Abstract
Alzheimer’s disease (AD) poses an ever-increasing public health concern as the population ages, affecting more than 6 million Americans. AD patients present with mood and sleep changes in the prodromal stages that may be partly driven by loss of monoaminergic neurons in the brainstem, but a causal relationship has not been firmly established. This is due in part to a dearth of animal models that recapitulate early AD neuropathology and symptoms. The goal of the present study was to evaluate depressive and anxiety-like behaviors in a mouse model of AD that overexpresses human wild-type tau (htau) prior to the onset of cognitive impairments and assess these behavior changes in relationship to tau pathology, neuroinflammation, and monoaminergic dysregulation in the dorsal raphe nucleus (DRN) and locus coeruleus (LC). We observed depressive-like behaviors at 4 months in both sexes and hyperlocomotion in male htau mice. Deficits in social interaction persisted at 6 months and were accompanied by an increase in anxiety-like behavior in males. The behavioral changes at 4 months coincided with a lower density of serotonergic (5-HT) neurons, downregulation of 5-HT markers, reduced excitability of 5-HT neurons, and hyperphosphorylated tau in the DRN. Inflammatory markers were also upregulated in the DRN along with protein kinases and transglutaminase 2, which may promote tau phosphorylation and aggregation. Loss of 5-HT innervation to the entorhinal cortex and dentate gyrus of the hippocampus was also observed and may have contributed to depressive-like behaviors. There was also reduced expression of noradrenergic markers in the LC along with elevated phospho-tau expression, but this did not translate to a functional change in neuronal excitability. In total, these results suggest that tau pathology in brainstem monoaminergic nuclei and the resulting loss of serotonergic and/or noradrenergic drive may underpin depressive- and anxiety-like behaviors in the early stages of AD.
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Introduction
Alzheimer’s disease (AD) is a devastating age-related neurodegenerative disease that afflicts a large proportion of individuals aged 65 and older[1]. Recent evidence suggests that neurofibrillary tangles (NFT) develop in brainstem nuclei including the dorsal raphe nucleus (DRN) and locus coeruleus (LC) before the hippocampus and cortex, which may lead to loss of monoaminergic neurons and neuropsychiatric symptoms (NPS) in the prodromal stages of AD [2,3,4,5,6,7,8,9,10,11,12,13]. The DRN contains a large population of serotonin (5-HT) neurons that project to the forebrain and regulate mood, sleep, and reward-seeking behaviors, all of which are perturbed in AD [14,15,16,17,18,19]. The LC is also associated with depressive and anxiety-like behaviors in both humans and rodent models [20, 21], with studies suggesting that even a minimal loss of noradrenergic (NA) neurons can lead to depressive behavior [22].
Perturbations in brainstem monoaminergic nuclei may drive prodromal neuropsychiatric symptoms in AD, but direct evidence linking brainstem neuropathology and monoaminergic depletion to specific behavioral changes in early AD is currently lacking. AD is usually diagnosed at later stages when significant neurodegeneration has already taken place, so there is a critical need to develop and characterize model systems that recapitulate the early stages of AD to help identify new biomarkers and therapeutic targets. The htau mouse model is a genetic cross between a mouse microtubule-associated protein tau (MAPT) tau knockout line [23] and the 8c line [24] that contains a wild-type human MAPT transgene under the tau promoter that results in expression of all six isoforms of human tau. These mice develop tau pathology in a more naturalistic fashion such that hippocampal dysfunction and memory deficits occur relatively late in life around 12 months of age [25]. These mice are cognitively intact at 4 months of age, but there is evidence of hyperphosphorylated tau in the DRN [26] which may be associated with altered serotonergic function and behavioral dysregulation reminiscent of prodromal AD. The goal of the present study was to develop a behavioral and neurochemical profile of htau mice at 4–6 months of age and determine whether they might be a useful model of prodromal AD. We first assessed htau mice for depressive and anxiety-like behaviors using a battery of tests including the elevated plus maze (EPM) and elevated zero maze (EZM), open field, social interaction test, sucrose preference test, and forced swim test. Monoaminergic neurons and glia in the DRN and LC were examined by immunohistochemistry, and ex vivo electrophysiology was used to assess functional changes in 5-HT and NA neurons. Expression of genes involved in monoamine biosynthesis and signaling, neuroinflammation, and proteostasis were also examined in brainstem tissues. Finally, serotonergic inputs to the entorhinal cortex (EC) and hippocampus may also be impacted by brainstem tau pathology and have been implicated in affective and cognitive changes in AD [27, 28]. We examined 5-HT immunoreactivity in the EC and serotonin transporter (SERT) immunoreactivity in the hippocampus, as well as mRNA expression of 5-HT receptors, tau-related genes, and inflammatory markers in these regions. Overall, our results suggest that tau accumulation in the brainstem coincides with monoaminergic dysfunction and NPS in the early stages of AD. Additionally, htau mice may be a useful model for testing therapeutic interventions aimed at ameliorating tau pathology in the brainstem and arresting neurodegeneration in early AD.
