Background
Lead (Pb) is a well-known neurotoxicant [
1,
2] that is still abundantly present in our environment despite the significant efforts to decrease its use and distribution in the environment [
3‐
5]. Pb targets multiple organ systems such as the nervous, hematopoietic, immune, renal, reproductive, endocrine, and skeletal. The central nervous system (CNS) is most sensitive to Pb toxicity, particularly during early development. In recent years, the role of immune system in Pb-induced neurotoxicity has emerged, and several inflammatory mediators, including cytokines and enzymes involved in inflammatory process, are known to play a significant role in neurotoxicity and neurodegeneration. In particular, Pb-induced astroglyosis and microgliosis have been shown to be involved in neurotoxictiy [
6,
7]. Compared to other systems, CNS is affected by doses of Pb as low as 7 μg/dL [
2]. Based on the evidence that Pb exposure at levels below the safety limit of 10 μg/dL causes neurological alteration in children [
2,
8], the US Centers for Disease Control and Prevention (CDC) recently revised the blood Pb level from 10 μg/dL, which was set in 1991, to 5 μg/dL as the reference level at which CDC recommends the initiation of public health actions [
9]. The mechanism(s) of Pb-induced neurotoxicity is thoroughly investigated and several biochemical targets and pathways involved in neurotoxicity have been identified. However, the mechanism by which these biochemical abnormalities lead to impairment of learning and memory is poorly understood at best.
Quinolinic acid (QA) is a metabolite of the kynurenine pathway (KP) of tryptophan metabolism and is a known excitotoxic compound acting through the activation of the
N-methyl-
d-aspartic acid receptor (NMDAR) [
8]. Approximately 95% of tryptophan is metabolized by the KP [
9]. QA is toxic to oligodendrocytes [
10] and neurons [
13,
14]. QA toxicity affects neurons located mainly in the hippocampus, striatum, and neocortex [
15]. In pathological concentration, QA promotes apoptosis in oligodendrocytes, neurons, and astrocytes [
16‐
18]. Increased levels of QA are involved in neurodegenerative and neurological disorders such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, AIDS-dementia, cerebral malaria, depression, and schizophrenia [
15,
19‐
22]. The mechanisms by which QA exerts its neurotoxic effects is through provoking enhanced intracellular calcium through over activation of NMDAR, augmented levels of extracellular glutamate, increased reactive oxygen species and reactive nitrogen species formation, decreased activity and expression of antioxidant systems, oxidative stress, stimulated protease activity, and cell death [
20,
23‐
25]. QA-induced neurotoxicity also involves destabilization of the cytoskeleton [
22] and energy depletion [
15].
The major and rate-limiting enzyme of KP, indoleamine-2,3-dioxygenase-I, (IDO-I), is expressed in astrocytes, microglia, and neurons. The expression of IDO-I is increased by inflammatory mediators and cytokines such as amyloid peptides, LPS, IL-1β, TNF-α, and IFN-γ [
11,
26,
27]. The other major enzyme of the KP that converts kynurenine (the first stable metabolite of KP) into QA is kynurenine 3-monooxygenase, which is also abundantly expressed in microglia, macrophages, and monocytes. The expression of this enzyme is also upregulated by pro-inflammatory mediators [
28]. Thus, a pro-inflammatory environment highly favors the generation of QA in the brain. In addition to the local activation of the KP and synthesis of QA in the CNS, kynurenine produced systemically can cross the blood brain barrier and can be converted into QA within the CNS [
29].
Both Pb and QA share several features of neurotoxicity. For example, in rats, both Pb [
30] and QA [
31‐
33] impair learning and memory. Similarly, both Pb and QA induce tau hyperphosphorylation [
22,
34]. Tau hyperphosphorylation is associated with memory loss in Alzheimer’s disease and other dementias like FTD-17. In addition, QA is produced in response to oxidative stress and is also a pro-oxidant and induces oxidative stress [
11,
23,
25‐
28]. Pb is known to be involved in oxidative stress. One of the reported mechanisms of QA neurotoxicity is increased accumulation of glutamate at the synapse by increasing its release from neurons and inhibiting its uptake by astrocytes [
15]. Similar to QA, Pb is also known to increase spontaneous release of glutamate and GABA from the presynaptic terminal of rat hippocampal neurons [
35].
