImageJ, showing a turn-on response when treated with Cu(gtsm) and a turn-off
response in the presence of BCS. Error bars denote standard derivation (SD; n = 3). Scale bar = 50 μm. (d) Confocal fluorescence microscopic
images of MEF ATP7A WT or KO cells treated with DMSO vehicle, 2 μM Cu(gtsm) for 30 min, or 200 μM BCS for overnight in DMEM/10%
FBS and washed with DMEM/10% FBS and PBS, followed by incubation with 1 μM CD649 in DPBS and then imaged after 15 min. (e) Average
cellular fluorescence intensity of CD649 determined from experiments performed in triplicate with λex = 633 nm. Error bars denote standard
derivation (SD; n = 3). Scale bar = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
on cell type-specific changes in labile copper offers a starting
point for further investigations of copper biology in the brain
and beyond to advance our understanding of transition metal
signaling.
promotes rapid reactivity at the carbamate unit of the acyl
imidazole and hydrolysis with a 7.1-fold increase in product
formation as found in LC chromatogram (Figure S1 of the
Supporting Information, SI). Likewise, liquid chromatograms
of the reaction between 5 μM CD649 and 2 equiv of Cu(I) in
PBS in the presence of 5 molar equiv of L-serine methyl ester
and 2 mM glutathione (GSH) showed complete conversion to
the copper-bound species and/or the copper-induced acyl
transfer reaction product within 1 h (Figure 1a).
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Copper-Directed
Acyl Imidazole (CD) Probes for Activity-Based Sensing
of Copper. To develop a copper-responsive activity-based
sensing probe that operates by proximal protein labeling, a
thioether NS2 motif fused to imidazole was chosen as the
copper recognition unit as inspired by previous copper-
selective fluorescent4,11−13,28−32,41−43,58,73−77 and MRI
probes48,51−53 as well as porous polymers for copper capture
and detection.78 The tripodal receptor 9 was synthesized in
three steps. Dicarboxylic acid silicon-rhodamine 6 and its NHS
ester 7 was prepared according to a reported procedure,79 and
NHS ester 7 was amide coupled to compound 10 to give
copper-directed acyl imidazole 649 (CD649, Scheme 1). The
coumarin derivative 11 was synthesized following published
literature protocols66 and conjugated to compound 9 to give
CD433 (Scheme 1). The final CD probes were characterized
After 30 min, the intact CD649 probe disappeared to afford
a product with a mass of 603 m/z (Figure 1b), corresponding
to the cleaved CD649 species with loss of the copper receptor
and another product with a mass of 893 m/z that corresponds
to CD649 coupled to GSH, with a retention time at 7.4 min.
(Figure 1c). Control experiments performed in the absence of
amino acids revealed that CD649 does indeed fragment upon
addition of copper ions; however, the resulting products were
not detected at the retention time of 7.4 min, supporting the
idea that nucleophilic amino acids are necessary to generate
the copper-responsive reaction products (Figure S3). This
activity-based sensing reaction is copper-selective, which is
supported by liquid chromatography trace of CD probes
incubated with other biologically relevant metal ions. Indeed,
comparing the LC traces of CD649 with 10 μM Na+, K+, Mg2+,
Ca2+, Fe3+, Co2+, Ni2+, Cu+, or Zn2+ with L-serine methyl ester
(25 μM) and 2 mM GSH in PBS, CD649 showed the
conversion to 7.4 min retention time products only with
copper addition (Figure 1d). After the metal selectivity was
evaluated in aqueous conditions, we proceeded to test the
copper-directed labeling strategy with lysozyme as a model
protein owing to its commercial availability in large quantities
(Figures 1e and S4). Lysozyme (1.55 μM, 50 μL) was
1
by H and 13C{1H} NMR, LC−MS, and high-resolution MS.