Materials and methods
Animals
Male and female C57BL/6J mice (Jackson Labs #000664) and htau +/- mice (Jackson Labs #005491) containing a transgene that encodes the human MAPT gene were used in this experiment. MAPT -/- (global tau knockout; Mapt < tm1(EGFP)Klt) mice were used as negative controls in RT-PCR and Western blot validation of tau splice isoforms. Mice were housed in a temperature-and humidity-controlled, AALAC-approved vivarium at the University of Iowa with ad libitum access to food and water.
Behavior
Anxiety- and depressive-like behaviors in C57BL/6J and htau +/- mice were evaluated at 4 and 6 months of age. The order in which behavioral tests were performed is depicted in Fig. 1A. All behavior tests were performed during the light phase of the light/dark cycle (9 am–3 pm) and were recorded with an overhead or side-view camera integrated with Media Recorder or Ethovision video tracking software (Noldus Information Tech, Inc.). Videos were scored by an experimenter blinded to animal genotypes. Unless otherwise noted, all behavior tests were scored using Ethovision.
Htau mice exhibit depressive-like behaviors at 4 and 6 months of age. A Experimental timeline of behavioral studies in htau and C57BL/6J mice. Behavior of 4-month-old htau and C57BL/6J mice in the B, C EPM, D, E open field, F social interaction test, G sucrose preference test and H, I forced swim test. Behavior of 6-month-old htau and C57BL/6J mice in the J, K EZM, L, M open field, N social interaction test, O sucrose preference test, P forced swim test and Q Barnes maze. These results indicate depressive-like behaviors in htau mice relative to C57BL/6J mice. *p < 0.05, **p < 0.01, ****p < 0.0001
Elevated plus maze
At 4 months of age, animals were tested in the EPM to evaluate anxiety-like behaviors [29]. Briefly, the maze was 60 cm above the floor and consisted of two open arms, two closed arms (5 × 35 cm), and a neutral starting zone (5 × 5 cm). Overhead LEDs were used to maintain lux in the open arms at 20 lux and < 5 lux in the closed arms. The closed arms had tall dark walls that allowed animals to hide. At the beginning of the test, animals were placed in the neutral zone and allowed to freely explore the maze for 5 min. Distance traveled, and time spent in the open arms vs closed arms were calculated using Ethovision XT14.
Elevated zero maze
At 6 months of age, animals were tested in the EZM to evaluate anxiety-like behaviors in a novel arena [30,31,32]. Briefly, the circular maze (outer diameter: 52 cm) was elevated 60 cm above the ground and consisted of two alternating open and closed corridors (width: 5 cm). Overhead LEDs maintained the lux in the open corridors at 20 lux and < 5 lux in the closed corridors. At the beginning of the test, animals were placed in the open corridor, facing the closed corridor, and allowed to freely explore the arena for 5 min. Behavior was recorded by an overhead camera. Distance traveled, and time spent in the open area vs closed corridors were calculated using Ethovision XT14. The open area preference and probability of entering the open areas were calculated to determine the anxiety-like behavior.
Open field test
Mice were placed in the corner of a 50 × 50 × 25 cm opaque plexiglass arena (20 lux) and allowed to freely explore the arena for 30 min. The open field test was performed at 4 and 6 months of age to evaluate locomotor and exploratory behavior. The total distance traveled (cm), time spent in the center of the arena, and time spent in the corners of the arena were measured through Ethovision XT14. The center of the open field was defined as the central 15% of the arena.