In this study, we investigated the role of QA in Pb-induced impairment of learning and memory. We hypothesize that Pb, being a prooxidant, increases the brain levels of QA, which subsequently results in neurotoxicity and impairment of learning and memory. The specific aims were investigating the effect of the following: (1) Pb exposure on QA level in blood and QA immunoreactivity in different regions of the brain; (2) Pb exposure during lactation on spatial learning and memory; (3) intraventricular infusion of QA on spatial learning and memory; and (4) intraventricular infusion of QA on the expression of various molecules involved in learning and memory. We report here that Pb exposure increased QA level in blood and QA immunoreactivity in the brain of rats and that QA infusion in the brain produced behavioral and biochemical changes that are largely similar to that produced by Pb exposure. These results support the hypothesis that increased QA production in response to Pb exposure is involved in learning and memory impairment.
Methods
Animals
Wistar rats were provided by the Animal Resources Center, Faculty of Medicine, Kuwait University. The animals were housed at constant temperature (21 ± 2 °C) and relative humidity (50 ± 10%) with a 12-h light/dark cycle (0700–1900 h). The animal maintenance and exposure were according to the protocol approved by the Animal Care and Use Committee of Kuwait University and the experimental protocol followed the ARRIVE guidelines for the care and use of laboratory animals.
Lead exposure protocol
At birth, pups were culled to 10 per liter and exposed to Pb via their dams’ drinking water (0.2% Pb acetate in deionized water) from postnatal day (PND) 1 to 21. Control group with similar number of pups was given deionized water. From PND21, both the groups (Pb-exposed and control) were given tap water until the termination of the study. In our previous experiments, similar exposure protocol produced a blood Pb level of 8.3
± 4.3 μg/dL in pups at PND21 [
36]. Rats from both groups were subjected to Morris water maze (MWM) test from PND30 and from PND45. Short-term memory (STM) was assessed by probe test (memory retention test) which was done 48 h after the last learning session. Long-term memory (LTM) was assessed by probe test on PND45 or PND60. After the LTM retention test (PND45 and PND60, respectively), animals were euthanized with CO
2. Thoracic cavities were opened, and blood was drawn from the right ventricle for measuring QA by ELISA.
ELISA for quinolinic acid
Blood was centrifuged at 2000×g for 15 min. Serum was transferred to Eppendorf tubes and stored at − 80 °C until analysis. The ELISA for QA was conducted in serum by using QA ELISA kit (Aviva Systems Biology Corporation, San Diego, CA, USA; Cat. No. OKCD02284). Standard and the samples (50 μl) were added to the wells pre-coated with specific antibody in a 96-well plate, and the assay was conducted as per the manufacturer’s instructions. The results were calculated by fitting the optical density into a four-parametric logistic curve.
Intraventricular infusion of quinolinic acid
Wistar rat pups (21-day old) were anesthetized with a mixture of ketamine (40 mg/kg) and xylocaine (5 mg/kg) and fixed in a stereotaxic frame. The coordinates were as follows: anteroposterior—3 mm behind bregma; lateral—3.6 mm from midline; and depth—3.8 mm from skull surface. Two holes were made, one for a steel cannula aimed at the right lateral ventricle and the other for the fixing screw. An Alzet Brain Infusion Kit (cannula and tubing) was used with Alzet 1007D osmotic minipump (Durect Corporation, CA, USA). QA (9 mM) prepared in sterile saline was infused in the right lateral ventricle for 7 days. Assembling and filling the osmatic minipump were done in a sterile culture hood. Filled osmotic minipumps were incubated in saline water bath maintained at 37 °C for 12 h to stabilize the pump and to check the flow. Rat pups infused with the same volume of sterile normal saline served as vehicle control (VC) group. Osmotic pumps were removed on 8th post implantation day. Spatial learning and memory was assessed by MWM test starting on PND30 and PND45 (two separate groups).