For labeling proximal proteins in live cells in a copper-
dependent manner, the CD probes should bind and react
through accessible nucleophilic amino acids. To evaluate the
reactivity and stability of the CD probes in vitro, the hydrolysis
reaction of CD433 in the presence or absence of Cu(I) was
monitored by liquid-chromatography in PBS buffer containing
glutathione (2 mM). CD433 alone is stable in aqueous
solutions as monitored for 4 h, but the introduction of copper
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incubated with biologically relevant metal ions for 30 min at
room temperature and then the premixed solutions were
further incubated with 1 μM of CD649 for 30 min. Fluorescent
gel analysis showed that CD649 selectively labels the protein in
case of Cu(I)/Cu(II) and the labeled band became
significantly diminished upon addition of BCS (structure is
shown at Figure 2b), supporting the notion that the
nucleophilic amino acids on a protein surface were labeled
with CD649 via acyl transfer reaction in the presence of copper
ions (Figure 1e). We further checked the CD649 selectivity
with HEK 293T lysates incubated with Cu(I) and Fe(II)
prepared in the bioglove box, which exhibits the comparable
labeling capability of CD649 toward Cu(I)/Cu(II) labeled
protein (Figure S5). To investigate any oxidation state
specificity of CD probes toward copper, lysozyme in PBS
was premixed with Cu(I)/Cu(II) for 5 min, followed by
incubation with CD649 for 2 h under a N2 atm so that no
oxidation of Cu(I) would occur. Similar fluorescence
intensities of labeled lysozyme were found in the presence of
Cu(I) and Cu(II) ions, respectively (Figure S10), indicating
that the CD probes are responsive toward both Cu(I) and
Cu(II) ions.
In addition, we found a linear increase in fluorescence
intensity of HEK 293T cell lysates labeled by CD649 with
increasing copper ion concentration, and a limit of detection is
found to be 0.864 nM (Figure S8). These data suggest that
protein labeling by the CD probes is highly sensitive and dose-
dependent toward copper. A dose-dependent increase in
fluorescence intensity of lysozyme labeled by CD649 was also
found with increasing copper, whereas changes in fluorescence
intensities with fixed copper ion concentrations and varying
lysozyme concentrations were much smaller and plateaued at
high lysozyme concentrations (Figure S9). In view of the much
higher concentrations of protein compared to CD probe in
biological environments, the effect of changes in protein
concentration on observed fluorescence should be minimal.
Finally, to evaluate the relative copper affinity of the CD
probes, CD433 was employed in a competitive copper binding
assay with the reported copper fluorescent probe, CSR1, whose
emission profile does not overlap with that of CD433.80,81 In
these experiments, CD433 (0−5 equiv) was continuously
added to a pH 7.0 buffer solution (25 mM HEPES) containing
the copper-complexed form of CSR1, which is reported to
have a dissociation constant (Kd) with Cu(I) of 2 × 10−13 M.59
The solution containing the probes was excited at 608 nm,
where CD433 does not absorb, and as the concentration of
CD433 was increased, the observed fluorescence intensity
derived from CSR1 significantly decreased, suggesting that
CD433 can compete with CSR1 for copper binding and is
capable of displacing the copper ion from this Si-rhodol sensor
(Figure S6). As CSR1 is capable of monitoring dynamic
changes in labile copper pools in live cells as shown by
identification of copper fluxes induced by lipolysis,59 this set of
experiments presages that the CD platform should have
sufficient affinity for copper in related cellular assays.
by preincubating with Cu(gtsm) to increase intracellular
copper levels and BCS to decrease intracellular copper levels.
As anticipated, we observed a robust 2-fold increase in
fluorescence intensity following treatment with 2 μM Cu-
(gtsm), with the labeling being distributed evenly across the
cytosol as compared to the punctate staining pattern observed
under basal conditions. In contrast, HEK 293T cells pretreated
with 200 μM BCS for 10 h to induce copper depletion
displayed a significant decrease in fluorescence intensity as
compared to control cells as shown with CD649 imaging
(Figure 2a), establishing that this ABS probe can monitor
changes in labile copper levels in live cells, with fairly low cell
toxicity as indicated by PrestoBlue assay (Figure S11).
Likewise, CD433 was incubated with HEK 293T cell lysates
in the presence of 1 equiv of Cu(I), and fluorescent intensity
was measured at different incubation times up to 120 min.
