Brain region-specific dopamine receptor changes and astrocyte activation influence tau pathology through CDK5 in Alzheimer's disease models

Abstract

Background

The abnormal extracellular accumulation of amyloid-β (Aβ) plaques and intracellular tau inclusions are hallmarks of early events in the pathogenesis of Alzheimer's disease (AD). Although growing evidence implicates neurotransmitter dysregulation in AD-associated neurodegeneration, the influence of these pathological hallmarks on dopaminergic signaling remains poorly understood. This study reports changes in dopamine receptor (DR), Aβ, and astrocyte distribution in the cortex and hippocampus of 5XFAD mice.

Objective

To investigate how Aβ pathology alters dopamine receptor subtype expression in the AD brain and neuronal models, and whether this contributes to tau phosphorylation and CDK5 activation.

Methods

We examined DR1-DR5 expression and localization in the cortex and hippocampus of 5XFAD mice using immunohistochemistry, qPCR, and western blot. SH-SY5Y cells were differentiated with retinoic acid and treated with Aβ₁₋₄₂; MC-65 cells produced endogenous Aβ via tetracycline withdrawal. DR1, DR2, and DR3 agonists were used to assess effects on cAMP, CDK5, and tau phosphorylation.

Results

In AD brains, Gs-coupled DR1 and DR5 were upregulated, while Gi-coupled DR2, DR3, and DR4 were downregulated at mRNA and protein levels. SH-SY5Y and MC-65 cells recapitulated these subtype-specific changes following Aβ exposure. In the cortex, receptor alterations were implicated in increased CDK5 and tau phosphorylation. DR activation modulated cAMP and kinase pathways in a receptor- and cell-specific manner. The cortex showed greater vulnerability to Aβ-associated degeneration, whereas the hippocampus was more susceptible to inflammation and tau pathology.

Conclusions

These findings reveal a role for DR subtypes in regulating tau phosphorylation and CDK5, with implications for AD-related cognitive dysfunction.

Introduction

Alzheimer's disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder characterized by memory loss and cognitive decline. ¹ The clinical symptoms of AD are heterogeneous. Pathologically, the disease is defined by two hallmark features: the extracellular deposition of amyloid-β (Aβ) plaques and the intracellular accumulation of hyperphosphorylated tau, forming neurofibrillary tangles (NFTs).^2,3 These features impair synaptic function and destabilize the cytoskeleton, leading to widespread neuronal loss. While the etiology of AD is traditionally linked to Aβ and tau, recent studies have suggested that perturbed neurotransmitter systems also play a crucial role in disease progression.^4,5 However, how these alterations interact with Aβ deposition and tau pathology remains poorly understood.

Cholinergic dysfunction has long been a focus in AD research, primarily due to the early degeneration of basal forebrain cholinergic neurons. ⁶ Recently, however, studies have focused on the dopaminergic (DAergic) system. Dopamine (DA) regulates movement, motivation, and executive functions, including reward, depression, and critically influences learning and memory.^7–9 Studies have implicated DAergic dysfunction as an upstream contributor to cognitive decline in human AD brains and animal models; however, the underlying mechanisms remain incompletely understood.^10–13 Moreover, early tau pathology and neuronal loss have been observed in the ventral tegmental area (VTA) and substantia nigra, key DAergic brain regions projecting to the cortex and hippocampus. Disruption of these two major DAergic projections, which are involved in cognition and emotional regulation, may contribute to neuropsychiatric symptoms, including apathy and depression.^13,14 Watamura et al. recently showed that stimulation of DA release from the VTA increases neprilysin activity and reduces Aβ deposition in cortical regions of transgenic mice. ¹⁵ However, the relationship between DAergic neurotransmission, Aβ pathology, and the progression of AD is still a matter of debate.

DA exerts its effects through five different dopamine receptors (DR1-5), members of the G protein-coupled receptors (GPCRs) family, classified into D1-like (DR1, DR5) and D2-like (DR2, DR3, DR4) families. D1-like receptors activate adenylyl cyclase and elevate intracellular cyclic adenosine monophosphate (cAMP), whereas D2-like receptors inhibit adenylyl cyclase and reduce cAMP levels. ¹⁶ Beyond cAMP regulation, DRs orchestrate multiple intracellular processes, modulating phospholipase C-inositol triphosphate signaling, Ca²⁺ and K⁺ ion channels, and Na⁺/K⁺ ATPase activity, which are critical for neuronal excitability, synaptic plasticity, and metabolic stability.^16,17 DRs are widely expressed in the central nervous system, with DR1 being the most abundant, followed by DR2, DR3, and DR5, whereas DR4 is the least expressed subtype. ¹⁸ Their spatial distribution across the cortex, hippocampus, and striatum underlies their roles in learning, memory, and executive function. Dysregulation of DR-mediated signaling disrupts kinase-phosphatase homeostasis, leading to aberrant tau phosphorylation, aggregation, and neuronal instability.^7,19–21 These findings suggest that DR dysfunction may directly contribute to cytoskeletal destabilization and tau-driven neurodegeneration, extending beyond traditional neurotransmission roles. Notably, hippocampal DR2 correlates with memory function in AD.^19,22,23 Donthamsetti et al. showed that DA increases cortical excitability by acting through the D2-like receptors, while D1-like receptors enhance cortical acetylcholine release. ²⁴ In line with this, we previously showed that the expression levels of DR1 and DR2 were reduced in human AD brain tissue compared to control subjects. ¹¹ DR5 was the only receptor subtype with increased expression in the AD frontal cortex compared to controls. Importantly, Koch et al. showed that DR2/DR3 agonists, such as rotigotine, may alleviate AD pathology by restoring cortical plasticity, highlighting the neuroprotective role of receptor-specific modulation. ⁷

Tau is a key microtubule-associated protein whose phosphorylation is tightly regulated by kinases such as cAMP-dependent protein kinase A, cyclin-dependent kinase 5 (CDK5), and glycogen synthase kinase 3.^25,26 Under physiological conditions, tau stabilizes microtubules and maintains cytoskeletal integrity; however, tau hyperphosphorylation disrupts microtubule binding, impairs synaptic plasticity, and contributes to neurodegeneration.^26–28 While studies have suggested DR involvement in tau regulation, direct experimental validation of how specific DR subtypes influence tau phosphorylation and CDK5 activity remains limited and warrants further investigation. We previously demonstrated that DR1 and DR2 orchestrate cytoskeletal stability by interacting with microtubule-associated proteins [MAP2 and β-III tubulin (Tuj1)], which directly influence neuritogenesis and synaptic integrity. ²⁹ Given that tau pathology in AD involves aberrant phosphorylation and cytoskeletal collapse, these observations raise the possibility that DR subtype signaling may be modulated by tau-related neurodegenerative processes through kinase-regulated pathways such as CDK5.

To determine whether Aβ, NFT, and tau phosphorylation (p-tau) in AD pathology are associated with DAergic neurotransmission in animal models and in vitro, we used 5XFAD transgenic (TG) and age-matched wild-type (WT) mice, as well as SH-SY5Y and MC-65 human neuronal cell models, exposed to exogenous or endogenous Aβ. We first characterized the expression of DR1-DR5 in the cortex and hippocampus of 5XFAD mice, revealing region- and subtype-specific alterations consistent with DAergic disruptions reported in AD. We recapitulated these in vivo findings in vitro using SH-SY5Y and MC-65 human neuronal cell lines under Aβ exposure. To functionally evaluate these receptor alterations, we selectively activated DR1, DR2, and DR3 in these cell models and examined their effects on p-tau and CDK5 expressions. Our results demonstrate that DR2 and DR3 activation attenuate p-tau and CDK5 expression in a cell-type-specific manner. These findings suggest a mechanistic link between DR-mediated signaling and tau pathology, implicating DR subtypes as potential therapeutic targets in AD-related neurodegeneration.