Spatial learning and memory testing
Pb-exposed and QA-infused rats, along with their respective controls, were subjected to the MWM test at PND30 (control, n = 10; Pb-exposed, n = 14; vehicle control, n = 7; QA infused, n = 8) and a separate group at PND45 (control, n = 9; Pb-exposed, n = 13; vehicle control, n = 10; QA infused, n = 11). The water maze apparatus consisted of a water tank of 2.0 m in diameter, divided into four virtual quadrants. A circular platform was submerged in one of the quadrant (target/platform quadrant). The rats were trained in the water maze in six sessions on four consecutive days (one session on the first and last day and two sessions on the 2nd and 3rd day). Each session consisted of four trials; each trial was of 120-s duration. Inter-trial interval was 60 s. In each trial, time taken to reach the hidden platform (escape latency) was measured and analyzed using EzVideo™ 5.70 Digital Video Tracking system (Accuscan Instruments, Inc., Columbus, OH, USA). Two days and 10 days after the last learning session, rats were subjected to memory retention test (probe test), for short-term (STM) and long-term memory (LTM), respectively. Each probe test session was of 30-s duration. Data on several parameters, i.e., platform quadrant (zone) time, platform quadrant entry latency, and distance traveled in the target/platform quadrant, were measured and analyzed using EzVideoTM 5.70 Digital Video Tracking system.
Western blotting
At the end of the experiment (PND45 and PND60, respectively for the two groups), animals were euthanized with CO
2 and then decapitated. Skull cap was removed, and the brain along with the skull base was bisected. Cerebral hemispheres were placed in pre-weighed Eppendorf tubes and were snap-frozen in liquid nitrogen and stored at − 80 °C till analyses. Cerebral hemispheres were homogenized in five volumes of RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 50 nM NaF, 1 mM sodium vanadate and protease inhibitor cocktail (Roche Diagnostic, Castle Hill, NSW, Australia) (PND30: vehicle control,
n = 6; QA infused,
n = 6) (PND45: vehicle control,
n = 6; QA infused,
n = 6). Protein in each sample was determined by the Bradford method and the homogenates were kept at − 80 °C till used. Protein (50 μg) from each sample was resolved on a 10% bis-tris SDS-PAGE (NuPAGE, Invitrogen, Carlsbad CA, USA) and transferred onto PVDF membrane. The membrane was blocked with 5% non-fat dry milk in TBS-Tween-20 for 1 h and incubated with specific antibodies (Table
1) overnight at 4 °C at a dilution of 1:500 or 1:1000 (Table
1). The membrane was rinsed with TBS-Tween-20 six times (10 min each). The membrane was then incubated with the HRP-conjugated appropriate secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at room temperature, washed as before and developed with the ECL kit (Thermo Scientific). Actin was used as a loading control. After developing the radiographic film, protein bands were quantified by Syngene Genetool software.