Social interaction test
The social interaction test was performed in 4- and 6-month-old mice, and was performed as previously described [33, 34]. Briefly, the animal was placed in the central chamber of a transparent 3-chamber arena (20 lux) and allowed to explore the environment for 10 min. Following this, a novel C57BL/6J mouse (stranger mouse) of the same sex and approximate age as the experimental mouse was placed in one of the side chambers under a metal cage. An empty metal cage was also placed in the alternate side chamber. The experimental mouse was allowed to explore the environment and interact with the stranger mouse for 10 min. The location of the stranger mouse was alternated between the right and left chamber to control for any side preferences. The total time spent interacting with the stranger mouse and the empty cage was scored.
Sucrose preference test
The sucrose preference test was performed over four days and used to evaluate the degree of anhedonia of htau mice at 4 and 6 months of age [35, 36]. Animals were transferred to PhenoTyper observation cages (Noldus) that were fitted with two sipper bottles and Lickometers (to measure the number of licks the animal makes to a bottle). Animals had ad libitum access to tap water or a 5% sucrose solution (ThermoFisher) for 1 h. The placement of sucrose and water bottles was alternated each day to control for any side preference. The number of approaches to the water bottle and the sucrose bottle was measured through Ethovision XT14.
Barnes maze
Spatial learning and cognitive deficits were evaluated in 6-month-old C57BL/6 J and htau mice as described previously [32, 37, 38]. The Barnes maze was a 5-day protocol [39], with environment habituation on day 1, training sessions on days 2 and 3, rest on day 4, and a probe trial on day 5. The maze was a gray circular arena (diameter: 91 cm), consisting of 20 equally divided holes, and was elevated 93 cm above the ground. The room was well lit and visual cues were present on the walls.
On Day 1, animals were guided to the predetermined ‘goal box’, which had been fitted with an escape chamber. In contrast, the remaining 19 (non-target holes) were not fitted with any chambers, and animals could see the ground below.
Over days 2 and 3, animals underwent a total of five training trials. A buzzer sound (~ 100 dB) was played while animals explored the environment. Once the animal found the goal box and entered the escape chamber, the buzzer was turned off and animals were allowed to rest for 1-min before being returned to a holding cage. If an animal did not find the goal box/escape chamber within a 2-min trial, they were guided to the escape chamber as they had been on Day 1. Inter-trial interval was 30 min. Animals had 3 training trials on day 2, and 2 trials on day 3.
On the probe day, the escape chamber was removed from the goal box. Animals were placed on the maze platform, and the buzzer sound was presented. Animals were allowed to explore the environment for 2 min. Behavior was recorded by an overhead camera.
The number of visits to the goal box & non-target holes was measured along with the latency to approach the goal box and time spent in the target quadrant.
Forced swim test
Depressive-like behavior was evaluated at 4 and 6 months in the forced swim test. Mice were gently placed in a tall cylinder filled with 24–25 °C tap water (32 cm height × 20 cm diameter, water height: 25 cm) for 6 min. After the test, animals were placed in a clean cage under a heating lamp for 5 min to warm them and allow them to dry off. Animal behavior was recorded from a side-view camera and analyzed with Ethovision XT14. The 6-min videos were divided into two bouts: a pretest (first 1–2 min) and a test (last 3–6 min) phase. The latency to the first immobile bout, frequency of immobile bouts, and duration of each immobile bout was evaluated.
Immunofluorescence
A cohort of C57BL/6 J and htau +/- mice were deeply anesthetized with tribromoethanol and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) to collect brains for immunofluorescence experiments. Brains were cryosectioned at 25 µm using a Leica cryostat (CM3050S, Leica, Germany). Slices were stored at 4 °C in a cryoprotectant solution. The immunofluorescence was performed as described previously [40, 41]. Briefly, for each region of interest (ROI), 3–4 slices were used across the rostral-caudal axis. Slices were washed in PBS and incubated in 0.5% Triton X-100/PBS for 30 min, blocked in 10% normal donkey serum in 0.1% Triton X-100/PBS, and then incubated with the respective primary and secondary antibodies (Table 1). For AT8 IF experiments, mouse-on-mouse blocking reagent (3% final volume; Vector Laboratories) was added to the blocking solution to reduce non-specific binding to endogenous mouse IgG. Slices were subsequently washed in PBS, mounted on glass slides, and coverslipped with Vectashield mounting media (Vector Laboratories, Inc.).