Methods

Cell culture and treatments

Human neuroblastoma SH-SY5Y cells (Sigma-Aldrich, Oakville, Canada) and human MC-65 cells (a kind gift from Dr G.M. Martin, Washington University) were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin under standard cell culture conditions (37°C, 5% CO2, humidified atmosphere).^29,30

To induce neuronal differentiation, SH-SY5Y cells were treated with 10 µM all-trans retinoic acid (RA, Sigma-Aldrich) for 5 days. After differentiation, SH-SY5Y cells were exposed to 1 µM Aβ₁₋₄₂ (Anaspec, Fremont, USA) for 24 h in the presence or absence of 1 µM selective DR agonists: SKF 38393 hydrobromide (DR1 agonist, Cat# 0922), Quinpirole hydrochloride (DR2 agonist, Cat# 1061), and (+)-PD 128907 hydrochloride (DR3 agonist, Cat# 1243). All DR agonists were purchased from Tocris Bioscience.

For MC-65 cells, Aβ production was induced by removing tetracycline for 24 h from the media, during which cells were treated with DR agonists at the same concentrations as above. Control groups consisted of differentiated SH-SY5Y cells and MC-65 cells maintained in medium containing 1 μg/mL tetracycline (to suppress Aβ expression).

Animal model

Male B6SJLF1 (carrier; 5XFAD) and age-matched non-carrier (WT) were purchased at 6 weeks of age from Jackson Laboratory/MMRRC and housed at the Multidisciplinary Biomedical Facility, University of British Columbia (n = 6/group). All animals were acclimatized for two weeks under controlled environmental conditions (22 ± 2°C; 50 ± 10% humidity; 12-h light/dark cycle) with ad libitum access to food and water. Both WT and TG groups were aged to 34 weeks, after which animals were euthanized, and their brain was isolated on ice. All animal procedures were approved by the UBC Animal Care Committee and complied with guidelines established by the Canadian Council on Animal Care (protocol #A22-0210).

Peroxidase immunohistochemistry

The subcellular distribution of DR subtypes in brain tissue was studied by peroxidase-based immunohistochemistry using the VECTASTAIN^® ABC Kit (Cat# PK-4001, Vector Laboratories), as previously described.^11,31 Immunolabeling of DR1 and DR2 was performed on free-floating coronal sections (30 µm) using a vibratome, whereas DR3, DR4, and DR5 were analyzed using paraffin-embedded sections prepared by Wax-it Histology Services (Vancouver, BC, Canada). 5 µm-thick paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked by incubating the sections in 0.3% hydrogen peroxide prepared in Tris-buffered saline (TBS, pH 7.4) for 15 min. After three rinses in TBS, sections were incubated in 5% normal goat serum (NGS) in TBS for 1 h at room temperature. Tissue sections were incubated overnight at 4°C with DR subtype-specific polyclonal antibodies (1:500) ¹¹ in TBS containing 1% NGS. Following three washes in TBS, sections were incubated for 1 h at room temperature with biotinylated goat anti-rabbit IgG (1:250), followed by a 30-min incubation with the avidin-biotin-peroxidase complex. Immunoreactivity was visualized using 0.05% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich) in water. The sections were then covered with a coverslip, and brightfield images were captured using a Leica DML microscope equipped with a CoolSNAP CCD camera. The specificity of antibodies was validated in the absence of primary antibodies, as described previously. ¹¹ Image composites were constructed and processed using Adobe Photoshop 2024.

Quantification of immunoreactivity

Quantitative measurement of immunoreactivity was conducted using ImageJ software.^30,31 For each animal, ten randomly selected non-overlapping regions of interest (ROIs) were selected from the cortex and hippocampus. Brightfield images were converted to 8-bit grayscale, and the mean signal intensity was measured within each ROI. The background intensity was subtracted to correct for nonspecific staining. The corrected values were expressed in arbitrary units (AUs). Data from at least three animals per group were analyzed, and group means were used for statistical comparisons.

Quantification of immunopositive neurons

Immunopositive neurons were quantified using a combination of manual counting and semi-quantitative neuron tracing using ImageJ. For each animal, ten randomly selected, non-overlapping fields were captured at 10× magnification from the cortex and hippocampus. A blinded observer manually counted neurons exhibiting distinct immunoreactivity; background signal was quantified and subtracted from intensity measurements. To corroborate these findings, the neuronal soma was also traced using the NeuronJ plugin within ImageJ software. Neuronal density was calculated as the number of immunopositive cells per square millimeter (mm²), and group means were used for statistical comparisons.

Immunocytochemistry

Differentiated SH-SY5Y cells were grown on glass coverslips and were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed cells were blocked for 1 h in 5% normal goat serum in PBS (pH 7.4), followed by overnight incubation at 4°C with rabbit polyclonal anti-DRs (1:300). ¹¹ Following PBS washes, cells were incubated for 2 h at room temperature with Cy3-conjugated secondary antibodies (1:500, Jackson ImmunoResearch, USA). Coverslips were mounted, and images were captured using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Germany). Image composites were constructed and processed using Adobe Photoshop 2024.

Western blot analysis

Protein lysates were prepared from SH-SY5Y cells, MC-65 cells, and mouse brain tissue using Cell Lysis Buffer (Cat# 9803S; Cell Signaling Technology), supplemented with a 1% protease and phosphatase inhibitor cocktail. For brain samples, animals were euthanized, and the cortex and hippocampus were rapidly dissected on ice. Tissues were homogenized in lysis buffer, followed by centrifugation at 10,000 × g for 10 min at 4°C to remove debris. The resulting supernatants were collected, and protein concentrations were determined using the Bradford assay. 12 µg of protein was resolved on SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore). Membranes were blocked with 5% BSA in TBS-T for 1 h and incubated overnight at 4°C with the following primary antibodies: Aβ (1:500, Cat# 8243S, Cell Signaling Technology), GFAP (1:1000, Cat# mab360, Millipore), p-tau Ser404 (1:1000, Cat# 20194, Cell Signaling Technology), p-tau Thr205 (1:1000, Cat# 49561, Cell Signaling Technology), total tau (1:1000, Cat# 46687, Cell Signaling Technology), CDK5 (1:1000, Cat#05-364, Millipore), and DR (1:500 ¹¹ ). Blots were then incubated with HRP-conjugated species-specific secondary antibodies (Jackson ImmunoResearch Laboratories) and developed using an HRP-chemiluminescence substrate and imaged using the FluorChem 8800 system (Alpha Innotech). β-actin or Vinculin (1:5000) was used as a loading control, and densitometric analysis was performed using FluorChem software.

Quantitative reverse transcription PCR

Total RNA was extracted from SH-SY5Y, MC-65, and mouse cortex samples using the RNeasy Mini Kit (Cat# 74104, Qiagen), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized using the Omniscript Reverse Transcription Kit (Cat# 205111, Qiagen) with Oligo-dT primers. qPCR was performed using SYBR Green PCR Master Mix (Cat# 4309155, Applied Biosystems, Thermo Fisher Scientific) on a StepOne Real-Time PCR System (Applied Biosystems). GAPDH was used as the reference gene, and relative expression was calculated using the 2^ΔΔCt method (primers listed in Supplemental Table 1). Each sample was run in triplicate, and data were collected from three independent biological replicates.

Quantification of intracellular cAMP levels

Intracellular cAMP levels were quantified using the Cyclic AMP XP^® Assay Kit (Cat# 4339; Cell Signaling Technology), following the manufacturer's protocol. Briefly, SH-SY5Y and MC-65 cells were plated in 12-well plates and treated with Aβ and DR agonists as described in the treatment section. Post-treatment, cells were lysed, and the total protein content of each lysate was determined using the Bradford assay. Absorbance was measured at 450 nm using a standard microplate reader, and cAMP concentrations were determined from a standard curve generated with known cAMP concentrations. All experiments were performed in triplicate across three biologically independent experiments.

Statistical analysis

All experiments were performed in triplicate, and data are presented as mean ± SD. Statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). For comparisons between two groups, an unpaired t-test was used. One-way or two-way ANOVA was applied as appropriate for multi-group comparisons, followed by Tukey's multiple comparisons test. Statistical significance was interpreted using following thresholds: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). In cell-based assays, ‘#’ indicates significant differences between control and Aβ-treated groups; ‘*’ denotes significant differences between Aβ and receptor agonist-treated groups. For the mouse brain data, ‘*’ represents significant differences between WT and TG animals. A p-value < 0.05 was considered statistically significant.