Table 1
Details of primary antibodies used for Western blot
PP1 (E-9)*, # | PP1 Catalytic subunit | Santa Cruz Biotech |
Anti-PP2A (Clone 1D6)*, # | PP2A Catalytic subunit | Merck Millipore |
Anti-Synaptophysin (Clone SVP-38)*, # | Synaptophysin | Sigma-Aldrich |
CREB (48H2)*, # | Total CREB-1 protein | Cell Signaling |
Phospho-CREB (Ser133) (87G3)*, # | Ser 133-phosphorylated CREB | Cell Signaling |
AT180*, ## | Pt231 | Invitrogen |
Anti-PSD95*, ## | Rat PSD95 | Sigma-Aldrich |
Anti-NR1*, ## | Amino acids 918–938 with N-terminally added lysine of the NR1 subunit of NMDAR | Sigma-Aldrich |
Anti-phospho-NR1 (Ser897)**, ## | Phospho-Ser897 of human NMDA Receptor | Cell signaling |
Anti-phospho-NR2B (Ser1303)**, ## | CKLRRQH(Ps)YDTFVD of NR2B | Merck Millipore |
Anti-NR2B**, ## | 1437–1456 of mature mouse NR2B | Merck Millipore |
Tau Antibody (A-10)*, # | Total tau | Santa Cruz Biotech |
Tissue processing for immunohistochemistry
At the end of probe test (PND45 for the PND30 group and PND60 for the PND45 group), rats were euthanized with CO2 and transcardially perfused with the fixative consisting of 4% paraformaldehyde and 0.1% glutaraldehyde in 100 mM phosphate buffer, pH 7.4 at 4 °C (PND45: control, n = 4; Pb-exposed, n = 4; PND60: control, n = 4; Pb-exposed, n = 4). Brains were transferred to cold fixative and kept in the fixative for 4–6 h at 4 °C. Tissues were washed overnight in cold phosphate buffer, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Sections were cut at a thickness of 6 μm on rotary microtome. Sections were deparaffinized, rehydrated, and washed with 10 mM phosphate buffered saline (PBS), pH 7.4 at room temperature. The sections were quenched for endogenous peroxidise activity and free aldehyde groups with 3% H2O2 in water and 50 mM glycine in PBS, respectively. The sections were then sequentially incubated in primary antibody (rabbit anti-quinolinic antibody, Cat. No. ab37106, Abcam, Cambridge, UK, 1:100 dilution), biotinylated secondary antibody, and avidin-biotinylated horseradish-peroxidase macromolecular complex (ABC kit, Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized using diaminobenzidine as chromogen. Sections were counterstained with hematoxylin prior to mounting. The sections incubated in normal serum instead of the primary antibody served as negative controls (results not shown). For quantification of QA immunoreactive cells, from each rat, four sections were selected. Number of immunoreactive cells in six randomly selected fields in each section for each region (Cortex, CA1, CA3, dentate gyrus and thalamus) were counted at × 40 magnification in an Olympus microscope (BH-2) fitted with Nikon digital camera (Nikon Digital Sight DS-Fi1). NIS-elements D2.20 software was used for quantification of immunoreactive cells. At × 40 objective, each field was 56,575 μm2 in area. Mean number of immunoreactive cells/field in each region in each rat was calculated. Finally, mean ± SEM was calculated for each group.
Statistical analysis
Data were expressed as Mean ± SD. Differences in the expression of various biochemical markers (by Western blot) and the levels of QA in the blood of VC and QA-exposed animals were analyzed by a Student’s t test for two independent samples with unequal variance. A p < 0.05 was considered statistically significant. For the spatial learning test, we averaged the escape latencies across the four trials for each rat. These means were then analyzed across the six sessions. A two-way repeated measures ANOVA was used for main effect (treatment comparisons) with sessions as the repeated measure and escape latency as the dependent variables. For the probe tests (spatial memory), group means were compared by Student’s t test for all parameters. Data were analyzed by SPSS version 23 for Windows (SPSS Inc., Chicago, IL, USA).
Discussion
In this study, we hypothesized that Pb-induced neurotoxicity involves, at least in part, increased levels of QA. Our results show that Pb exposure not only resulted in significant increase in the number of QA-immunoreactive neurons in the brain but also increased QA levels in serum. To our knowledge, this is the first study reporting increased QA production in response to Pb exposure. Pb, a pro-oxidant metal, may increase QA levels by microglial and astroglial activation. Microglial and astroglial activation in the brain, and in particular in the hippocampus, has been reported in Pb-exposed rats and mice. This microglial and astroglial activation was associated with increased expression of pro-inflammatory cytokines like IL-1β, IL-6, and TNF-α [
37‐
43]. Similarly, in vitro Pb exposure of BV-2 mouse microglia resulted in increased expression of pro-inflammatory cytokines (TNF-α, IL-6, MCP-1) and pro-inflammatory enzyme COX-2 [
44]. Thus, the generation of pro-inflammatory mediators and cytokines by Pb seems a logical mechanism for increased production of QA.