Those experiments show that the integrated fluorescence
linearly increased and the fluorescence was quenched by
preincubation with BCS (Figure S7), which confirms the
copper-responsiveness of this probe. Next, we sought to
evaluate the ability of CD649 to assay labile copper pools in
genetic models of copper misregulation. In particular, we
focused on matched cell lines with different levels of ATP7A, a
copper-translocating P-type ATPase that regulates cellular
copper pools by mediating cellular copper egress and secretory
function. Indeed, mutations in the copper transporters ATP7A
and ATP7B are at the heart of genetic disorders of copper
homeostasis.26,82−84 In particular, Menkes disease is derived
from a loss of ATP7A function with systemic copper
misregulation leads to hyperaccumulation of liver copper.85
As anticipated, CD649-stained ATP7A knockout (KO) cells
showed significantly higher levels of fluorescence compared to
control ATP7A wild-type (WT) cells, revealing that CD649
can indeed detect elevated levels of labile copper in ATP7A
KO cells, presumably due to the deficiency in cellular copper
export (Figure 2d,e). Furthermore, both WT and KO cells
showed an increase in fluorescence signal following incubation
with Cu(gtsm) in a time- and dose-dependent manner, which
can be attenuated by the addition of the copper chelator BCS
(Figure 2b). These data validate that CD649 can detect
changes in labile copper levels with pharmacological and/or
genetic manipulation.
CD649 Enables Activity-Based Sensing of Copper in
Major Brain Cell Types. After confirming that CD649 can
monitor changes in labile copper pools in mammalian cell line
models, we then moved on to apply this new chemical tool to
probe labile copper pools relevant to the brain and central
nervous system. Indeed, copper homeostasis has profound
effects in the brain as a neuromodulator in healthy
states12,14,86−89 and as a mediator of neuroinflammation and
neurotoxicity in aging and disease states.6,14,90−93 In this
context, previous work from our laboratory using a
combination of X-ray fluorescence microscopy (XRFM) and
a reversible binding-based BODIPY fluorescent probe for
copper (CS3) identified that neuronal activity triggers
significant movements of labile copper pools from the soma
to peripheral neuronal processes.13 Here, we sought to expand
on this observation by providing foundational information on
labile copper pools in three main types of cells in the brain for
comparison: neurons, astrocytes, and microglia. Starting with
neurons, CD649 revealed punctate staining localized primarily
to the soma of cultured primary hippocampal neurons in their
resting state (Figure 3b). Activation and depolarization of
Live-Cell Imaging of Labile Copper Pools with CD649
Under Situations of Copper Elevation or Depletion.
After establishing that CD probes are sensitive to copper with
high metal selectivity and can label a model protein by SDS-
PAGE analysis with a fluorogenic readout, we next tested the
ability of this ABS platform to respond to changes in labile
copper levels in living cells. Specifically, we applied CD649 to
live HEK 293T cells and cellular copper levels were perturbed
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Figure 3. CD649 can image translocation of labile copper pools from cell bodies to projections in cultured primary neurons upon chemical/
electrical stimulation. (a) Depiction of observed copper redistributions in neurons before and after depolarization by various stimuli. (b)
Fluorescence images of primary cultured hippocampal neurons stained with 1 μM CD649 in the resting state and after depolarization by KCl (50
mM), 50 μM glutamate and 5 μM glycine (Glu/Gly), or electrostimulation (3000 evoked action potentials at a frequency of 20 Hz). (c)
Percentage of neurons showing copper pools residing at distances greater than one cell body away from soma. The percentage of such neurons
increases significantly after depolarization. Scale bar is 20 μm. **p < 0.01.
Figure 4. Fluorescence imaging of labile copper pools in human astrocytes (HA) and microglia SIMA9 under various inflammatory responses using
CD649. (a) Confocal fluorescence microscopic images of astrocytes treated by PBS control or a combination of TNF- α and IL-1 α (both at 20 ng/
mL) up to 48 h and stained with 5 μM CD649 in HBSS for 30 min, fixed and then imaged. Scale bar = 25 μm. (b) Average cellular fluorescence
intensity of CD649 determined from experiments in (a) performed in triplicate with λex = 633 nm. Error bars denote standard deviation (SD; n =
3) (c) ICP−MS measurement to determine total cellular 63Cu levels in astrocytes under TNF-α and IL-1α inflammatory stimuli (with
normalization of different cell numbers by total cellular 31P level). Error bars denote SD; n = 2. (d) Confocal fluorescence microscopic images of
microglia stimulated by PBS control, lipopolysaccharide (LPS, 0.1 μg/mL), or CpG oligodeoxynucleotides (ODN, 5 μg/mL). Five μM CD649 in
HBSS was incubated subsequently for 30 min and then imaged after fixation. Scale bar = 25 μm. (e) Average cellular fluorescence intensity of
CD649 determined from experiments in (d) performed in triplicate with λex = 633 nm. Error bars denote SD; n = 5 different images from the
triplicate experiments. (f) ICP−MS measurement to determine total cellular 63Cu levels in microglia under various inflammatory stimuli (with
normalization of different cell numbers by total cellular 31P level). Error bars denote SD; n = 2. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p <
0.0001.