Results

Brain region-specific Aβ deposition and reactive astrogliosis in the cortex and hippocampus of 5XFAD mice

Given that Aβ accumulation and NFTs are major causes of neurodegeneration and cognitive decline in AD, we first validated Aβ deposition and GFAP-positive astrocytes in brain regions linked to AD, including the cortex and hippocampus, using the 5XFAD mouse model, comparing it to age-matched WT mice. No Aβ immunoreactivity was detected in the cortex or hippocampus of WT animals (Figure 1 I, panels a-e). In contrast, immunohistochemical distribution revealed widespread deposition of Aβ plaques in 5XFAD mice, particularly in the deep cortical layers and cingulate cortex (Figure 1 I, panels f-h). Within the hippocampus, Aβ aggregates were enriched in the stratum oriens (SO) of CA1, adjacent to the corpus callosum and stratum lacunosum-moleculare (SLM). We observed enhanced Aβ deposition in the molecular layer (ML) and hilus of the dentate gyrus (DG), regions critical for synaptic integration and memory (Figure 1 I, panels i-j). Western blot analysis of cortical and hippocampal lysates confirmed the presence of Aβ (∼10 kDa) in 5XFAD brains, with no detectable expression in WT brain tissue. Densitometric analysis showed a significant elevation in Aβ plaques in these brain regions of 5XFAD brain, corroborating histological evidence of plaque burden (Figure 1 III).

Figure 1.

Photomicrographs of cortex and hippocampus showing amyloid deposits and astrocytes in WT and 5XFAD TG mouse brain. (I) In comparison to WT (I panels a-e), the 5XFAD TG mouse brain (I panels f-j) shows increased accumulation of Aβ in the cortex and hippocampus. Note the enhanced expression of Aβ in the deep layers of the cingulate cortex (CG; I, panels f, g, and h). In the hippocampus, increased Aβ deposition is observed in the stratum oriens (SO) and hilus of the dentate gyrus (DG) (I, f). High-magnification images (I, panels h and j) show extracellular Aβ plaques. (II) Photomicrographs of GFAP-positive astrocytes in the cortex and hippocampus of WT (II, panels a-e) and TG (II, panels f-j) mice. In the TG brain, increased GFAP immunoreactivity and astrocyte hypertrophy are observed in CA2 and the hilus of DG (panels f, i, and j). (III and IV) Immunoblot analysis shows increased expression of Aβ and GFAP in cortical and hippocampal lysates from TG mice compared to WT. β-actin and vinculin were used as loading controls. (V) Quantification of GFAP-positive astrocytes in WT and TG brains. Scale bars: (a, f) = 100 μm; (b, d, g, i) = 20 μm. Arrows indicate extracellular Aβ plaques or GFAP-positive astrocytes. Data are presented as mean ± SD (n = 6); *p < 0.05 versus WT.

Given the functional changes in astrocytes and the established association between Aβ pathology, inflammation, and tau hyperphosphorylation, we next examined astrocytic changes using GFAP immunohistochemistry and western blot analysis. While GFAP-positive astrocytes were present in both genotypes, we observed distinct morphological changes and spatial distribution in 5XFAD mice. In WT brains, GFAP-positive astrocytes exhibited thin, evenly distributed processes, indicating a homeostatic phenotype (Figure 1 II, panels a-e). In contrast, 5XFAD brains exhibited hypertrophic astrocytes, particularly in Aβ-rich areas including the deep cortical layers, SO, SLM, and ML (Figure 1 II, panels f-j). These observations uncovered a region-specific spatial overlap between amyloid pathology and astrocytic activation. Western blot analysis confirmed GFAP upregulation at ∼50 kDa in 5XFAD brains, consistent with a reactive astrocytic immunoreactivity (Figure 1 IV). To determine whether this increase reflected astrocyte proliferation, we next quantified GFAP-positive cells in the cortex and hippocampus of 5XFAD and WT mice. As shown in Figure 1 (V), the quantitative analysis of astrocytes revealed a 230% increase in cortex and 80% increase in hippocampus of 5XFAD mice. Together, our results are consistent with the hypothesis that an increased number of astrocytes is the potential source of inflammation in AD.

Gs-coupled dopamine receptor subtypes are upregulated in 5XFAD mice

To determine whether Aβ deposition and increased numbers of GFAP-positive astrocytes in AD mice are associated with changes in subcellular distribution and expression of DR subtypes, we examined DR1 and DR5 expression in the cortex and hippocampus of WT and 5XFAD mice using immunohistochemistry, western blotting, and qPCR.

Enhanced cortical DR1 expression in 5XFAD mice

DR1-like immunoreactivity was widely distributed across cortical regions and displayed significant upregulation in 5XFAD mice compared to WT mice (Figure 2, I and II; panels a-c). Compared to WT, immunohistochemical analysis revealed a significant upregulation of DR1-positive neurons and enhanced DR1-staining in the cortex of 5XFAD mice. In WT brains, DR1 expression was primarily localized to cortical layers II-V, with strong labeling in pyramidal neurons (arrows). In contrast, 5XFAD mice exhibited elevated DR1 staining across all cortical layers. High-magnification images further confirmed increased DR1-like immunoreactivity in both the soma and proximal dendrites of cortical pyramidal neurons in 5XFAD mice (arrows, Figure 2, II panel c).

Figure 2.

DR1 expression and neuronal density in the cortex and hippocampus of WT and 5XFAD TG mice. As compared to WT (I, panels a-l), DR1 expression is increased in the cortical layers (II, panels b, c) and different hippocampal regions [CA1 (e, f), CA2 (g, h), CA3 (i, j), and DG (k, l)] of 5XFAD TG mice (II, panels a-l). Note enhanced DR1 staining in the DG of the TG brain (II, panel k and l). (III) Immunoblot analysis shows significant upregulation of DR1 in cortical lysates from TG mice, with no significant change in the hippocampus. Vinculin was used as a loading control. (IV) Cortical tissue from TG mice shows elevated DR1 mRNA levels. (V) Quantification of DR1 immunoreactivity and number of DR1-positive neurons (VI) in the cortex and hippocampus of TG compared to WT mice. Scale bars: (a, d) = 100 μm; (b, e, g, j, k) = 20 μm. Data are presented as mean ± SD (n = 6); *p < 0.05 versus WT. Arrows indicate DR1-positive neurons; arrowheads indicate DR1 immunoreactivity in neuronal processes.

Changes in DR1 distribution in hippocampus of 5XFAD mice

DR1 immunostaining was observed throughout the hippocampus in both genotypes, but 5XFAD mice showed a region-specific increase in DR1-like immunoreactivity relative to WT (Figure 2, panels Id and IId). In WT mice, moderate DR1 staining was observed in pyramidal neurons of CA1 and CA3, with mild expression in CA2 and weak staining in the hilus of the DG (arrows, Figure 2, I panel e-l). In 5XFAD mice, DR1 expression was enhanced in CA1 and CA3 neurons, accompanied by aberrant extension of DR1-positive dendritic processes into the SR (arrowhead, Figure 2, II panels f and j). DR1 expression in the SO was comparable between genotypes. In the DG, 5XFAD mice exhibited significantly stronger DR1 immunoreactivity in the granule cell layer (GL) and hilus (arrows), while the ML remained moderately expressed (Figure 2, II panels k and l).

DR1 protein and mRNA expression are elevated in 5XFAD brain

To corroborate the changes observed via immunohistochemistry, we assessed DR1 expression by western blot in cortical and hippocampal lysates. As shown in Figure 2 III, DR1 protein levels were significantly elevated in the cortex of 5XFAD mice compared to WT mice. In the hippocampus, DR1 levels were upregulated, albeit not significantly. Consistent with protein data, qPCR analysis revealed a significant increase in DR1 mRNA levels in 5XFAD mice relative to WT mice, suggesting transcriptional upregulation of DR1 expression in response to AD-related pathology (Figure 2 IV).

Quantification of staining intensity and neuronal density in ad-relevant regions

Next, we assessed the staining intensity and changes in the neuronal cell population exhibiting DR1-like immunoreactivity in WT and TG mice. As shown in Figure 2 V, DR1 intensity per neuron was significantly increased in the cortex and the CA1, CA3, and DG regions of 5XFAD mice, while CA2 remained unchanged. Additionally, the total number of DR1-positive neurons was significantly higher in both the cortex and hippocampus of 5XFAD mice compared to WT (Figure 2 VI). This increase in DR1 immunoreactivity intensity suggests a functional reorganization of dopaminergic signaling in AD pathology, potentially contributing to circuit dysfunction.