We then tested the effect of QA, infused directly into the ventricle, on learning and memory to investigate if Pb exposure and QA produce similar impairment of learning and memory. It is assumed that QA infused into the right ventricle will be circulated throughout the brain and will mimic increased levels of QA in the brain caused by Pb exposure. Our results show that similar to Pb-exposure, QA infusion resulted in significant impairment of learning at PND30 but not at PND45. This lack of effect of learning impairment in the PND45 rats may be explained by the clearance of Pb or QA from the brain over time. Pb exposure significantly impaired STM at both PND30 and PND45. QA infusion also impaired STM; however, the impairment was only statistically significant at PND45. The lack of statistical significance in memory impairment at PND30 could be explained by the small number of rats (7–8 in the QA group) in these experiments. The lack of effect of Pb or QA on learning at PND45 but significant impairment of STM at this age suggest that effects of both Pb and QA on STM are long-lasting compared to their effect on learning. It may also suggest that the effects of both Pb and QA on learning process and memory recall are different. The prolonged effect on memory recall, but not learning, may be due to the adverse effects of Pb and QA on molecules, receptors and specific structural components (e.g. synapses, dendritic field, dendritic spines) concerned with memory recall. Alternatively, Pb and QA may affect the memory consolidation process itself. Overall, QA infusion mimics the effects of exposure of Pb through dams’ drinking water, in producing similar effects on learning and STM, supporting our hypothesis of QA involvement in Pb-induced neurotoxicity.
A similar QA infusion protocol in adult Sprague-Dawley rats resulted in learning impairment and short-term working memory deficits [
32]. The working memory deficits caused by QA infusion in this study lasted for up to 3 weeks after the termination of infusion, and these results conform to our findings. Cognitive deficits caused by QA infusion in the brain of Sprague-Dawley rats were prevented by simultaneous subcutaneous infusion of memantine, an NMDA receptor antagonist [
33], suggesting that QA impaired learning and memory through NMDAR activation. In other studies, a single injection of QA into the striatum of rats also caused significant memory impairment [
31,
45].
The effects of Pb on NMDAR have been extensively studied, and it has been reported that Pb affects NMDAR function by modulating the expression and the subunit composition of the receptor (Reviewed by Rahman [
1]). Furthermore, these effects seem to be brain region-specific. For example, Pb exposure decreased the expression of NR1 and NR2B subunits in hippocampal neurons, whereas in cortical neurons, no effect was seen on the expression of NR1 and the expression of NR2B was significantly increased [
46,
47]. We investigated if QA infusion in the brain would produce similar effects on the NR1 and NR2B subunit expression. We did not find any changes in the protein levels of NR1 at either PND45 or PND60. On the other hand, the expression of NR2B was not affected at PND45, but was significantly decreased by QA infusion in the brain lysate of PND60 rats. The decrease in NR2B content in the brain may be due to the normal ontogenic changes in the brain. During early postnatal stage, the expression of NR2B containing NMDAR predominates. A developmental switch occurs later, which is accompanied by an increase in the NR2A-containing receptors and a subsequent decrease in NR2B containing receptors [
41]. NR2B-containing NMDAR are more abundant in the brain as these are expressed both in synapses and outside the synapses, compared to NR2A containing NMDAR, which are predominantly located in synapses [
48]. An increase in the synaptic NR2B-containing receptor without any change in the overall cellular level of NR2B in response to Pb exposure has been reported [
47,
49]. In the Zebrafish embryo, Pb decreased the expression of NR1 and had no effect on the expression of NR2B [
50]. Further research is needed to elucidate the temporal and brain-region-specific changes in the expression of these subunits in response to QA administration.