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these cultures by 50 mM KCl for 2 min,13 or glutamate/glycine
(50 μM/5 μM),8 or electrostimulated (3000 evoked action
potentials at a frequency of 20 Hz),94,95 followed by staining
with 1 μM CD649, showed a significant redistribution of labile
copper pools from somatic cell bodies to peripheral locations,
in agreement with previous XRFM/CS3 data.13 The
percentage of CD649-stained neurons showing redistributed
copper ions at least one cell body away from the center of the
neuron was calculated, revealing that depolarized neurons have
a 2-fold increase relative to resting neurons (Figure 3c),
consistent with soma to dendrite copper pool redistribution.
We also applied CD649 to real-time monitoring of labile
copper pools in hippocampal neurons, showing punctate
localization of labile copper in somatic cell bodies in neurons
in their resting state with loss of these puncta upon application
of a depolarization stimulus (Figure S12).
CONCLUSIONS
■
To close, we have described the design, synthesis, and
characterization of a unique platform for activity-based sensing
(ABS) of copper by exploiting copper-dependent bioconjuga-
tion chemistry of acyl imidazole electrophiles. CD probes
respond in a copper-dependent manner by increased covalent
labeling of the dye with proximal proteins in cells at sites with
elevated labile copper pools, which minimizes dye diffusion
away from copper hotspots and preserves spatial information.
This ABS strategy utilizes direct copper binding to trigger a
fluorogenic response and does not require a metal- and
oxygen-dependent reaction, offering an advantage over
previous TPA-derived ABS probes. CD649 is capable of
imaging changes in labile copper pools in HEK 293T and MEF
cells with pharmacological copper supplementation and
depletion, as well as detect elevations in endogenous levels
of labile copper accumulation caused by knockout of the
ATP7A copper exporter in MEF cell models. Moreover, we
applied CD649 to characterize labile copper pools in the three
main cell types in the brain: neurons, astrocytes, and microglia.
In cultured primary hippocampal neurons, CD649 can track
redistributions of labile copper pools from somatic cell bodies
to peripheral processes upon depolarization. This probe also
reveals distinct cell-specific responses in labile copper
dynamics with inflammatory stimuli, where inflammation
triggers relocation of labile copper in astrocytes and a labile
copper increase in microglia. We speculate that the opposing
dynamics of labile copper in these two cell types may
contribute to neuroinflammatory reactions, where labile copper
contraction in astrocytes is compensated by labile copper
expansion in microglia as signal to communicate between cell
types. Such crosstalk could serve as a neuroprotective
mechanism in neurodegenerative disease. Current efforts are
geared toward expanding the palette of ABS probes that
operate by dual sensing/bioconjugation mechanisms for dual
imaging/proteomics purposes, as well as applying CD649 and
related chemical tools to decipher the biology of metals in the
brain and beyond. We anticipate that such activity-based
reagents will be of value in providing foundational information
on the continuum between metal signaling and metal
metabolism.
Next, we sought to characterize labile copper pools in
astrocyte and microglia, as these cell types in the brain
influence neurogenesis, angiogenesis, immune response, and
synaptogenesis.96 In particular, astrocytes have been reported
as key regulators of brain copper homeostasis as they can
efficiently acquire, store, and export copper owing to their high
resistance to copper toxicity.96,97 However, microglia are the
major resident immune cells in the brain, producing factors
that influence surrounding astrocytes and neurons and
promoting neuron survival as well as synaptic pruning,97−99,96
with less known about their copper biology. As both of these
cell types are involved in inflammation, we utilized CD649 to
probe changes in labile copper pools in human astrocyte (HA)
and microglia SIMA9 cell lines in response to inflammatory
stimuli. Cells were pretreated with inflammatory activators
such as tumor necrosis factor alpha (TNF-α), interleukin-1
alpha (IL-1α), lipopolysaccharide (LPS), or CpG oligodeox-
ynucleotides (ODN), washed and then stained with 5 μM
CD649. CD649 revealed labile copper pools localized in a
punctate pattern within human astrocytes in their resting state.
