The subventricular zone (SVZ) is the largest neurogenic region in adult mammalian brain. It lines the wall of lateral ventricles, and harbors adult neural stem/progenitor cells (NSPCs). The NSPCs in the SVZ remain largely quiescent; but upon activation, NSPCs differentiate into neuroblasts and enter the rostral migratory stream (RMS), migrating toward their destination brain regions. The olfactory bulb (OB) is the primary destination for these SVZ-derived migratory neuroblasts [1
]. Within the OB, newly arrived neuroblasts further differentiate into granule cells and periglomerular cells in the granule cell layer (GCL) and glomerular layer (GL), respectively. Importantly, these two types of replenishable interneurons by SVZ neurogenesis regulate the normal olfactory function as they form synapses with the OB projection neurons that receive signals from the olfactory sensory neurons in the olfactory epithelium. Through these interneuron connections, the signal is further projected to the cortex for olfactory sensation. Thus, any adverse alterations in the SVZ adult neurogenesis can lead to changes in new neuron supply in the OB, ultimately impairing the smell function [1
Anosmia (i.e., partial or full loss of smell) is a frequently reported non-specific symptom that precedes by years the hallmark neuropathology in Alzheimer’s diseases (AD) and Parkinson’s disease (PD) [2
]. Evidence in the literature has identified a compromised SVZ adult neurogenesis in patients and animal models of AD and PD [3
]. However, the mechanisms by which the SVZ adult neurogenesis contributes to the neurodegeneration is largely unknown. Interestingly, metal dyshomeostasis, especially these essential elements such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn), has been associated with the aberrant SVZ and ensuing neurodegenerative alterations. For example, reports suggest that dyshomeostasis of Cu, Zn, Fe, and Mn in the brain not only accelerates the neurodegeneration [9
], but also causes disrupted SVZ adult neurogenesis [15
]. Recent studies even proposed that in COVID-19 patients observed with abnormally high metal concentrations, an aberrantly activated SVZ adult neurogenesis may cause a grey matter volume enlargement in OB [19
]. However, the relationship between patients’ metal dyshomeostasis and abnormal SVZ adult neurogenesis has yet to be elucidated.
Among the essential trace elements, several lines of evidence support Cu as a suppressor of SVZ adult neurogenesis. First, reports by both synchrotron X-ray fluorescence (XRF) and atomic absorption spectrometry (AAS) discovered a selective Cu enrichment in the SVZ in an age-dependent manner [21
]. Cu was concentrated within GFAP(+) cells in the SVZ, which represent largely NSPCs [21
]. Second, toxicological studies showed that a decreased Cu level in the SVZ after Mn overexposure abnormally increased NSPC proliferation, albeit with a declined new neuron production at OB; the latter seemed likely owing to an arrested migration of newborn neuroblasts in RMS [16
]. Third, directly delivering Cu ions to the cerebrospinal fluid (CSF) by intracerebroventricular (ICV) injections apparently suppressed the NSPC proliferation in the SVZ [16
]. Finally, a recent report by this lab modeling systemic Cu overload in rats further revealed that the SVZ adult neurogenesis was significantly downregulated in brains of Cu-overladed animals [24
]. These observations prompted us to propose a Cu-dependent regulatory mechanism in the SVZ, where a naturally high Cu level in the SVZ may suppress the adult neurogenesis in a state of “switching-off”, whereas a reduced SVZ Cu may “switch on” the neurogenesis, thus increasing the proliferation, migration, and differentiation of NSPCs along the SVZ-RMS-OB axis. This hypothesis became the subject of this study for experimental testing.
To investigate Cu participation in biological processes, there is a need to use the agents that specifically modulate Cu homeostasis. D-penicillamine (D-Pen), a selective Cu chelating agent, has been extensively used in research as well as in clinics, and proven to be capable of effectively removing Cu ions in body fluids and lowering Cu burden in patients [25
]. D-Pen (trade name Cuprimine) has been clinically used to treat Wilson’s disease, an inherited genetic disorder that causes abnormal Cu accumulation in the body and leads to severe brain and hepatic symptoms [26
]. D-Pen can form a stable, water-soluble complex with Cu intracellularly and extracellularly, allowing a rapid urinary excretion of Cu from the body [26
]. Thus, D-Pen is one of the most frequently used Cu-chelating agents in Cu biological and toxicological research.
The main purpose of this study was to understand the role of Cu in regulating adult neurogenesis in the SVZ. To reach this goal, we developed an SVZ-derived in vitro neurosphere model and used the unique Cu chelating agent D-Pen to elucidate how Cu status determined the proliferation, migration, and differentiation of NPSCs in neurospheres. We then extended the research in live animals by establishing an in situ intracerebroventricular (ICV) D-Pen infusion model, followed by tracing the newly generated neuroblasts/neurons in the SVZ-RMS-OB axis using immunohistochemistry (IHC) approach. Finally, we investigated the expression levels of Cu regulatory proteins in the SVZ and choroid plexus tissues using IHC. The results of this study provide the firsthand evidence to support a regulatory role of Cu in adult neurogenesis in the SVZ.