CREB is a transcription factor for many NMDAR activity-dependent immediate early genes which play an essential role in learning and memory [
51,
52]. CREB plays an essential role in the propagation of signal to the nucleus by linking NMDAR activation to the expression of genes necessary for synaptic plasticity [
53]. CREB is phosphorylated in response to NMDAR activation and induction of long-term potentiation (LTP). CREB phosphorylation at S
133 facilitates the recruitment of CREB-binding proteins and the assembly of transcriptionally active complex at the start site of CRE containing genes [
54]. We looked at the effect of QA infusion on the expression of CREB and its phosphorylation at S
133. No effect was seen on the total level of CREB or its phosphorylation at S
133 in response to QA infusion. Similar to this, no effect of Pb exposure was seen on the total level of CREB, but in contrast to the effect of QA, Pb exposure decreased the phosphorylation of CREB [
49,
55].
Synaptophysin is a major protein of synaptic vesicles and plays an important role in synaptic transmission, stabilization, and plasticity [
56,
57]. It is involved in neurotransmitter release and synaptic vesicle cycle and thus is used as marker of synaptic terminals [
58,
59]. A decrease in the expression of synaptophysin in rats and mice exposed to Pb has been reported, and this decrease in synaptophysin level was suggested to be a potential mechanism of Pb neurotoxicity [
47,
60‐
62]. On the other hand, Gassowska et al. [
63] reported increased synaptophysin expression in the cerebellum of rat pups perinatally exposed to Pb. In our study, QA infusion did not affect the level of synaptophysin in the whole brain lysate of rats. Similar to our results, intra-striatal injection of QA into rat brain showed no change in the expression of synaptophysin [
64]. Thus, the effect of QA on synaptophysin appears to be different from the effect of Pb exposure.
At the postsynaptic site, neurotransmitter receptors, signaling enzymes, and cytoskeletal proteins are clustered along with scaffolding proteins in a structure called postsynaptic density [
65]. PSD-95 is an important component of postsynaptic density which is highly expressed in glutamatergic synapses along with NMDAR [
63]. PSD-95 increases the number and the size of dendritic spines and contributes to synaptic stabilization and plasticity [
66‐
68]. Reduction of PSD-95 in neurodegenerative diseases of the brain, such as Alzheimer’s disease and Parkinson’s disease, has been reported [
69‐
71]. In rat pups, perinatal Pb exposure decreased the expression of PSD-95 in the forebrain, cortex, and cerebellum, but increased its expression in the hippocampus [
63]. In mice, developmental Pb exposure also resulted in decreased mRNA and protein levels of PSD-95 at PND40 [
62]. We observed a significant decrease in PSD-95 level in the brain in response to QA infusion at PND45 but not at PND60. The lack of effect at PND60 is likely due to the clearance of QA and its effects from the brain with time. In a previous study, prenatal inhibition of the KP by Ro61-8048 administration into the dams, which inhibits the enzyme kynurenine-3-monoxygenase and shifts the pathway in favor of kynurenic acid, resulted in increased expression of PSD-95 in the PND21 rat pups [
72]. These results are parallel to our findings. As PSD-95 is a marker for synapses, we speculate a decrease in the number of synapses in the brain of QA-infused rats. We have previously shown a decrease in the number of synapses in the hippocampus of rat pups developmentally exposed to Pb [
30]. Overall, the effect of QA infusion on PSD-95 expression is similar to the effect of Pb exposure on PSD-95. The effect of QA infusion on the number of synapses, particularly in the hippocampus, needs to be investigated.