Cortical DR5 immunoreactivity is elevated in 5XFAD mice

As shown in Figure 3 I (panels a-c), DR5-like immunoreactivity in the WT cortex was sparse and weakly distributed across all cortical layers, with no laminar distinction. High-magnification images (Figure 3 I panels b-c) revealed weak DR5 staining, primarily localized to the neuronal soma (arrow). In 5XFAD mice, although DR5 distribution remained sparsely distributed in the cortical brain region, the intensity of DR5 immunoreactivity was markedly enhanced, particularly at the cell membrane (arrow, Figure 3 II panels b-c).

Figure 3.

DR5 expression is upregulated in the cortex and hippocampus of 5XFAD TG mice. (I and II) Representative images show DR5 immunoreactivity in the cortex and hippocampus of WT (I, panels a-l) and TG (II, panels a-l) brains. As compared to WT, 5XFAD mice observed enhanced DR5 immunostaining in both the cortex (panels b, c) and hippocampus subfields [CA1 (e, f), CA3 (i, j), and DG (k, l)]. (III) Western blot analysis shows increased DR5 protein expression in the hippocampus, with no significant change in the cortex. β-actin was used as a loading control. (IV) q-PCR reveals significant upregulation of DR5 transcript levels in TG cortex. (V) Quantification of DR5 immunoreactivity indicates increased intensity in the cortex and all hippocampal subregions, except CA2. (VI) Box plots show an increased number of DR5-positive neurons per mm² in both the cortex and the hippocampus of TG mice. Scale bars: (a, d) = 100 μm; (b, e, g, j, k) = 20 μm. Data are presented as mean ± SD (n = 6); *p < 0.05 versus WT. Arrows indicate DR5-positive cell bodies; arrowheads indicate DR5 labeling in neuronal processes.

Immunohistochemical localization of DR5 in the hippocampus of 5XFAD mice

In the hippocampus of WT mice, DR5 immunoreactivity was low, with limited expression in pyramidal neurons of CA1 and CA3 and minimal labeling in DG (Figure 3 I panels d-l). In 5XFAD mice (Figure 3 II panels d-l), CA1 neurons showed enhanced DR5 immunoreactivity in SP (arrow), with receptor distribution extending into dendrite-like processes in SR (arrowhead). While DR5 expression in CA2 was unchanged, CA3 displayed a pronounced upregulation in receptor expression. Additionally, the DG exhibited enhanced DR5-positive GL cells, particularly within the hilus (arrow).

DR5 protein and transcript levels are upregulated in 5XFAD brain

Having observed a distinct spatial pattern of DR5 immunoreactivity, we next performed western blot and qPCR analyses to quantify DR5 protein and mRNA expression. Western blot analysis (Figure 3 III) showed significant upregulation of DR5 protein in hippocampal lysates from TG mice, with no significant difference observed in cortical tissue. In contrast, qPCR analysis (Figure 3 IV) demonstrated significantly elevated DR5 mRNA in the cortex of 5XFAD mice compared to WT mice, suggesting transcriptional upregulation. These findings support the concept that DR5 regulation in 5XFAD mice involves both transcriptional and post-translational mechanisms that alter receptor localization and may contribute to aberrant dopaminergic signaling in AD.

Quantification of DR5-positive neurons confirms region-specific upregulation in 5XFAD mice

Quantitative analysis of DR5 immunoreactivity intensity revealed a significant increase in cortex, CA1, CA3, and DG of 5XFAD mice, with no change in CA2 (Figure 3 V). Additionally, the total number of DR5-positive neurons was higher in both the cortex and hippocampus of 5XFAD mice compared to WT controls (Figure 3 VI).

Taken together, this atypical redistribution of DR1-like receptors may reflect compensatory alterations in receptor signaling, possibly driven by increased excitatory drive, impaired inhibitory balance, or maladaptive synaptic plasticity.

Region-specific loss of Gi-coupled dopamine receptors in 5XFAD mice

Having observed a significant upregulation of DR1 and DR5 in 5XFAD mice, we next examined whether the impact on Gi-coupled receptors (DR2, 3, and 4) was comparable or distinct from that of Gs-coupled DRs. These receptors are critical regulators of synaptic plasticity and inhibitory neurotransmission in the AD brain. Immunohistochemistry, western immunoblot, and qPCR analyses revealed receptor subtype-specific downregulation with regional variations between the cortex and hippocampus in 5XFAD mice relative to WT.

DR2 immunoreactivity in the cortex of 5XFAD mice shows reduced perisomatic localization and neuronal loss

In the cortex, DR2-positive neurons were widely distributed in cortical layers II to V in WT mice (Figure 4, panel Ia). DR2 immunoreactivity was predominantly localized to the cytoplasm, with distinct membrane-associated staining around the neuronal soma and extending into processes (arrowhead), indicating receptor distribution in perisomatic and neuritic compartments (Figure 4, panels Ib, c; arrows). In contrast, DR2 immunoreactivity in 5XFAD mice appeared more diffuse and weaker (arrows, Figure 4, panel IIa-c). Neuronal cells appeared smaller, possibly reflecting apoptotic stress or synaptic dysfunction. DR2-expressing neurons were also less densely distributed, particularly in layers II and V, suggesting disrupted dopaminergic network integrity (Figure 4, panel IIa).

Figure 4.

DR2 expression is selectively diminished in cortex but preserved in hippocampus of 5XFAD TG mice. (I and II) Representative images show DR2 immunoreactivity in the cortex and hippocampus of WT mice (I, panels a-l) and TG (II, panels a-l). In WT mice, DR2 is detected at the cell surface in deep cortical layers (b, c), pyramidal neurons of CA1 (e, f), CA2 (g, h), CA3 (i, j), and DG interneurons (k, l). In TG brain, DR2 immunostaining is reduced as compared to WT. (III) Immunoblot analysis shows decreased DR2 protein levels in the cortex of TG mice, with no significant change in the hippocampus. Vinculin was used as the loading control. (IV) q-PCR reveals significantly lower DR2 mRNA expression in TG cortex. (V) Quantification of DR2-positive neuronal intensity confirms a reduction in cortex and CA3 of TG mice (VI) Box plots show reduced density of DR2-positive neurons in the cortex but not the hippocampus of TG mice. Scale bars: (a, d) = 100 μm; (b, e, g, j, k) = 20 μm. Data are presented as mean ± SD (n = 6); *p < 0.05 versus WT. Arrows indicate DR2-positive neurons; arrowheads present neuronal processes.

Changes in subcellular expression of DR2 in the hippocampus of 5XFAD mice

To assess the regional variation of DR2 expression in the hippocampus, we examined its subcellular distribution. In WT animals, strong DR2 expressions were observed in neuronal processes and dendrites (arrowhead) within the SP of CA1 and CA2, whereas CA3 exhibited minimal DR2 staining (arrow, Figure 4 I panel e-j). In 5XFAD mice, DR2-positive interneurons were absent from the SO, where they expressed moderate staining in WT. DR2 immunoreactivity in the GL of the DG remained comparable between groups. However, there was a significant reduction of DR2-positive neurons in the hilus of the DG in 5XFAD mice when compared to WT (Figure 4 II panels k and l, arrow). These findings suggest that DG granule neurons are relatively resistant to DR2 dysregulation, whereas CA3 pyramidal neurons and the hilus of DG exhibit vulnerability.

Western blot and qPCR confirm cortical DR2 downregulation at protein and transcript levels

To determine whether subcellular changes in DR2 localization corresponded to the receptor expression and mRNA, we performed western blot and qPCR analyses. Immunoblotting revealed a significant decrease in expression of DR2 at the protein levels in cortical lysates of 5XFAD mice compared to WT controls (Figure 4 III). In contrast, hippocampal DR2 protein levels remained unchanged. Cortical downregulation of DR2 was corroborated at the transcriptional level, showing a corresponding decrease in DR2 mRNA in 5XFAD cortex (Figure 4 IV). These results suggest that DR2 loss in 5XFAD mice may result from transcriptional dysregulation and impaired receptor stability within cortical circuits.