Observations from the current study clearly establish that the Cu status in the SVZ, the largest germinal region in adult brains, plays a critical role in regulating proliferation, migration, and differentiation of adult NPSCs along the SVZ-RMS-OB axis. This conclusion is supported by several lines of evidence. First, our in vitro neurosphere studies revealed that increasing Cu levels in the culture medium significantly suppressed NPSC’s differentiation and migration, while chelating Cu by D-Pen greatly reduced cellular Cu burden and restored cells’ differentiation and migration. Second, in vivo ICV infusion of D-Pen in mice, either by short-term (7-day) or long-term (28-day) infusion, selectively reduced Cu concentrations in the SVZ; this reduction significantly upregulated the proliferation of NPSC in SVZ and facilitated neuroblast migration in RMS. Third, long-term low-dose D-Pen infusion increased new neuron production in OB, especially in the GCL, without depleting the NSPC pool in the SVZ, suggesting the impact of the Cu status on cell differentiation in OB which may affect the olfactory function. Fourth, treatment with D-Pen in both of our in vitro and in vivo studies significantly influenced the expression of regulatory proteins participating in NPSC’s differentiation and migration, as well as cellular Cu transport and storage along the SVZ-RMS-OB axis. Finally, our data also revealed that infusion of D-Pen to the lateral ventricles greatly increased the expressions of CTR1 in the choroid plexus, suggesting a potential contribution of the choroid plexus in regulating the Cu level in the SVZ.
A well-characterized in vitro SVZ-derived neurosphere model is essential to study the SVZ adult neurogenesis. Existing protocols have described workflows to isolate adult NSPCs [34
]; however, these protocols allow only for simple qualitative assessment, but not for quantitative analyses of neuronal lineage differentiation and migration. The workflow established in this report fills these gaps in the following two critical steps. First, we adapted Leibovitz’s L-15 medium for freshly extracted brain in subsequent microdissection of SVZ. Compared to simple inorganic salts-based PBS and HBSS, L-15 medium contains amino acids, vitamins, and inorganic salts, which prevent the cell death of NSPCs upon the immediate isolation from the live brain tissues and ultimately facilitate the neurosphere formation. Second, poly-L-ornithine (PLO) has been used in the literature, often in combination with laminin, to promote the survival, migration, and differentiation of neurospheres derived from embryonic brain through ERK pathway [36
]. Our protocol used PLO but replaced laminin with a polymer-made bottom to improve cell attachment. By comparing the migration area and final neuron production, it was evident that this approach greatly improved cell growth, even in comparison with embryonic neurospheres [36
]. These improvements yielded nearly uniform neurospheres with an average 216.8 ± 5.4 µm (RSD% 2.5, n =10) in the diameter (Supplementary Figure S1
), giving the confidence in subsequent quantitative analyses of neurosphere dynamics between different treatments.
Applying D-Pen in our in vitro and in vivo experiments proved to be useful in reducing cellular and tissue Cu levels. Experiments with Phen Green, a dye specific to identifying the labile Cu pool which is different from the protein-bound Cu pool [39
], indicated a reduced level of bioactive Cu in neurospheres following D-Pen treatment. Although D-Pen treatment by itself cannot completely remove Cu from the culture medium, the depleted intracellular labile Cu pool by D-Pen apparently effectively modulated adult neurogenesis by increasing neurosphere differentiation and migration (Figure 3
A), and in cases of excess Cu in culture medium, it reversed Cu’s suppression of adult neurogenesis and Cu’s modulation of pertinent NSC regulatory factors (Figure 4
and Figure 5
). Our results are consistent with the literature reports that only the labile, bioactive Cu ions mediate the Cu-related cellular activities [40
Our recent work has established that systematic Cu disorders in adult rats can alter SVZ neurogenesis, leading to neurochemical imbalance in the SVZ-RMS-OB axis [24
]. The Cu concentrations along the SVZ-RMS-OB axis in that study change significantly in both Cu-deficient and Cu-overload animals. However, the possibility of an overall systemic Cu dyshomeostasis on the SVZ-RMS-OB axis cannot be ruled out. Hence, this study adopted the ICV infusion approach by locally infusing D-Pen directly into the CSF in brain ventricle, making it possible to investigate the impact of CSF Cu homeostasis on the SVZ adult neurogenesis, under the assumption that D-Pen would selectively reduce Cu concentrations in SVZ, a ventricular region bathing in the CSF. Our AAS data showed that the ICV infusion of D-Pen indeed dose-dependently reduced Cu concentrations in the SVZ; but it did not affect other tested brain regions such as RMS, OB, hippocampus, and frontal cortex, except for striatum where Cu levels were increased. Noticeably, the low-dose D-Pen resulted in a marginal reduction of SVZ Cu (13.1%), which was not statistically significant (p
= 0.19), as compared to a significant reduction in the high dose D-Pen infusion (21.4%). This marginal reduction could be due to the increased Cu level in striatum, an adjacent region to the SVZ. In fact, the SVZ is a thin layer covering striatum; it was possible that the process in isolating SVZ tissue may carry minor account of striatal tissue. Since D-Pen ICV infusion caused an elevated Cu in striatum, a minor amount of contaminated striatal tissue could confound the results of SVZ Cu, which would have shown a greater Cu reduction after low-dose D-Pen infusion. Importantly, the fact that no Cu level changes were found in other tested brain regions including RMS and OB suggests a local action of D-Pen directly on SVZ, which influenced adult neurogenesis in the SVZ-RMS-OB axis.