Confocal z-stacks (1 µm) were captured on an Olympus FV3000 laser scanning confocal microscope (20 sections/z-stack) and converted to maximum projection images using Image J software. Images were analyzed by trained researchers blind to experimental conditions to obtain cell counts per unit area, % immunoreactive area, and optical density using ImageJ. The optical density was estimated by first converting images to an 8-bit grayscale image and performing a background correction. Optical density calibration was performed with a 21-step tablet (available from ImageJ) using the Rodbard function. Following optical density calibration, mean grayscale values were recorded from the ROIs with an effort to avoid artifacts. Percent (%) immunoreactive area was performed on the ROIs of thresholded images. For each image, the ROI was drawn according to the shape of the region based on the mouse brain reference atlas of Paxinos & Franklin [42] and the monoaminergic signal. ROIs of the nuclei were drawn based on the reference atlas and IF immunoreactivity for each image, and then applied to all channels (AT8, Iba-1, or GFAP).
Ex vivo electrophysiology
Brain slice preparation
Deeply anesthetized mice were transcardially perfused with ice-cold, oxygenated modified artificial cerebrospinal fluid (aCSF) containing the following (in mm): 110 choline-Cl, 2.5 KCl, 7 MgSO4, 0.5 CaCl2, 1.25 NaH2PO4, 26.2 NaHCO3, 25 glucose, 11.6 Na-ascorbate, 2 thiourea, and 3.1 Na-pyruvate (pH: 7.3–7.4; osmolality: 300–310 mOsmol/kg). Then their brains were quickly dissected and coronal slices containing the DRN or LC (300 μm) were obtained using a vibratome (VT1200S; Leica Biosystems, Wetzlar, Germany). The brain slices recovered at 34 °C for 30 min in a chamber containing the choline-Cl-based aCSF described above and continuously bubbled with 95% O2/5% CO2. After the initial recovery, brain slices were transferred to and held in a different modified aCSF at room temperature, saturated with 95% O2/5% CO2, for at least 1 h before recordings started. The holding aCSF contained the following (in mm): 92 NaCl, 2.5 KCl, 2 MgSO4, 2 CaCl2, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na-ascorbate, 2 thiourea, and 3 Na-pyruvate (pH: 7.3–7.4; 300–310 mOsmol/kg).
Ex vivo electrophysiological recordings
During recordings, slices were continuously perfused (2 ml/min) with standard aCSF containing (in mm): 124 NaCl, 4 KCl, 1.2 MgSO4, 2 CaCl2, 1 NaH2PO4, 26 NaHCO3, and 11 glucose (300–310 mOsmol/kg), saturated with 95% O2/5% CO2 and maintained at 30 ± 1 °C. Patch electrodes (3–5 MΩ) were filled with a solution containing (in mm): 135 K-gluconate, 5 NaCl, 2 MgCl2, 10 HEPES, 0.6 EGTA, 4 Na2-ATP, and 0.4 Na2-GTP (pH: 7.3; 288–292 mOsmol/kg). In order to identify 5-HT or NA neurons post hoc by immunofluorescence, biocytin (2 mg/ml; Tocris Bioscience, Bristol, UK) was added into the internal solution. Only tryptophan hydroxylase 2 (TPH2)-positive neurons from the DRN and tyrosine hydroxylase (TH)-positive neurons from the LC were included in data analysis. Neurons were visualized via an upright microscope (BX51W1; Olympus, Tokyo, Japan) accompanied by a differential interference contrast imaging system. Membrane currents were amplified with a Multiclamp 700 B amplifier (Molecular Devices, San Jose, CA, USA), filtered at 3 kHz, and sampled at 20 kHz with a Digidata 1550B digitizer (Molecular Devices). Data were acquired via the pClamp 11 software (Molecular Devices). Access resistance was monitored online and changes greater than 20% would lead to discontinuation of the recordings.