Quantification confirms reduced DR2 signal and neuron number in cortex and hippocampus

ImageJ quantification showed a significant reduction in DR2 staining intensity in the cortex of 5XFAD mice (Figure 4 V) and a moderate decrease in CA3, with no significant changes in CA1, CA2, or DG. The number of DR2-positive neurons per mm² was also significantly lower in the cortex of 5XFAD mice compared to WT (Figure 4 VI), while hippocampal neuron counts remained unchanged. These findings support the conclusion that DR2 is selectively downregulated in the cortex and CA3 region of the hippocampus, highlighting the regional vulnerability of Gi-coupled DAergic signaling in AD pathology.

DR3 expression is significantly diminished in the cortex of 5XFAD mice

Brain sections from WT and 5XFAD mice revealed selective and region-specific alterations in DR3-like immunoreactivity in the cortical and hippocampal regions. In WT cortex, DR3 expression was prominent in layers II-V, with punctate, membrane-associated staining patterns observed in both pyramidal and non-pyramidal neurons (arrow, Figure 5 I panel a-c). In contrast, 5XFAD mice showed a marked loss of DR3 immunostaining across the cortex, with receptor signal diffusely distributed into the cytoplasm and reduced membrane localization (Figure 5 II panels a-c, arrow). The absence of membrane-associated DR3 signal indicates receptor internalization into the cytoplasm, likely resulting from neuronal degeneration.

Figure 5.

DR3 expression is decreased in the cortex and hippocampus of 5XFAD TG mice compared to WT. (I and II) DR3-like immunoreactivity is distributed in different cortical layers and hippocampal subregions in WT (I, panels a-l) and TG mice (II, panels a-l). Strong DR3-positive staining is observed in the deep cortical layers (b, c), pyramidal neurons of CA1 (e, f), CA2 (g, h), CA3 (i, j), and DG interneurons (k, l), which is significantly reduced in TG mice. (III) Immunoblot analysis shows decreased DR3 protein expression in cortex and hippocampus of TG compared to WT. β-actin was used as the loading control. (IV) q-PCR confirms downregulation of DR3 transcript in cortical tissue. (V) Quantification of DR3 immunoreactivity reveals decreased staining intensity in cortex and all hippocampal subfields in TG mice. (VI) Box plots show reduced density of DR3-positive neurons in both cortex and hippocampus. Scale bars: (a, d) = 100 μm; (b, e, g, j, k) = 20 μm. Data are presented as mean ± SD (n = 6); *p < 0.05 versus WT. Arrows indicate DR3-positive neurons; arrowheads indicate DR3 immunoreactivity in neuronal processes.

5XFAD mice exhibited widespread downregulation of DR3 expression in the hippocampus

In the hippocampus of WT mice, DR3 immunoreactivity was evident in pyramidal neurons within the SP as well as in GL of the DG (Figure 5 I panels d-l). The receptor was localized to both somatic and dendritic compartments; high magnification images showing DR3-like immunoreactivity extending into apical processes (arrowhead) within the SR (Figure 5 I panel f, h, j). Sparsely distributed DR3-positive interneurons were also visible in the SO. In 5XFAD mice, DR3 expression was markedly reduced in all hippocampal regions (arrow, Figure 5 II panels d-l). Moderate DR3 staining was observed in neuronal processes from a limited neuronal cell displaying receptor immunoreactivity at the cell surface in the SP of the CA1 region (arrow, Figure 5 II panel f). This uniform suppression of DR3 across the hippocampus suggests a broad dopaminergic deficit impacting multiple subregions.

Western blot and qPCR reveal transcriptional and translational loss of DR3 in 5XFAD brain

To validate the immunohistochemical observations, western blot and qPCR were performed to assess DR3 in cortical and hippocampal tissue. DR3 protein level revealed a significant reduction in 5XFAD mice compared to WT controls (Figure 5 III). Consistently, qPCR analysis showed a significant decrease in DR3 mRNA levels in the cortex (Figure 5 IV), indicating that transcriptional repression contributes to the observed protein loss. Together, these findings highlight that DR3 downregulation may play a critical role in the synaptic and cognitive deficits associated with AD pathology.

Quantitative image analysis confirms DR3 neuronal loss in cortex and hippocampus

Quantification of DR3 immunoreactivity using ImageJ revealed a significant reduction in both staining intensity and DR3-positive neuron count in the cortex and hippocampus of 5XFAD mice. Scatter plot analysis showed a substantial decrease in DR3 signal intensity across all hippocampal fields and cortex (Figure 5 V). Furthermore, DR3-positive neuron density was reduced by nearly 50% in both cortex and hippocampus of 5XFAD mice relative to WT (Figure 5 VI), underscoring widespread DR3 suppression. These results indicate that DR3 is one of the most uniformly downregulated DR subtypes in 5XFAD mice, with widespread loss across both cortical and hippocampal regions. The spatial deficit implicates DR3 downregulation as a major contributor to the DAergic dysfunction underlying cognitive decline in AD.

Immunohistochemical distribution of DR4 in the cortex is downregulated in 5XFAD Mice

In WT mice, peroxidase labeling revealed that DR4-positive neurons were widely distributed across cortical layers, with the highest expression in layer IV, followed by layers V and VI (Figure 6 I panels a-c). Layers II and III displayed a discrete population of DR4-positive neurons, while layer I was devoid of staining. DR4-like immunoreactivity was uniform, confined to neuronal soma, with a strong signal at the apical end of pyramidal neurons (Figure 6 I panel c, arrows). On the other hand, in the 5XFAD brain, DR4-positive neurons displayed a comparable distribution pattern to WT mice, but with less immunoreactivity (Figure 6 II panels a-c, arrows). Layers I and deep layer IV exhibited sparse DR4-positive neurons.

Figure 6.

Differential regulation of DR4 in cortex and hippocampus of 5XFAD TG mice. (I and II) Representative images show DR4 immunoreactivity in the cortex and hippocampus of WT (I, panels a-l) and TG (II, panels a-l) brains. DR4-like staining is observed in cortical layers (panels b, c) and different hippocampal regions, CA1 (e, f), CA2 (g, h), CA3 (i, j), and dentate gyrus (k, l). In TG brain, DR4 immunoreactivity was downregulated in cortex, with differential expression in hippocampal subfield. (III) Western blot analysis shows reduced DR4 protein expression in the hippocampus of TG mice, with no change in the cortex. β-actin was used as the housekeeping control. (IV) q-PCR analysis shows no significant difference in DR4 transcript levels between WT and TG groups. (V) Quantification of DR4-positive immunoreactivity reveals reduced DR4 immunostaining in the cortex and CA1, CA2 subfields. (VI) Box plots show reduced density of DR4-positive neurons in cortex but not in hippocampus. Scale bars: (a, d) = 100 μm; (b, e, g, j, k) = 20 μm. Data are presented as mean ± SD (n = 6); *p < 0.05 versus WT. Arrows mark DR4-immunoreactive neurons; arrowheads highlight labeling in processes.

DR4 expression in the hippocampus exhibits region-specific alterations in 5XFAD mice

DR4 immunoreactivity was widely expressed in different hippocampus regions, including CA1-CA3 and DG of WT mice (Figure 6 I panels d-l). DR4-like immunoreactivity was mainly expressed in SP and a few neuronal cell bodies in SO and SR (arrow, Figure 6 I panels e-h). The neuronal processes ending in SR also exhibit mild DR4-like immunoreactivity (arrowhead). DR4-positive neurons were detected in both the hilus and GL of DG (Figure 6 I panels k and l). In contrast, 5XFAD mice exhibited a comparable distribution pattern; however, receptor immunoreactivity was reduced in DR4-positive neurons located in CA1 and CA2, particularly within SP and SR (arrow, Figure 6 II panels e-h). Conversely, DR4 staining was upregulated in CA3 and DG regions of 5XFAD mice (arrow, Figure 6 II panels i-l), indicating region-dependent differences in DR4 immunoreactivity within the hippocampus.

Western blot and qPCR analysis of DR4 expression in 5XFAD cortex and hippocampus

Western blot analysis revealed no significant changes in DR4 protein levels in the cortex between 5XFAD and WT mice (Figure 6 III). However, a moderate reduction in DR4 expression was detected in the hippocampus of 5XFAD mice. qPCR analysis of DR4 mRNA expression showed no significant difference in the cortex (Figure 6 IV), indicating stable transcriptional regulation in this region.