One cycle SVZ adult neurogenesis takes approximately 28 days in mice [41
]. Under this timeframe, the neuroblasts differentiated from SVZ NSPCs migrate in the RMS and subsequently reach OB, where they ultimately mature into two types of interneurons in two sub-regions of OB, i.e., PGCs in GL and GCs in the GCL. Our characterization of the SVZ-RMS-OB axis suggested that the low-dose D-Pen, but not the high-dose, facilitated the neurogenesis along the SVZ-RMS-OB axis. It was evident that following the short-term, 7-day infusion, more Ki67(+)/Nestin(+) cells emerged in the SVZ in the D-Pen (low) group. The observation was in line with the findings in RMS and OB, i.e., more BrdU(+)/DCX(+) neuroblasts observed in the OB following short-term, low-dose D-Pen infusion. However, for short-term, high-dose D-Pen treatment, more of such cells were found in RMS with fewer detected in the OB, implying that the high-dose D-Pen infusion, by yet undefined mechanism(s), arrested newly differentiated neuroblast cells in RMS. Similarly, in the long-term 28-day infusion, the D-Pen at low dose, but not the high dose, significantly increased newly generated BrdU(+)/NeuN(+) mature neurons in OB, especially in GCL. These findings suggest a dose-specific effect of D-Pen chelation of Cu in the CSF and/or SVZ on altering neurogenesis in the SVZ-RMS-OB axis, and only the low-dose D-Pen activates the SVZ neurogenesis.
Stimulating the generation of adult NSPCs is not uncommon; but an aberrant activation often leads to a depleted NSPC pool in SVZ [42
]. By examining the ratio of activated NSCs (aNSC) over the quiescent NSCs (qNSC), we found the low-dose D-Pen infusion increased the NSPC activation without eliciting notable alterations in the total NSC numbers, suggesting that the low-dose D-Pen (with a mild Cu reduction) did not deplete the NSPC pool. However, challenging the SVZ with the high-dose D-Pen seemed likely to deplete the NSPC pool, as evidenced by fewer GFAP(+)/CD133(+)-stained NSCs along the lateral ventricular wall. Taking together the overall dynamics of NSPCs in SVZ, RMS and OB, a mild reduction of Cu in the SVZ by the low-dose D-Pen treatment appeared to significantly increase newborn neurons in the OB, yet not at the cost of depleting the NSPC pool in the origin of SVZ. Since a depleted NSC pool, no matter partially or fully, can repopulate over a period of time [45
], it would be interesting to investigate whether and how a prolonged D-Pen chelation may alter the NSC pool in the SVZ in our future experiments.
Our data also showed the cluster-like enrichment of CTR1 and MT3, two critical proteins in regulating cellular Cu homeostasis, along the lateral ventricular wall in the SVZ, and their expressions were altered after D-Pen Cu chelation. These observations are in a good agreement with reports by our group and others [17
]. Nonetheless, the current study revealed that ICV D-Pen infusion, while not changing the average CTR1 fluorescent intensity in the SVZ clusters, caused a translocation of CTR1 clusters from the cytosol toward the cell membrane facing the CSF. As CTR1 possesses the high capacity in taking up the extracellular Cu [47
], this observation may reflect a higher demand of SVZ for Cu present in the CSF upon Cu chelation by D-Pen. In addition, quantitative analyses of CTR1 in striatum revealed that, upon Cu chelation, striatal CTR1 was increased in a dose-dependent manner (Figure 9
), which may account for increased Cu levels following D-Pen treatment as shown by the AAS data.
Data in this study also showed an upregulated MT3 expression in the SVZ following ICV D-Pen infusion in a dose-dependent manner, which is consistent with the result from the systemic Cu-deficiency model by this lab [24
]. This may indicate a compensatory mechanism by which the Cu-depleted cells demanded more Cu for intracellular storage. In addition, unlike other MT isoforms, MT3 reportedly acts as a growth inhibitory factor (GIF) in astrocytes, and a decreased astroglia GIF level was associated with neuronal loss in AD brains [48
]. Thus, an increased MT3 may act as the neuronal modulatory GIF to modulate the SVZ cells. This hypothesis, however, needs further testing.