To examine neuronal intrinsic excitability, recordings were conducted in current clamp mode. The DRN 5-HT neurons we patched were not spontaneously firing, and we performed recordings at resting membrane potential (RMP) and at − 70 mV holding potential to offset the variation of RMP. Input resistance was assessed by the change in membrane potential upon − 100 pA hyperpolarizing current injection. Rheobase reflected the minimal current needed to evoke action potentials (AP). Numbers of evoked APs were recorded after injecting depolarizing currents for 250 ms at 10 pA incremental steps (0–200 pA).
All the LC NA neurons were spontaneously firing, which we continuously recorded for 10 min and analyzed their firing profiles based on the last 5 min: firing frequency, mean amplitude of action potentials, variation of firing timing (assessed by coefficient of variation: CV = mean/standard deviation of inter-firing intervals). Because the NA neurons were spontaneously firing, it was impossible to assess their intrinsic excitability at RMP. Therefore, we slightly hyperpolarized the neurons by holding them at − 55 mV and performed the same recordings as in DRN 5-HT neurons.
AP characteristics were measured using pClamp, version 10 (Clampfit 10.7; Molecular Devices; RRID: SCR_011323). Many AP parameters require a clearly defined baseline in order to be assessed accurately, and the parameters are dependent on current injection magnitude. Due to differences in excitability between neurons, the current injection steps of 120 pA (held at − 70 mV) for the DR and 140 pA (held at − 55 mV) for the LC had the largest sample size for the comparison of AP characteristics that are dependent upon verifiable baseline. The metrics of ‘maximum decay slope’ and ‘time to achieve maximum decay slope’ are not dependent upon the correct identification of the recording baseline, so we examined these metrics simultaneously across all current injection steps.
Reverse transcriptase-quantitative PCR
DRN, LC, EC, dorsal hippocampus (DHP) and ventral hippocampus (VHP) tissues were micro-punched from C57BL/6J and htau +/- brains, and total RNA was isolated as described previously [41]. Briefly, the RNA was extracted from the tissue using the TRIzol reagent method. The DNA contaminants from the extracted RNA were eliminated using a DNA-free™ DNA Removal Kit (Life Technologies, USA). The concentration and purity of RNA were checked using a NanoDrop 1000 spectrophotometer and the RNA was reverse transcribed to cDNA. RT-qPCR for the target genes was performed using SYBR green qPCR master mix (Bio-Rad Laboratories, USA) and specified primers (Table 3) on a CFX96™ Real-time-PCR System (Bio-Rad Laboratories, USA). The thermal profile used for RT-qPCR was 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, followed by a melt curve analysis profile (60 °C to 95 °C in 0.5 °C increments at a rate of 5 s/step). Fold changes in the mRNA levels were determined for each gene after normalizing with β-actin Ct values using the fold change 2−ΔΔCT method [43]. Results are represented as fold changes in the mRNA levels (± SEM).
Western blot
A separate group of C57BL/6J and htau +/- mice were decapitated under isoflurane anesthesia to collect brains for Western blot experiments as reported [41]. Briefly, DRN and LC brain regions were dissected for Western blots of ptau (AH36; pSer202/pThr205), total tau (HT7), TPH2, TH, indoleamine 2,3 dioxygenase 1 (IDO1), transglutaminase 2 (TGM2), β-actin, and GAPDH. Total proteins were isolated using RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were quantified using the BCA method and an equal amount of protein was resolved in 10% SDS-PAGE gel and transferred to a PVDF membrane (0.2 µm; Millipore) for immunoblotting. The blots were blocked with Starting Block T20 (TBS) Blocking Buffer (ThermoFisher Scientific) for 20 min at 37 °C and incubated overnight with primary antibodies specific to AH36, HT7, TPH2, TH, IDO1, TGM2, β-actin, and GAPDH at 4 °C (Table 2). Further, the blots were incubated with fluorescent or HRP-conjugated secondary antibodies (Table 2) as per the manufacturer's instructions. The blots were imaged and acquired using the LI-COR Odyssey Imager (LI-COR Inc.) at 700 and 800 nm. Protein bands were quantified using ImageJ software (National Institutes of Health) and the average relative density of the TPH2, TH, IDO1, and TGM2 was determined after normalization to β-actin or GAPDH. Results are represented as a mean relative density of the protein levels (± SEM).