Quantification of DR4-positive neurons reveals opposing patterns in cortex and hippocampal subfields

Quantitative analysis of DR4-positive neurons and immunoreactivity intensity was calculated using ImageJ. Immunostaining intensity analysis revealed a significant reduction in DR4 signal in the cortex and in CA1 and CA2 regions, in contrast to elevated DR4 immunoreactivity in CA3 and DG of 5XFAD brains (Figure 6 V). The number of DR4-positive neurons per mm² was significantly reduced in the cortex of TG compared to WT (Figure 6 VI), whereas hippocampal DR4-positive neuron counts remained unchanged. These results indicate that DR4 expression levels appear modestly altered; the receptor undergoes pronounced changes in subcellular distribution in AD pathology, particularly within the hippocampus.

Together, these results suggest that astrocyte activation and increased Aβ deposition with receptor-specific changes in the 5XFAD brain are critical in impaired cognitive function and memory loss in AD.

Increased tau phosphorylation and CDK5 expression in the cortex of 5XFAD mice

Previous studies have implicated tau phosphorylation and CDK5 in the pathogenesis of neurodegenerative disorders, particularly through the cleavage of its activator p35 to p25, which leads to cytoskeletal disruption and tau hyperphosphorylation.^32,33 We next examined total tau expression and isoform-specific phosphorylation in the cortex of 5XFAD mice. Phosphorylation of tau at Thr205 and Ser404 in the cortex of 5XFAD mice was significantly increased (Figure 7A, B). In contrast, total tau (t-tau) levels were reduced in the 5XFAD cortex compared to WT (Figure 7C). To determine whether CDK5 expression is under the influence of Aβ burden and tau phosphorylation in AD brain, we examined CDK5 levels in the cortex of 5XFAD mice. As shown in Figure 7D, CDK5 expression was significantly increased in the cortical tissue lysate of the 5XFAD when compared to WT controls.

Figure 7.

Upregulation of tau phosphorylation and CDK5 expression in the cortex of 5XFAD TG mice. (A and B) Western blot analysis of cortical lysates from WT and 5XFAD mice showing a significant increase in phosphorylated tau at Thr205 and Ser404, normalized to Vinculin. (C) Total tau levels are reduced in TG cortex compared to WT. (D) CDK5 expression is significantly upregulated in TG cortex. Bar graphs represent densitometric quantification (mean ± SD, n = 4), *p < 0.05 versus WT.

Changes in dopamine receptor expression in SH-SY5Y and MC-65 cells

Having observed distinct region-specific alterations in DR subtype distribution in the cortex and hippocampus of 5XFAD mice, we examined whether the presence of Aβ, either exogenously or endogenously, modulates DR expression in neuronal cell models. To investigate this, we used differentiated SH-SY5Y cells treated with exogenous Aβ₁₋₄₂ (1 µM, 24 h) and MC-65 cells, in which intracellular Aβ accumulation was induced by tetracycline withdrawal (24 h). These two Aβ models recapitulate extracellular and intracellular Aβ pathologies, respectively. DR1-DR5 expression and subcellular distribution were assessed using immunocytochemistry, western blotting, and qPCR. Due to technical limitations, immunocytochemistry was not performed in MC-65 cells.

Aβ differentially regulates Gs-coupled dopamine receptors 1 and 5 in SH-SY5Y and MC-65 cells

Differentiated SH-SY5Y cells were treated with Aβ₁-₄₂ and evaluated for DR1 and DR5 expression at the protein and RNA levels. In control differentiated cells, DR1 immunoreactivity was observed at the plasma membrane and along proximal and apical neurites (Figure 8A, arrows). Following Aβ treatment, DR1-like immunoreactivity was increased at the cell surface (Figure 8A, arrowheads). This enhanced membrane localization was accompanied by elevated DR1 protein levels in cell lysates, as confirmed by western blotting in SH-SY5Y cells upon treatment with Aβ (Figure 8B). In MC-65 cells, DR1 expression also increased upon tetracycline withdrawal (Aβ induction), albeit to a lesser extent compared to SH-SY5Y cells (Figure 8C), indicating a model-dependent variability in DR1 response.

Figure 8.

Differential regulation of DR1 and DR5 in response to Aβ exposure in SH-SY5Y and MC-65 cells. (A) Immunofluorescence images show DR1 (upper panel) and DR5 (lower panel) localization in differentiated SH-SY5Y cells treated with RA alone or RA + Aβ. In RA + Aβ-treated cells, both DR1 and DR5 display enhanced surface expression. Note: loss of neurites with Aβ treatment and upregulation of receptors. Supplemental Figure 1A, B shows fluorescence intensity quantification. (B-E) Western blot analysis reveals increased DR1 expression in SH-SY5Y (B) and MC-65 (C) cells following Aβ exposure, with no significant change in DR5 levels (D, E). β-actin was used as the loading control. Bar graphs show densitometric analysis of DR1 and DR5. (F, G) qPCR analysis shows increased DR1 mRNA levels in SH-SY5Y and MC-65 cells, while DR5 transcript levels remain unchanged. Arrows indicate receptor localization in neurites, while arrowheads denote surface expression at the soma. Scale bar: 20 μm. Data are presented as mean ± SD (n = 3); *p < 0.05 versus Aβ.

In comparison to DR1, DR5 showed increased surface localization in Aβ-treated SH-SY5Y cells (Figure 8A, arrow and arrowhead), but total DR5 protein levels in lysates remained unchanged in both SH-SY5Y and MC-65 cells (Figure 8D, E). To validate these findings at the transcription level, qPCR analysis was performed for DR1 and DR5. In SH-SY5Y cells, DR1 mRNA was significantly upregulated following Aβ treatment (Figure 8F), consistent with increased protein expression. MC-65 cells also showed an elevated DR1 mRNA (Figure 8G), recapitulating the protein-level changes. In contrast, DR5 mRNA levels remained unchanged in both SH-SY5Y and MC-65 cells (Figure 8F, G), corroborating the western blot results. Supplemental Figure 1A and 1B show quantitative analysis of fluorescence staining in control and Aβ-treated cells.

Aβ induces differential downregulation of Gi-coupled dopamine receptors in SH-SY5Y and MC-65 cells

We have previously demonstrated that DRs regulate neuritogenesis and interact with MAPs in a receptor subtype-specific manner. ²⁹ Moreover, studies also support that Gs-coupled receptors respond differently to neurotoxic stimuli compared to Gi-coupled receptors. Given the regulatory role of Gi-coupled DRs in synaptic signaling and the previously observed Aβ-induced upregulation of DR1 and DR5, we therefore examined the expression of DR2, DR3, and DR4 in SH-SY5Y and MC-65 cells following exposure to Aβ.

In SH-SY5Y cells, DR2 immunoreactivity was localized to neurites with a distinct puncta pattern of staining at the plasma membrane (Figure 9A, arrows). Upon Aβ treatment, DR2 immunoreactivity was reduced at the cell surface (Figure 9A, arrowhead). However, western blot analysis revealed no significant difference in DR2 protein levels between control and Aβ-treated SH-SY5Y or MC-65 cells (Figure 9B, C). In comparison to control, DR3-like immunoreactivity was increased at the cell surface in response to Aβ-treated SH-SY5Y cells (Figure 9A, arrows). This was consistent with a significant increase in DR3 expression in SH-SY5Y cell lysates (Figure 9D). However, no significant changes in DR3 expression were observed in MC-65 cells (Figure 9E). Immunofluorescence localization showed loss of DR4 immunostaining in Aβ-treated SH-SY5Y cells when compared to control. Reduced expression of DR4 was also observed in tissue lysate prepared from Aβ-treated SH-SY5Y and MC-65 cells by immunoblot analysis (Figure 9F, G). Supplemental Figure 2A-C shows quantitative analysis of fluorescence staining in control and Aβ-treated cells.

Figure 9.