Interestingly, our IHC data demonstrated that neither CTR1 nor MT3 clusters completely overlapped with GFAP and/or Nestin. This suggests that certain cell types not studied in this report may occupy the CTR1 and MT3; more importantly these yet-to-be-defined cell types may also greatly contribute to Cu enrichment in SVZ. Given the cluster-like distribution pattern of CTR1 and MT3, we hypothesize that other Cu-related proteins, including but not limited to DMT1, ATP7A, and ATP7B, may also follow this expression pattern in SVZ. We believe that the single-cell RNA-seq technique can be used in our future studies to identify the cell types of interest and to solve this “cluster” puzzle.
The choroid plexus plays a critical role in regulating Cu homeostasis in the CSF [49
]. The current finding on a significant increase of CTR1 in the choroid plexus after D-Pen chelation of Cu is in a good agreement with the literature reports by systematic Cu deficiency induced by restricting dietary Cu [24
]. Given the anatomical closeness of the choroid plexus to the SVZ in brain ventricle and the fluid communication between the two via the CSF, it is reasonable to postulate that changes in Cu-regulatory functions in the choroid plexus may modulate adult neurogenesis in nearby SVZ. Again, this hypothesis deserves extensive experimental testing.
How then does Cu regulate the adult neurogenesis in the SVZ? Extensive reports in the literature have linked Cu to neuronal differentiation, synaptic signal transduction, and neurodegeneration [24
]. However, a role of Cu in neurogenesis, no matter in early postnatal phase or in adult brain, remains elusive; even less is known about the molecular regulatory mechanism taking place in the SVZ-RMS-OB axis. Several transcription factors and molecules are known to stimulate the NSPC proliferation (e.g., b-catenin, Notch1, and Shh), facilitate the differentiation of NSPCs to neuroblasts (e.g., Dlx-2), and guide the direction of newborn neuroblast migration (e.g., SLIT1, SLIT2, ROBO1, and ROBO2) [29
]. The qPCR data from our in vitro neurosphere studies clearly showed that the expression of some of these critical transcription factors was sensitive to Cu levels in the surrounding environment. Moreover, the Cu-induced alteration in mRNA expressions of these neurogenesis regulators could be alleviated by a mild D-Pen chelation. Thus, we postulate that Cu may regulate the adult neurogenesis in SVZ by acting on these critical neurogenesis modulators. This assumption, however, does not exclude Cu interactions with other yet-to-be-identified factors that participate in SVZ adult neurogenesis, as Cu is a biologically highly active trace element. This hypothesis deserves further investigation.
The other interesting observation from this study is that only a mild reduction of Cu by the low-dose D-Pen treatment activates neurogenesis in adult brain. Cu is an essential metal to brain cells, and yet any excess or deficiency of this metal causes neurotoxicity. Thus, an elegant balance is necessary in maintaining cellular Cu levels through Cu uptake, storage, and export (Zheng and Monnot, 2012). Interestingly, such processes were largely supported by organelles, especially Golgi apparatus and mitochondria [58
]. Compared to SVZ neuroblast cells, NSPCs possess larger Golgi apparatus and more mitochondria [61
]. Golgi apparatus facilitates intracellular Cu trafficking and exports excessive Cu through ATP7A and ATP7B [50
] and mitochondria produce the energy required for NPSCs’ proliferation through the Cu-dependent tricarboxylic acid (TCA) cycle [62
]. The low-dose D-Pen treatment may remove the Cu-related suppression of cellular modulators in adult neurogenesis, but not lead to a Cu deficiency-associated cytotoxicity, so that a mild Cu chelation may activate the neurogenesis process. In contrast, the high-dose D-Pen chelation of Cu may create a condition similar to Cu deficiency, which subsequently disrupts normal functions of mitochondria in NPSCs [64
]. Interestingly, this assumption was supported by our finding that, when treated with higher concentrations of D-Pen, neurons differentiated from neurospheres exhibited extensive NeuN translocation from nucleus to cytosol, which was not elicited by D-Pen at 20 µM (Figure 3
); the observation suggests that high-dose D-Pen caused cytotoxicity in NSPCs by disrupting the normal neural differentiation. Indeed, the reports from human Parkinson’s brain and animal PD models suggest that Cu deficiency is associated with PD pathology, impaired SOD1 and COX activities, and increased cellular oxidative stress [65
]. Thus, it is possible that by over-chelation of intracellular labile Cu ions, the high-dose D-Pen treatment may generate the cytotoxicity that overshows its benefit. Taken together, Cu chelation in the SVZ can lead to increased adult neurogenesis; but the magnitude of the chelation needs to be well controlled.