Stimulation with TNF-α and IL-1α triggers a loss of punctate
staining over increased inflammatory response (Figure 4a and
b). The decrease in fluorescence signals measured by CD649
was supported by complementary ICP−MS measurements of
total cellular copper to phosphorus ratios showing expected
decreases in the response to TNF-α and IL-1α inflammatory
stimuli (Figure 4c), suggesting a reduction and/or relocaliza-
tion of labile copper pools. In contrast, microglia SIMA9 cells
showed an increase in their labile copper pools in the response
to inflammatory stimuli such as LPS and ODN compared to
their basal state as monitored by CD649 imaging (Figure 4d
and e). The increases in labile copper-dependent fluorescence
signals were also supported by complementary ICP−MS
measurements of total cellular copper (Figure 4f). The
observed increase in the microglial labile copper pool under
inflammatory stimuli is in line with a previous study reporting
increased expression of ATP7A of the microglia cells in the
response to the pro-inflammatory response.100−102 The rise in
ATP7A leads to increased copper uptake and elevated
expression of the CTR1 copper importer to expand the overall
labile copper pool.101 Taken together, these data showcase the
utility of CD649 for activity-based sensing of copper applied to
reveal basic information on labile copper pools and their
distinct dynamics in various brain cell types.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c05727.
Experimental details including synthesis and character-
ization, metal reactivity and selectivity of CD probes,
copper binding assay of CD433, and real-time imaging
of cellular copper pools (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Christopher J. Chang − Departments of Chemistry, Molecular
and Cell Biology, and Helen Wills Neuroscience Institute,
University of California, Berkeley, California 94720, United
States; orcid.org/0000-0001-5732-9497;
Email: chrischang@berkeley.edu
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Article
(4) Chang, C. J. Searching for Harmony in Transition-Metal
Signaling. Nat. Chem. Biol. 2015, 11 (10), 744−747.
Authors
Sumin Lee − Departments of Chemistry, University of
California, Berkeley, California 94720, United States;
orcid.org/0000-0003-4179-7952
Clive Yik-Sham Chung − Departments of Chemistry, University
of California, Berkeley, California 94720, United States; School
of Biomedical Sciences and Department of Pathology, The
University of Hong Kong, Hong Kong, P.R. China;
orcid.org/0000-0003-1382-719X
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Pei Liu − Departments of Chemistry, University of California,
Berkeley, California 94720, United States; orcid.org/0000-
0002-4667-1139
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Laura Craciun − Molecular and Cell Biology, University of
California, Berkeley, California 94720, United States
Yuki Nishikawa − Department of Synthetic Chemistry and
Biological Chemistry, Graduate School of Engineering, Kyoto
University, Kyoto 615-8510, Japan; ERATO Innovative
Molecular Technology for Neuroscience Project, Japan Science
and Technology Agency (JST), Kyoto 615-8530, Japan
Kevin J. Bruemmer − Departments of Chemistry, University of
California, Berkeley, California 94720, United States;
orcid.org/0000-0003-3416-5615
Itaru Hamachi − Department of Synthetic Chemistry and
Biological Chemistry, Graduate School of Engineering, Kyoto
University, Kyoto 615-8510, Japan; ERATO Innovative
Molecular Technology for Neuroscience Project, Japan Science
and Technology Agency (JST), Kyoto 615-8530, Japan;
orcid.org/0000-0002-3327-3916
Kaoru Saijo − Molecular and Cell Biology and Helen Wills
Neuroscience Institute, University of California, Berkeley,
California 94720, United States; orcid.org/0000-0003-
1656-1753
Evan W. Miller − Departments of Chemistry, Molecular and
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Author Contributions
○These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH (GM 79465 to C.J.C. and R35 GM119855
to E.W.M.) for support of this work. C.Y.-S.C thanks the
Croucher Foundation for a postdoctoral fellowship. P.L. was
supported by a graduate fellowship from A*STAR, Singapore.
K.S. thanks the NIH (1R01HD09203) and Pew Scholarship.
We thank Alison Killilea, Carissa Tasto, and Molly Fischer of
the UC Berkeley Cell Culture Facility for cell culture support
and thank Stefan Schmollinger at Sabeeha Merchant lab at UC
Berkeley for ICP-MS measurement.
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