Aβ-induced changes in subcellular distribution and expression of DR2, DR3, and DR4 in SH-SY5Y and MC-65 cells. (A) Representative photomicrographs showing subcellular distribution of DR2 (upper panel), DR3 (middle panel), and DR4 (lower panel) in control and Aβ-treated SHSY5Y cells. DR2 and DR4 immunoreactivity were downregulated, whereas DR3 showed increased membrane localization upon Aβ treatment. Arrows indicate receptor localization in neurites, while arrowheads denote surface expression at the soma. Supplemental Figure 2A-C shows quantification of fluorescence staining intensity. (B, C) Western blot analysis reveals no change in DR2 levels. (D, E) DR3 is upregulated in SH-SY5Y cells but unchanged in MC-65 cells. (F, G) DR4 protein expression is significantly decreased in both SH-SY5Y and MC-65 cells following Aβ exposure. β-actin was used as the loading control. (H, I) qPCR analysis shows no significant change in DR2, DR3 and DR4 mRNA levels in SH-SY5Y cells, but significant downregulation in MC-65 cells. Scale bar: 20 μm. Data are presented as mean ± SD (n = 3); *p < 0.05 versus Aβ.

Consistent with protein expression, the mRNA expression for DR2, 3, and 4 subtypes was not significantly changed in Aβ-treated SH-SY5Y cells (Figure 9H). In contrast, mRNA expression of DR subtypes was significantly decreased in MC-65 cells in response to Aβ-induced toxicity (Figure 9I). Taken together, the results described here suggest differential regulation of DR subtypes in response to endogenous and exogenous Aβ-induced toxicity.

Activation of dopamine receptors differentially regulates Aβ-induced increase in cAMP, tau hyperphosphorylation, and CDK5 activation in AD models

AD is characterized not only by Aβ accumulation and NFT formation, but also by hyperphosphorylation of tau, a microtubule-associated protein critical for axonal stability and synaptic plasticity. Previous studies have shown that Aβ can promote tau phosphorylation and NFT formation in dystrophic axons. ³⁴ Furthermore, coupling DR subtypes to Gs and Gi proteins and consequent distinct regulation of second messenger cAMP exerts a determinant role in regulating DR-mediated signaling pathways.

Aβ-induced cAMP elevation is modulated by DR1 and DR2 agonists in SH-SY5Y and MC-65 cells

To evaluate whether selective DR activation modulates Aβ-induced toxicity, we first assessed intracellular cAMP levels in SH-SY5Y and MC-65 cells treated with Aβ alone or in combination with subtype-specific DR agonists. Consistent with previous findings, cAMP levels were significantly enhanced upon treatment with Aβ in both cell lines. In SH-SY5Y cells, Aβ treatment increased cAMP by 28.5% compared to control. When compared to Aβ treatment, the DR1 agonist further increased cAMP by 19.5%, whereas the DR2 agonist reduced it by 15% (Figure 10A). A similar but more pronounced inhibitory effect of the DR2 agonist was observed in MC-65 cells, where Aβ increased cAMP by 33% compared to control; DR1 agonist induced a further 7.3% increase, while DR2 agonist reduced cAMP levels by 37.8% (Figure 10B) when compared to Aβ alone, suggesting enhanced Gi-coupling efficiency under endogenously released Aβ-induced toxicity.

Figure 10.

Dopamine receptor subtype-specific agonists modulate Aβ-induced increased cAMP, Tau phosphorylation, and CDK5 expression. (A, B) Intracellular cAMP levels were significantly elevated following Aβ exposure in SH-SY5Y and MC-65 cells and were differentially modulated by DR subtype agonists. (C-E) In SH-SY5Y cells, Aβ treatment increases p-tau (T205) while total tau remains unchanged. DR3 agonist attenuated Aβ-induced elevation of tau phosphorylation. (F-H) In MC-65 cells, Aβ enhances p-tau (T205, S404), whereas total tau is downregulated. DR2 agonist significantly attenuates Aβ-induced tau dysregulation at both epitopes. (I and J) Quantification of CDK5 expression in SH-SY5Y (I) and MC-65 (J) cells shows a significant increase following Aβ treatment, which is attenuated by DR2 (MC-65 cells) and DR3 agonists (SH-SY5Y cells). Data are presented as mean ± SD (n = 3); # indicates significance versus control, * indicates significance versus Aβ.

DR2 and DR3 agonists exert cell-type specific modulation of Aβ-induced tau phosphorylation in SH-SY5Y and MC-65 cells

Having characterized DR subtype alterations in the 5XFAD mouse brain and in SH-SY5Y and MC-65 cells following Aβ exposure, we investigated receptor-specific modulation of cAMP signaling and its impact on tau pathology. In SH-SY5Y and MC-65 cells, Aβ exposure increased p-Tau levels at T205 compared to controls (Figure 10C, F), which was reduced by DR3 in SH-SY5Y cells and DR2 and DR3 agonists in MC-65 cells. In SH-SY5Y cells, levels of p-tau at S404 remained unchanged after DR agonist treatments as compared to the Aβ condition (Figure 10D), whereas in MC-65 cells, the DR2 agonist significantly reduced Aβ-induced p-tau (S404) (Figure 10G). T-tau was downregulated in MC-65 cells after Aβ exposure (Figure 10H) but remained stable in SH-SY5Y cells across all conditions (Figure 10E). These findings indicate that DR2 and DR3 agonists may protect against Aβ-induced tau pathology, particularly at phosphorylation-prone epitopes; however, effects were cell-and receptor-type specific.

Expression of CDK5 in Aβ-induced toxicity in SH-SY5Y and MC-65 cells

Since CDK5 is a key kinase implicated in pathological tau phosphorylation, we next examined CDK5 expression in Aβ-exposed SH-SY5Y and MC-65 cells, and its modulation by DR subtype agonists. In SH-SY5Y cells, CDK5 was elevated following Aβ treatment and further enhanced by DR1 and DR2 agonists, while DR3 agonist reversed this effect (Figure 10I). In contrast, MC-65 cells showed elevated CDK5 in response to Aβ, which was significantly suppressed by DR2 agonist treatment, whereas DR1 and DR3 agonists had no significant effect (Figure 10J).

Increased Aβ accumulation and GFAP-positive astrocytes in the 5XFAD mice brain are selectively correlated with DR subtypes

Previous studies have demonstrated functional and pathological correlations among Aβ, NFT formation, inflammation, and tau phosphorylation in AD brains. To investigate the potential link between DAergic neurotransmission changes and AD pathophysiology, we examined whether DR subtypes are expressed in close vicinity to Aβ. In comparison to WT mice, brain samples from 5XFAD mice showed high expression of Aβ deposition in the hippocampus and cortex. WT mice showed no Aβ deposition in any brain regions. Aβ and GFAP were predominantly detected in the cingulate cortex, as well as in the SO and DG regions of the hippocampus (Figure 1). Within these plaque-rich and GFAP-positive areas (Figure 1), DR1 and DR5 were significantly upregulated (Figures 2 and 3). In contrast, DR2 and DR3 were significantly downregulated in these regions, potentially disrupting inhibitory modulation and contributing to neuroinflammatory responses. Importantly, DR1 and DR5 were also upregulated in the ML, where GFAP was particularly elevated, suggesting that DAergic signaling alterations align with localized astrocytic activation in the hippocampus. Biochemical evidence from SH-SY5Y and MC-65 cells of Aβ-induced neurotoxicity further supports the role of DA in AD pathogenesis. As shown in Figure 10, DR subtypes, selective agonists blocked Aβ-induced tau phosphorylation and expression of CDK5.

Discussion

The present study investigates DAergic alterations in the cortex and hippocampus of 5XFAD mice, as well as in SH-SY5Y and MC-65 neuronal models after Aβ exposure, thereby advancing our understanding of DAergic dysregulation in AD pathophysiology. Although changes in DAergic transmission during AD progression are well documented, it remains unclear whether these changes arise directly from Aβ deposition, tau phosphorylation, and NFT formation, and whether they ultimately contribute to cognitive impairment. Moreover, the precise mechanisms linking DAergic neurotransmission to AD pathology are not fully understood.