Statistical analysis
All data were analyzed with GraphPad Prism version 9 (GraphPad Software Inc, La Jolla, CA). Outliers were identified and removed using a ROUT test (Q = 1%). Experiments comparing two groups were analyzed with a Student’s t-test, with p < 0.05. Where variance between groups was unequal, a Welch’s t-test was performed (p < 0.05). Behavior experiments were analyzed using a two-way ANOVA and Bonferroni corrections for post hoc analyses. Data are reported and graphically represented as means ± standard error of the mean (SEM).
Results
Htau mice exhibit depressive-like behaviors by 4 months and anxiety-like behaviors by 6 months of age
In this study, we used htau +/- mice (Jackson Labs strain # 005491) that had been backcrossed to a mouse tau knockout line (Mapt < tm1(EGFP)Klt). These mice are on a C57BL/6J background, so C57BL/6J mice were used as controls throughout this study as in previous reports [25, 26, 44]. Htau mice express all six isoforms of human tau including the 3R and 4R isoforms and are thought to represent a more naturalistic model of AD with late-onset cognitive impairment [25] (Additional file 1: Fig. S1). We asked whether htau mice exhibit behavioral phenotypes reminiscent of prodromal AD by 4 months of age, which coincides with the appearance of hyperphosphorylated tau in the DRN but prior to the onset of cognitive impairments [25, 26]. Male and female htau and C57BL/6J mice were tested in the EPM, open field, social interaction test, sucrose preference test, and forced swim test at this time point (Fig. 1A). We found a significant interaction between sex and genotype on time spent in the open arms of the EPM at 4 months (F1,53 = 4.92, p < 0.05), but post-hoc comparisons between htau and C57BL/6J mice were non-significant in both males and females. No group differences in locomotor activity were observed in this assay (Fig. 1B, C). There was a main effect of sex in time spent in the center of the open field (F1,55 = 12.25, p < 0.001) but no sex x genotype interaction, suggesting that female mice generally exhibited more anxiety-like behavior in this test. Htau mice also exhibited hyperlocomotion in the open field (Main effect genotype: F1,55 = 8.32, p < 0.01) although Bonferroni post-tests were only significant in males (t55 = 2.77, p < 0.05) (Fig. 1D, E). Interestingly, both sexes exhibited significant depressive-like behaviors in the social interaction test (Main effect of genotype: F1,57 = 74.37, p < 0.0001; Bonferroni post-tests t57 = 7.32, p < 0.01 for males and t57 = 5.06, p < 0.0001 for females) (Fig. 1F), whereas sucrose preference was only reduced in male mice (Main effect genotype: F1,56 = 5.89, p < 0.05; Bonferroni post-test: t56 = 2.65, p < 0.05) (Fig. 1G). Overall, there was no effect of genotype on immobility time or latency to immobility in the forced swim test, but female mice spent more time immobile than males (Main effect of sex: F1,57 = 4.22, p < 0.05) (Fig. 1H, I).
We then examined anxiety and depressive-like behaviors in these mice at 6 months of age. Here we found a significant effect of genotype in time spent in the open area of the EZM (F1,56 = 12.21, p < 0.001), with male htau mice exhibiting a significant reduction in open area time (Bonferroni post-tests: t56 = 3.11, p < 0.01) (Fig. 1J). Locomotor activity did not significantly differ between groups in this test (Fig. 1K). There was also a significant effect of genotype in time spent in the center of the open field (F1,55 = 5.15, p < 0.05) that is suggestive of enhanced anxiety-like behavior, although post-hoc comparisons were non-significant in both sexes (Fig. 1L). Locomotor activity in the open field did not differ significantly between groups, which contrasted with the hyperlocomotive phenotype observed at 4 months (Fig. 1M). Depressive-like behaviors in the social interaction test persisted at 6 months in both sexes (Main effect of genotype: F1,50 = 94.73, p < 0.0001; Bonferroni post-tests: t50 = 8.23 for males and t50 = 5.70 for females) (Fig. 1N), although the decrease in the sucrose preference noted at 4 months had resolved by this time point (Fig. 1O). Similar to what we observed at 4 months, there was no effect of genotype in the forced swim test at 6 months, although female mice continued to spend more time inactive than males (Main effect of sex: F1,57 = 5.76, p < 0.05) (Fig. 1P). There was also no effect of sex or genotype on spatial memory in the Barnes Maze at 6 months (Fig. 1Q), which is consistent with previous reports that memory impairments in htau mice only become apparent at 12 months of age [25].