4. Materials and Methods
Chemical reagents were purchased from the following sources: copper chloride dihydrate (MW 170.48 g/mol, purity > 99%), D-penicillamine (MW 149.21 g/mol, purity > 98%), 5-bromo-2′-deoxyuridine (Bromodeoxyuridine, BrdU), heparin sodium, and paraformaldehyde (PFA) from Sigma Aldrich (St. Louis, MO, USA); Nunclo Sphera 96-well, U-shaped-bottom microplate, wide bore pipette tips, neurobasal plus medium, B-27 plus supplement, GlutaMAX supplement, gentamicin (50 mg/mL), trypsin-EDTA (0.05%), defined trypsin inhibitor, DNAse I, epidermal growth factor (EGF), fibroblast growth factors (FGF), fetal bovine serum (FBS), Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) (A-11008), Alexa Fluor 568-conjugated goat anti-chicken IgY (H + L) (A-11041), Cy5-conjugated goat anti-rat IgG (H + L) (A-10525), Phen Green (cell permeant) from Thermo Scientific (Waltham, MA, USA); Apoptosis assay kit from Abcam (Cambridge, MA, USA); normal goat serum (NGS) from Jackson ImmunoResearch (West Grove, PA, USA); 24 Well Plate with #1.5 glass-like polymer coverslip bottom from Cellvis (Mountain View, CA, USA); Cultrex ready-to-use poly-L-ornithine solution from R&D Systems (Minneapolis, MN, USA); and Triton X-100 from Bio-Rad (Hercules, CA, USA). All reagents were of analytical grade, HPLC grade, or the best available pharmaceutical grade.
Osmotic pumps (model 1007D, model 1004) and brain infusion kit 3 were purchased from DURECT Corporation (Cupertino, CA, USA); and Gluture topical tissue adhesive from MWI Animal Health (Boise, ID, USA). Information regarding primary antibodies, host species and dilution factors is provided in Supplementary Table S1
C57BL/6 mice aged 3 months old were purchased from Envigo Inc. (Indianapolis, IN). Upon arrival, animals were housed in a temperature-controlled room under a 12 h-light/12 h-dark cycle and allowed to acclimate for one week prior to experimentation. Animals had free access to distilled-deionized water and Purina semi-purified rodent chow (Purina Mills TestDiet, Richmond, IN, USA) ad libitum. The study was conducted in compliance with standard animal use practices and approved by the Animal Care and Use Committee of Purdue University (PACUC No. 1112000526).
4.3. Experimental Design
The Experiment 1 was to develop and characterize an in vitro neurosphere model for investigation of SVZ adult neurogenesis. This model allowed us to test D-Pen’s efficacy in mitigating Cu-induced neurosphere disruption as well as the underlying mechanisms.
The Experiment 2 extended our in vitro studies to in vivo, to establish an ICV infusion animal model to explore how D-Pen administration locally by ICV infusion altered the brain Cu levels and to observe the ensuing adult neurogenesis along the SVZ-RMS-OB axis in response to short- and long-term D-Pen infusion at two doses. Within the long-term infusion timeframe, we also investigated the changes of the homeostasis of NSPC pool, and the expression patterns of CTR1 and MT3 by IHC, two critical Cu regulatory proteins, in the SVZ.
Finally, the Experiment 3 extended our in vitro and in vivo studies on SVZ to investigate whether D-Pen infusion in vivo altered the expressions of Cu regulatory proteins (i.e., CTR1 and MT3) in the choroid plexus (CP), a blood-rich tissue in brain ventricles nearby the SVZ and regulating Cu transport in the CSF [49
4.4. Establishment of an In Vitro Neurosphere Model for Adult Neurogenesis Studies
To establish an in vitro model of neurospheres for Experiment 1, three types of culture medium were prepared as follows. First, the neurosphere background medium (Medium-1) was prepared by mixing 48.5 mL Neurobasal Plus Medium with 1 mL B-27 Plus supplement, 0.5 mL GlutaMAX, and 50 μL Gentamicin (50 mg/mL). Of note, B-27 Plus supplement consists of a cocktail of antioxidants, which creates a reducing environment to improve adult NSPCs’ viability and their long-term survival. Second, the growth factor-enriched neurosphere induction medium (Medium-2) was prepared based on the Medium-1 by adding 2 μL EGF (0.1 mg/mL), 2 μL FGF (0.1 mg/mL), and 2 μL heparin stock solutions (10 mg/mL) per 10 mL Medium-1. Finally, the neurosphere differentiation medium (Medium-3) was prepared by adding 0.1 mL FBS into 10 mL of Medium-1 without the growth factor.