Whereas, LTM involves protein synthesis, growth, and formation of new synapses [
73‐
75], STM is regulated by covalent modification of proteins in the presynaptic or postsynaptic structures [
76‐
78]. Reversible protein phosphorylation is one of the major covalent modifications involved in this process and is regulated by the balance between protein kinases and protein phosphatases. Of the protein phosphatases, serine/threonine protein phosphatases PP1 and PP2A are the major phosphatases in the brain, accounting for over 90% of the total mammalian brain protein phosphatase activity [
78]. Any alteration in the activity of these two phosphatases may significantly affect the phosphorylation state of proteins. A decrease in the activities of these phosphatases has been implicated in Alzheimer’s disease. [
79]. On the other hand, overactivation of these phosphatases are also reported to be involved in learning and memory impairment (reviewed by Rahman et al. [
1,
30]). PP1 and PP2A are located in physical proximity to NMDAR [
80]. Following excitatory neurotransmission and Ca
2+ influx, NMDAR are phosphorylated and then rapidly dephosphorylated. This reversible phosphorylation controls synaptic strength, memory formation, and storage by the induction of LTP or long-term depression (LTD) [
81]. Phosphorylated NMDAR have enhanced channel openings, and the consequent increase in Ca
2+ influx is implicated in the induction of LTP [
82‐
85]. Dephosphorylation of the NMDAR on the other hand is implicated in the induction of LTD [
86,
87]. Stimulation of NMDAR activates PP1 and PP2A [
88]. Downstream of the NMDAR, these phosphatases dephosphorylate CREB [
89] and thereby reduce CREB-mediated gene expression [
90‐
93].
Pb is known to dysregulate serine/threonine protein phosphatases in the brain. We have previously reported that early postnatal Pb exposure resulted in increased PP1 but decreased PP2A levels in the brain of rat pups at PND21 [
34]. We also reported a decrease in PP2A levels in the hippocampus of rats pups at PND30 [
28]. We therefore investigated the effect of QA infusion on the levels of these phosphatases in the brain. PP1 expression was decreased only in the PND60 rats, whereas the expression of PP2A was decreased at both PND45 and PND60. Parallel to these results, we have previously reported a decrease in total brain phosphatase activity and in PP2A activity by QA in a dose-dependent manner in cultured human neurons. We also showed a decrease in the expression of both PP1 and PP2A in a dose-dependent manner [
22]. An increase in the phosphorylation level of low molecular weight neurofilament subunit in neurons and the glial fibrillary acidic protein in astrocytes, which is consistent with decreased phosphatase activity, has been reported in response to a single injection of QA in the brain [
25].
Tau is a microtubule associated protein which is required for the maintenance of intact microtubule structure. Disruption of microtubules has been shown to be associated with memory loss and neuronal death [
94]. Abnormally hyperphosphorylated tau has not only less affinity for binding with microtubules, but also sequesters normal tau and other microtubule-associated proteins and causes disassembly of microtubules [
95]. Tau hyperphosphorylation and its subsequent accumulation as paired helical filaments are implicated in neurodegenerative diseases and learning and memory impairment. Pb causes hyperphosphorylation of tau both in vitro [
96] and in vivo in Pb-exposed animals [
34]. In this study, we showed that QA infusion resulted in the phosphorylation of tau at T
231, without affecting total tau. We have previously reported a dose-dependent increase in the phosphorylation of tau at several residues including T
231 in cultured human neurons exposed to QA [
20]. Tau at this site was also phosphorylated by Pb in our previous studies [
34,
96]. Hyperphosphorylation of tau at T
231 results in tau self-assembly [
97]. QA-induced NMDAR activation appears to be the mechanism of this increased tau phosphorylation, as both glutamate and NMDA increased tau phosphorylation at sites similar to QA. Furthermore, MK-801, an NMDAR antagonist, inhibited tau phosphorylation at AT-180 site [
22,
98]. These results clearly indicate that both Pb and QA have similar effects on tau phosphorylation, further supporting our hypothesis of QA involvement in Pb-induced neurotoxicity.