Monoaminergic depletion and hyperphosphorylated tau in the brainstem at 4 months
Next, we investigated whether loss of monoaminergic neurons in the DRN or LC at the 4-month mark might account for these behavioral phenotypes. We focused on males in these experiments as they had the most robust behavioral phenotypes at 4 months of age. All histological experiments were performed in separate cohorts of mice to avoid any confounds of behavioral testing on monoamine levels. The DRN was subdivided into rostral, mid and caudal subregions, which were previously found to have distinct forebrain projections and behavioral outputs [19, 45,46, Tau accumulation in the DRN and subsequent loss of monoaminergic drive may promote depressive-like behaviors in the prodromal phase of AD prior to the onset of cognitive decline. Further studies in AD mouse models are needed to define a causal relationship between DRN or LC neuropathology and specific AD symptoms.Conclusion
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional file 1.
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Acknowledgements
We would like to thank Gloria Lee for providing RD3 and RD4 antibodies for Western blot analysis and Gabrielle Bierlein-De La Rosa for technical assistance with behavioral studies.
Funding
This work was supported by R01 AA028931, R01 AG070841, R00 AA024215, and K99 AA024215 to C.A.M. We also thank the Carver Trust and the Williams-Cannon Foundation for their support. K.K. was supported by T32 DK112751, S.P. was supported by T32 GM067795, and L.K. was supported by T32 NS045549.
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CAM, KK and NB designed the studies and wrote the manuscript with editorial help from MH and TA. KK performed behavioral and histological studies and analysis. NB also performed tissue collection and RT-qPCR analysis of gene expression in the DRN and LC and assisted with Western blot experiments. GG performed histological experiments, confocal microscopy and analysis of tau pathology in the DRN, LC, EC, and hippocampus. RW performed slice electrophysiology experiments and analysis. GPS performed Western blot experiments and analysis for histology studies. LK provided additional analysis of electrophysiological data. SP provided conceptual input into the design of histological studies and assisted with imaging and analysis. SMT performed RNA isolation and RT-qPCR analysis in EC and hippocampal tissues. MH and TA also provided intellectual input into experimental design and interpreting the results. All authors read and approved the final manuscript.
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Supplementary Information
Additional file 1: Supplementary Methods
: RT-PCR quantification and tau splice isoforms; Detection of tau isoforms in whole brain lysates. Supplementary Figure 1: Validation of htau mice showing presence of insoluble 3R tau isoform in whole brain lysates at 12 months of age. (A) Mouse and human tau splice isoforms in C57BL/6J, htau +/- and MAPT -/- mice. The human-specific primers only amplified products in htau +/- while mouse-specific primers only amplified products in C57BL/6J mice. (B) (i) Protein expression of human tau (HT7) in total protein extracts and (ii) 3R and 4R tau isoforms in Sarkosyl soluble and insoluble fractions from C57BL/6J, htau +/- and MAPT -/- mice. Only htau +/- mice contained human-specific tau (HT7) and the 3R tau isoform in detergent insoluble fractions. Both htau +/- and C57 mice contained the 4R isoform in soluble fractions. Supplementary Figure 2: Orthogonal images confirming hyperphosphorylated tau in 5-HT neurons in the DRN. (A) Atlas plate depicting the mid region of the DRN that was used to obtain orthogonal planes. (B-D) Representative orthogonal planes showing colocalization between 5-HT (cyan) and ptau (red) in the DRN. (E) Atlas plate depicting the mid region of the LC that