A 3-month-old mouse was anesthetized by intraperitoneal (ip.) injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). The whole brain was extracted and washed in ice-cold Leibovitz’s L-15 medium to remove excessive blood. The brain was then transferred to a 100 mm culture dish pre-filled with 10 mL ice-cold Leibovitz’s L-15 medium for microdissection. With the brain’s ventral aspect facing upward, a coronal cut was made by a razor blade at the optical chiasm illustrated by the dash line in Figure 1
A. To dissect and collect the SVZ fraction, the rostral portion was placed against the dish with the coronal cut facing upward. Following removal of the septum, the Dumont curved #5/45 forceps was used to isolate the SVZ, a thin layer of tissue lining the lateral ventricle marked by the dash circle in Figure 1
The isolated SVZ fraction was physically dissociated by mincing them gently with a scalpel blade for 1 min until no large tissues remained. The gentle mincing was necessary to avoid damaging the petri dish’s plastic material. The minced tissue was resuspended in 2 mL pre-warmed 0.05% Trypsin-EDTA and transferred to a 15-mL centrifuge tube. The preparation was incubated in a water bath at 37 °C for 7 min. Since the tissues tended to sink to the bottom of the tube, the tube was tapped every 1~2 min to facilitate the digestion during the incubation. This was followed by adding 2 mL of Defined Trypsin Inhibitor containing DNAse I at 0.01 mg/mL to terminate the digestion. After centrifugation at 300× g for 5 min, the pellet was resuspended in 1 mL of the neurobasal background medium and gently triturated with a 1-mL pipette tip up and down for 7 times. By adding another 4 mL of the Medium-1, the suspension was filtered through a 40-μm sterile cell strainer to remove the undissociated tissue chunks. This filtrate containing single cells or small aggregates was then centrifuged at 300× g for 5 min, followed by resuspending the pellet in 12 mL of the inductive Medium-2. The prepared suspension was seeded in one 12-well culture dish, with 1 mL suspension per well. The culture was maintained under 37 °C with 5% CO2 for 6 days.
The formation of neurospheres was recorded by tracking their daily growth (Figure 1
C). Small neurospheres (diameter ~20 μm) were first observed on Day 3; larger neurospheres gradually formed in the days followed. On Day 6, neurospheres with desirable sizes (diameter 100~200 μm) became visible and afloat in the medium. During the preliminary experiments, it was noticed that extending the culture duration beyond 6 days caused the attachment of neurospheres to the bottom of the dish with ensuing spontaneous differentiation or the core of neurospheres turning dark. Therefore, the optimal culture duration to acquire high-quality primary neurospheres was 6 days.
On Day 6, the primary neurospheres were gently aspirated without disturbing the adherent debris through wide bore pipette tips to a 15 mL centrifuge tube. Following centrifugation at 300× g
for 5 min, primary neurospheres were collected and then incubated with 0.05% Trypsin-EDTA for 2 min in a water bath at 37 °C in order to dissociate them into single cells; this process was necessary because the size of the original primary neurospheres varied widely, and the debris present in the culture adversely affected cell’s differentiation (Figure 1
C). The dissociated cells were reseeded at 2.5 × 104
cells/mL in a Nunclon™ Sphera™ 96-well U-shaped-bottom microplate (100 µL cell suspension per well). Following 2-day culture in Medium-2, a stable and robust neurosphere was formed in each dish, with an average diameter of 216.8 ± 5.37 µm (n = 10) (Figure 1
D and Supplementary Figure S1
). These premium neurospheres were ready for subsequent characterization studies and other experimentation.
4.5. Characterization of Neurospheres by Immunofluorescence
To characterize the proliferation of the formed neurospheres in Figure 1
D, BrdU was added to the culture medium at a final concentration of 10 µM and the incubation was continued for 3 h. The neurospheres were gently aspirated, washed twice with PBS, and fixed in 4% PFA in PBS for 10 min at room temperature, followed by 2× PBS washes. Neurospheres were then incubated in 2 N HCl for 30 min under 37 °C to hydrolyze the DNA. The mixture was neutralized in 0.1 M sodium borate buffer (pH 8.5) for 30 min at room temperature. After two washes with PBS, samples were blocked and permeabilized in PBST containing 5% normal goat serum and 0.3% TritonX-100. The preparation was further incubated with primary antibodies against BrdU, Nestin, DCX, and/or NeuN at 4 °C overnight. Following 3 washes with PBST, neurospheres were incubated with fluorophore-conjugated secondary antibodies (1:500) at room temperature for 1 h protected from light. After another 3 PBST washes, neurospheres were mounted on microscope slides with mounting medium and coverslips for direct characterization by fluorescent microscopy.
Another set of fresh neurospheres (Figure 1
D) were then seeded on a PLO-coated surface in the differentiation Medium-3 to allow for attachment, migration, and finally differentiation into mature neurons. After 7 days in culture, the differentiated neurospheres were characterized by immunofluorescence as described above and using primary antibodies against NeuN and Nestin.
4.6. Quantification of Apoptosis in Formed Neurospheres
An apoptosis detection kit was used to examine whether neurospheres, formed under the current protocol, had a low level of apoptosis. Briefly, differentiated neurospheres were washed twice with the assay buffer provided by the kit, followed by incubation in the assay buffer containing Apopxin Green Indicator (Ex/Em = 490/525 nm) at room temperature for 1 h. After two washes with the assay buffer, images were captured under a fluorescent microscope for green apoptotic signals. The differentiated neurospheres treated with hydrogen peroxide at 1.6 mM for 30 min were used as the positive control for apoptotic signals.