Neurotransmitter changes in neurodegenerative diseases are widely studied. Several studies have linked DAergic dysfunction with cognitive impairment and memory loss in AD.^13,35,36 However, a systematic evaluation of DR subtypes in different yet complementary AD models remains unexplored. Here, we identify a close association between DRs, Aβ deposition, and GFAP-positive astrocyte activation in 5XFAD mice. Specifically, we observed upregulation of Gs-coupled receptors (DR1, DR5) and downregulation of Gi-coupled receptors (DR2–DR4) as neurochemical markers associated with cognitive deficits and memory impairment in AD. To investigate Aβ accumulation and related neurotoxicity, we used two distinct in vitro models: differentiated SH-SY5Y cells exposed to exogenous Aβ, and MC-65 cells with endogenous Aβ accumulation. Given the established link between AD histopathological markers and CDK5, we also examined tau phosphorylation and CDK5 activation in these models. Our study provides the first comprehensive characterization of DR subtype-specific subcellular distributions in both 5XFAD mice and in vitro cell models. This multi-model approach revealed consistent patterns of receptor dysregulation across models as well as AD model-specific variations, highlighting differential susceptibility to Aβ pathology and associated DAergic disruptions. Region-specific analyses further revealed that the cortex is highly susceptible to Aβ deposit-mediated neurodegeneration. In contrast, the hippocampus is relatively more vulnerable to inflammation and hyperphosphorylated tau in AD, driven by increased GFAP-positive astrocytes.

Previous work has shown reduced DR expression and impaired DA signaling due to neuronal loss in the VTA. ¹³ Our findings extend these observations by detailing subcellular DR subtype alterations in the cortex and hippocampus. Consistent with previous studies indicating that DR1 and DR2 contribute to learning and memory, while DR3, DR4, and DR5 are more broadly implicated in cognition,^37,38 our data highlight reduced DR2 expression as a critical factor in hippocampal dysfunction. In agreement with,^7,19,21 our results underscore the role of DR2 in AD pathogenesis. In contrast, DR1 upregulation in AD may represent a compensatory response to DR2 loss, revealing a previously underexplored role for DR1 in DAergic remodeling in Aβ pathology. In human autopsy brain tissue, DR1-DR4 expression is decreased, except for DR5, which is elevated. ¹¹ Similarly, the loss of DR3 and DR4 in TG mice aligns with studies suggesting their contribution to cognitive function.^39,40 Interestingly, we observed increased DR5 expression in the hippocampus, potentially linked to AD-related cognitive impairment.

In 5XFAD mice, DR1 upregulation, unlike its reduction in the human AD cortex, ¹¹ suggests that DR1 is transiently upregulated by Aβ-induced excitotoxicity. However, as AD progresses, DR1 expressions are downregulated due to neuronal degeneration. Importantly, DR1-like receptor activation modulates cholinergic excitability, promoting acetylcholine release from basal forebrain neurons projecting to the cortex.^41,42 This indicates that early upregulation of DR1 in AD models may support synaptic function, but as neurodegeneration advances, compensatory mechanisms fail, leading to receptor loss. DR5 expression was likewise elevated in 5XFAD brains, with enrichment of DR5 in reactive astrocytes in AD tissue​, ¹¹ suggesting a glial response. Previous studies have documented the expression of both DR1 and DR2 in astrocytes, particularly activation has been shown to suppress neuroinflammation via upregulation of CRYAB (αB-crystallin) and inhibition of pro-inflammatory cytokine expression in the substantia nigra.^43,44 Supporting this, we observed a clear regional overlap between increased GFAP immunoreactivity and altered DR expression in the cortex and hippocampus of 5XFAD mice. This spatial overlap supports the likelihood that astrocytic DRs are a critical determinant in AD pathology, by reducing inflammasome activity and modulating extracellular glutamate levels, thus maintaining synaptic homeostasis and reducing excitotoxicity. Previous studies have established the critical role of DR2 in maintaining hippocampal circuit function, with reduced DR2 signaling impairing synaptic transmission and cognitive performance.^22,45,46 Notably, diminished DR2 activity has been reported to perturb synaptic efficacy, specifically at the CA3-CA1 pathway, contributing to memory deficits. ²² Although DR3 dysfunction is less studied in AD, region-specific compensatory changes in neurodegenerative conditions and suppression of hippocampal gamma oscillation support its role in AD pathology.^47–49 Moreover, studies suggest that DR3 is expressed in both glutamatergic and GABAergic neurons and colocalizes with DR1 and DR2 in a region- and age-dependent manner, may disrupt DAergic signaling and excitatory-inhibitory balance when lost in AD, exacerbating pathology.^50,51 The selective increase in DR4-positive cells in CA3 and DG may reflect a compensatory response to DR2 loss elsewhere. This reciprocal regulation, consistent with, ⁵² suggests that DRs undergo homeostatic cross-regulation to preserve functional equilibrium. These findings align with reports of severe DR4 depletion in the human AD cortex and indicate that DR4, a D2-like Gi-coupled receptor, is susceptible to Aβ-driven dysregulation. Furthermore, Wang et al. showed that DR4 modulates NMDA receptor activity in the prefrontal cortex via protein phosphatase-dependent pathways, a process disrupted in regions with DR4 loss. ⁵³ Thus, DR4 downregulation may impair the synaptic connections in AD, while its selective upregulation in CA3 and DG likely represents an adaptive mechanism to sustain network function.

Consistent with changes in DR subtypes observed in vivo, we found similar patterns of DR subtype expression in our in vitro model of AD. These findings suggest that Aβ pathology induces receptor-specific changes in expression and differentially regulates DR subtypes recruitment to the plasma membrane, with a greater effect in SH-SY5Y cells than in MC-65 cells. This pattern mirrors our observations in 5XFAD mice, highlighting DR1 dysregulation as a key player in Aβ-driven neurodegeneration. Additionally, our cAMP data revealed functional divergence between DR1 and DR2 in Aβ-induced neurotoxicity. We have previously shown that Aβ treatment in differentiated SH-SY5Y cells elevates intracellular calcium and calpain activity. ⁵⁴ Consistent with these observations, we observed increased cAMP following Aβ exposure. Moreover, activation of DR1, a Gs-coupled receptor, further enhanced Aβ-induced cAMP accumulation, which will likely facilitate calpain activation and the conversion of p35 to p25, thereby increasing CDK5 activity and driving tau hyperphosphorylation. Conversely, activation of DR2, a Gi-coupled receptor, reduced cAMP accumulation, conferring neuroprotection against Aβ toxicity. These results underscore the opposing roles of DR1 and DR2 in modulating cAMP-mediated signaling pathways that influence neuronal survival in AD.

Previous studies have demonstrated a critical role of CDK5 in synaptic activity and DA signaling, as well as its association with the pathogenesis of AD. Overactivated CDK5 results in phosphorylation of tau, promoting NFT formation and apoptosis. ²⁸ Consistent with these findings, we observed increased CDK5 expression and hyperphosphorylation of tau in SH-SY5Y and MC-65 cells in response to Aβ, which were mitigated by selective DR subtype activation. These results suggest a protective role for specific DR subtypes, potentially acting at early stages to prevent widespread tau pathology and neuronal loss. CDK5 activation leads to tau phosphorylation and apoptosis, contributing to neurodegeneration in AD, and selective DR subtype activation may prevent these processes. Upregulation of DR1 subtype or downregulation of DR2 subtype in 5XFAD mice may underlie the observed increase in intracellular calcium, facilitating calpain-mediated cleavage of p35 into the more stable fragment p25, thereby driving aberrant CDK5 activation. These receptor-level dysregulations establish a kinase-dominant signaling environment that promotes tau pathology. Future studies are needed to elucidate the molecular details of the mechanism involved in DA-mediated neuroprotection. Additionally, inhibition of p-tau and CDK expression following activation of DR subtypes suggests that DR subtype changes may precede Aβ deposition and tau phosphorylation in AD brain, supporting prior observations. ¹³

In conclusion, we found an opposing pattern of subcellular distribution and mRNA/protein expression for DR subtypes coupled to Gs and Gi in 5XFAD mice, and two in vitro AD models: differentiated SH-SY5Y and MC-65 cells. These findings provide mechanistic insights into the role of DA signaling in AD and highlight receptor-targeted modulation as a potential therapeutic avenue beyond Aβ- and tau-focused strategies. DR subtypes are abundantly expressed in the brain and form a heteromeric complex with other members of the GPCRs, modulating several downstream signaling pathways. Thus, DA signaling pathways, which are regulated in an age-dependent manner, may play a significant role in memory and cognitive function, offering a promising therapeutic target for AD pathogenesis.