was used to obtain orthogonal planes. (F-H) Representative orthogonal planes showing colocalization between TH (cyan) and ptau (red) in the LC. Supplementary Figure 3: Hyperphosphorylated tau and monoaminergic depletion in the brainstem of female htau mice at 4 months. (A) Representative confocal images of 5-HT immunostaining (20X; scale bar = 200 µm) and 5-HT/AT8 co-staining (60X; scale bar = 50 µm) in the DRN of C57BL/6J and htau +/- mice. (B) Atlas plate depicting one of the DRN regions analyzed (mid DRN) (C) Representative orthogonal image showing colocalizaton of ptau with 5-HT neurons in the DRN (100X; scale bar = 20 µm). (D) Histogram of 5-HT cell counts/mm2, (E) 5-HT immunoreactive area (%), (F) ptau (AT8) optical density in subregions of the DRN and (G) Correlation analysis between 5-HT IR area and AT8 optical density in the DRN (H) Representative confocal images of TH immunostaining (20X; scale bar = 200 µm) and TH/AT8 colocalization (60X; scale bar = 50 µm) in the LC of C57BL/6J and htau +/- mice. (I) Atlas plate depicting one of the LC regions analyzed (mid LC) (J) Representative orthogonal image showing colocalization of ptau with TH neurons in the LC (100X; scale bar = 20 µm). (K) Histogram of TH cell counts/mm2, (L) TH immunoreactive area, (M) ptau (AT8) optical density in subregions of the LC, and (N) Correlation analysis between TH immunoreactive area and AT8 optical density in the mid LC. *p < 0.05, **p < 0.01, ***p < 0.001. Supplementary Figure 4: Glial activation in the DRN and LC in htau mice at 4 months. (A) Representative confocal images of Iba-1 and GFAP immunostaining in the DRN of C57 and htau +/- mice (60X; scale bar = 50 µm). (B) Iba-1+ cell counts/mm2, (C) Iba-1 optical density, and (D) Iba-1 immunoreactive area in subregions of the DRN. (E) GFAP+ cells/mm2, (F) GFAP optical density, and (G) GFAP immunoreactive area in subregions of the DRN. (H) Representative confocal images of Iba-1 and GFAP immunostaining in the LC of C57 and htau +/- mice. (I) Iba-1+ cells/mm2, (J) Iba-1 optical density, and (K) Iba-1 immunoreactive area in subregions of the LC. (L) GFAP+ cells/mm2, (M) GFAP optical density, and (N) GFAP immunoreactive area in subregions of the LC. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01. White arrows denote Iba-1 +or GFAP+ cell bodies. Supplementary Figure 5: Reduced 5-HT innervation of the entorhinal cortex in htau mice at 4 months. Representative confocal images of 5-HT fiber immunostaining (40x; scale bar = 50 um) in C57 and htau mice. (A) Atlas representation of a section of the EC used for analysis (in red). (B) Representative confocal images of 5-HT staining in the EC of C57BL/6J and htau mice at 40X. (C) Histogram of % 5-HT IR area in subregions of the EC. (D) Representative confocal images of 5-HT staining in the EC at 100X. (E) Atlas representation of CA1 region of the hippocampus used in the analysis (in red). (F) Representative confocal image of SERT staining in the CA1 region of the hippocampus in C57BL/6J and htau mice. (G) Histogram of % SERT IR area in subregions of the hippocampus (H) Representative confocal images of SERT staining in the CA1 region of the hippocampus at 100X. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01. Supplementary Table 1: List of fold change values obtained from RT-qPCR of genes tested in the dorsal raphe nucleus, locus coeruleus, dorsal and ventral hippocampus, and entorhinal cortex of C57 and htau (*p < 0.05, **p < 0.01, ***p < 0.001).
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Khan, K.M., Balasubramanian, N., Gaudencio, G. et al. Human tau-overexpressing mice recapitulate brainstem involvement and neuropsychiatric features of early Alzheimer’s disease. acta neuropathol commun 11, 57 (2023). https://doi.org/10.1186/s40478-023-01546-5
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DOI: https://doi.org/10.1186/s40478-023-01546-5