4.7. Influence of Cu on Neurosphere Dynamics and D-Pen Intervention
To investigate how Cu exposure affected SVZ adult neurogenesis in vitro, neurospheres were cultured in the differentiation Medium-3 containing the final Cu concentrations at 1.0, 10.0, and 100.0 μM. The Cu treatment was terminated on day 7, and neurospheres were characterized by measuring the migration area and counting NeuN(+) mature neurons per neurosphere. For D-Pen intervention studies, the Medium 3 containing D-Pen alone at 20, 50, and 100 µM was used to culture neurospheres for 7 days, followed by assessment of neurosphere growth. From the above dose-response studies, the ideal concentrations for Cu and D-Pen were selected to investigate whether the presence of D-Pen in culture medium alleviated Cu-induced suppression in neurosphere growth. In these experiments, neurospheres grew in culture medium with (10 µM Cu) or without Cu overnight, followed by incubation with the medium supplemented with 20 µM D-Pen.
4.8. Estimation of Intracellular Cu Levels by Phen Green
Phen Green is a widely used fluorescent Cu sensor to estimate intracellular Cu levels in live cells. To determine intracellular Cu levels, the solid Phen Green (1 mg) was dissolved in 1 mL chloroform; the solution was aliquoted to separate vials and then the chloroform was allowed to evaporate to complete dryness in a light-proof fume hood. The dried vials with solid Phen Green were sealed and stored at −20 °C. Upon use, the chemical was resolubilized with 10 µL DMSO for live cell Cu imaging. Of note, the intensity of the Phen Green fluorescence correlates inversely to intracellular Cu levels due to Cu’s ability to quench its green fluorescence. In other words, neurospheres with a higher intracellular Cu level would emit less green fluorescence.
To quantify intracellular Cu status, neurospheres were cultured with the control medium, Cu (10 µM) medium, or Cu (10 µM) plus D-Pen (20 µM) medium for 7 days. These preparations were washed with PBS three times to completely remove the medium components. A serum-free background medium containing 5 µM Phen Green was added to neurospheres and incubation continued at 37 °C for 30 min. The Phen Green-loaded neurospheres were washed with PBS three times to remove unbound Phen Green; the samples were imaged under Nikon Confocal Microscope C1 (Tokyo, Japan). Average fluorescent intensity of a neurosphere was quantified by ImageJ. The intracellular Cu levels in neurospheres were estimated by the equation of (Fct − F)/Fct, where Fct and F represent the average fluorescence intensity of control neurospheres and experimental neurospheres, respectively.
4.9. Quantitative Polymerase Chain Reaction (qPCR)
Expression of mRNAs encoding key proteins in regulating Cu homeostasis and SVZ adult neurogenesis were quantified in neurospheres by qPCR. Following the 7-day culture in the control medium, Cu (10 µM) medium or Cu (10 µM) plus D-Pen (20 µM) medium, total RNA was isolated from neurospheres using TRIzol Reagent as per manufacturer’s instructions. An aliquot of RNA (0.5 µg) was reverse-transcribed into cDNA using the BioRad iScript cDNA synthesis kit. After addition of specific primers, iTaq Universal SYBR Green Supermix was used to quantify the fluorescence during the amplifications. The qPCR program was run by the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with an initial 3 min denaturation at 95 °C, followed by 40 cycles of 30 s denaturation at 95 °C, 10 s gradient 55.0–65.0 °C, and a 30 s extension at 72 °C. Dissociation curves were examined to verify that the majority of detected fluorescence was derived from the labeling of specific PCR products. Each qPCR reaction was run in duplicate. Relative mRNA expressions were calculated using 2−ΔΔCt
method with beta-actin as the reference gene. The forward and reverse primers for genes of interest in this study were designed by Primer Express 3.0 software and listed in Supplementary Table S2
4.10. Intracerebroventricular (ICV) Infusion of D-Pen by Alzet Osmotic Pumps
The ICV infusion used in Experiment 2 was graphically illustrated in Figure 6
A. Briefly, osmotic pumps were loaded with D-Pen dissolved in saline as per manufacturer’s instructions; pumps loaded with saline were used as controls. Specifically, the pump model 1007D and model 1004 were manufactured to infuse for 7 days at 0.5 µL/h and for 28 days at 0.11 µL/h, respectively. The D-Pen solution was prepared to administer at low (0.075 μg D-Pen/h) or high (0.75 μg D-Pen/h) dose during the two above infusion durations. The loaded pumps were connected to the Alzet brain infusion kit 3 to establish a pump-tubing-cannulation system. Prior to surgical cannulation, the pumps were primed by incubating in saline under 37 °C overnight (pump model 1007D) or 48 h (pump model 1004), after which the primed pumps were surgically implanted as follows.
Mice that had received three daily doses of BrdU at an interval of approximately 24 h (50 mg/kg, ip.) were individually placed in an anesthesia-induction chamber with the oxygen flow rate adjusted at 0.3 L/min and the isoflurane concentration at 3%. Once fully anesthetized, verified by no response upon a toe pinch, the head was fixed onto a stereotaxic device (KOPF Model 1900) with consistent isoflurane flow through the nosecone. Eye ointment was applied onto each eye, and hair was shaved from the eye level down to top of shoulder. A midline incision was made from eye level down to the shoulder area. A curved hemostat was then slowly inserted into the back skin of the animal with the curved end faced up to create a subcutaneous space for pump insertion. The pump was then gently inserted into the subcutaneous space and the skin was closed. The cannula holder was stereotaxically adjusted above the bregma. The coordination for the ventricular insertion was at 1.0 mm left and 0.2 mm posterior to the bregma (lateral/medial: 1.0 mm; anterior/posterior: −0.2 mm). The cannula’s stainless-steel tube was allowed to penetrate the skull (2.5 mm in depth) and tightly glued onto the skull for 15 s. The wound was closed by applying Gluture topical tissue adhesive alongside the cut. Lidocaine hydrochloride jelly and triple antibiotics ointment were topically applied on the wound, followed by a subcutaneous administration of ketoprofen (5 mg/kg). The postoperative mouse was placed on a heat pad, and typically recovered within 15 min after surgery. The pump-implanted mice were housed separately, with their conditions tightly monitored throughout the ICV infusion.
4.11. Determination of Cu Concentrations by Atomic Absorption Spectrometry (AAS)
Upon completion of ICV infusions of D-Pen or saline, mice were anesthetized and transcardially perfused with 20 mL ice-cold PBS and 20 mL ice-cold 4% PFA in PBS sequentially. A successful perfusion was confirmed by a tremor upon PFA perfusion. One half of the brain was used for IHC studies as described below. The other half of the brain was dissected for AAS analysis of Cu levels. Brain regions for AAS included SVZ, RMS, OB, hippocampus (HP), striatum (ST), and frontal cortex (FC). The SVZ tissue that covers the surface of the lateral ventricle was carefully cut off by a microdissection spring scissors under a microscope. Brain tissues were digested by ultrapure nitric acid in the MARSX press microwave-accelerated reaction system. Agilent Technologies 200 Series SpectrAA with GTA 120 graphite tube atomizer was used to quantify Cu concentrations. Digested samples were diluted properly to keep each reading within the linear range (0–50 µg/L). The detection limit of Cu by this AAS assay was 0.9 ng/mL. The intra-day and inter-day precisions of the method for Cu were 1.6% and 3.7%, respectively [69
4.12. Brain Slice Preparation and Immunohistochemistry (IHC)
The PFA-perfused half brain was further fixed in 20 mL 4% PFA in PBS under 4 °C overnight with slight agitation. Following 3× PBS washes, the fixed brain was dehydrated in 30% sucrose solution in PBS under 4 °C. Brains were expected to sink within 72 h. The dehydrated brains were then coronally or sagittally cut by a microtome into 40-µm brain slices. Specifically, coronal slices were serially placed in a 12-well plate to study changes in SVZ, OB, or RMS in the coronal view; while sagittal slices were serially harvested into a 6-well plate to investigate RMS changes. Each well accounted for 1/12 or 1/6 of the total brain sections, respectively. The brain slices were preserved in cryopreservation medium (30% sucrose, 1% polyvinylpyrrolidone, 30% ethylene glycol in 0.1 M phosphate buffer) under −20 °C.
For IHC analyses, brain slices were picked up by a paintbrush and washed in PBS 3 times to remove sucrose. For samples for BrdU analyses, slices were incubated in 2 N HCl for 30 min under 37 °C to hydrolyze the DNA. The acid-treated slices were then neutralized by 0.1 M boric solution (pH 8.5) for 30 min. Following a PBS wash, these slices were blocked in PBST containing 5% normal goat serum and 0.3% TritonX-100 for 1 h at room temperature. Blocked slices were then incubated with primary antibodies overnight at 4 °C. Following 3× PBST washes, slices were then incubated with fluorophore-conjugated secondary antibodies (1:500) for 1 h at room temperature in the dark. After 3 PBST washes, slices were counterstained with DAPI and then mounted onto microscope slides with mounting medium and coverslips. The IHC images were captured by Nikon A1Rsi Confocal system. Z-stack scanning and large-image stitching were conducted for brain slice 3D reconstruction and the subsequent cell counting. Information about antibodies used in this section was provided in Supplementary Table S1
4.13. Statistical Analysis
Data in this report were presented as mean ± standard deviation (SD). Statistical analyses of the differences among groups were carried out by one-way ANOVA with post hoc comparisons by the Dunnett’s test. Statistical column graphs were generated by GraphPad Prism 8 (San Diego, CA, USA), and all the statistical analyses were conducted using the embedded statistical programs. Differences were considered significant if p values were equal or less than 0.05.