Intranasal Paclitaxel Alters Alzheimer’s Disease Phenotypic Features in 3xTg-AD Mice

Abstract

Background:

Microtubule stabilizing drugs, commonly used as anti-cancer therapeutics, have been proposed for treatment of Alzheimer’s disease (AD); however, many do not cross the blood-brain barrier.

Objective:

This research investigated if paclitaxel (PTX) delivered via the intranasal (IN) route could alter the phenotypic progression of AD in 3xTg-AD mice.

Methods:

We administered intranasal PTX in 3XTg-AD mice (3xTg-AD n = 15, 10 weeks and n = 10, 44 weeks, PTX: 0.6 mg/kg or 0.9%saline (SAL)) at 2-week intervals. After treatment, 3XTg-AD mice underwent manganese-enhanced magnetic resonance imaging to measure in vivo axonal transport. In a separate 3XTg-AD cohort, PTX-treated mice were tested in a radial water tread maze at 52 weeks of age after four treatments, and at 72 weeks of age, anxiety was assessed by an elevated-plus maze after 14 total treatments.

Results:

PTX increased axonal transport rates in treated 3XTg-AD compared to controls (p≤0.003). Further investigation using an in vitro neuron model of Aβ-induced axonal transport disruption confirmed PTX prevented axonal transport deficits. Confocal microscopy after treatment found fewer phospho-tau containing neurons (5.25±3.8 versus 8.33±2.5, p < 0.04) in the CA1, altered microglia, and reduced reactive astrocytes. PTX improved performance of 3xTg-AD on the water tread maze compared to controls and not significantly different from WT (Day 5, 143.8±43 versus 91.5±77s and Day 12, 138.3±52 versus 107.7±75s for SAL versus PTX). Elevated plus maze revealed that PTX-treated 3xTg-AD mice spent more time exploring open arms (Open arm 129.1±80 versus 20.9±31s for PTX versus SAL, p≤0.05).

Conclusion:

Taken collectively, these findings indicate that intranasal-administered microtubule-stabilizing drugs may offer a potential therapeutic option for treating AD.

Keywords

Alzheimer’s disease axonal transport cognitive impairment intranasal drug administration microtubule stabilization

INTRODUCTION

Microtubule (MT) stabilizing drugs, which are commonly used as anti-cancer therapeutics, have been proposed for treatment of Alzheimer’s disease (AD) and other tauopathies [1 –3]. In tumors, microtubule-stabilizing drugs inhibit cellular mitosis by preventing microtubule disassembly [4, 5]. In neurons, microtubules are organized into fibers, which support the cytoskeleton and are critical to maintain neuronal functions. A rationale, which has been proposed previously for this therapeutic strategy in AD, is that altering neuronal microtubule dynamic stability can support cytoskeletal integrity and help preserve neuronal functions including axonal transport [1].

Previous studies have suggested that microtubule stabilizers alter neurodegeneration, as well as preserve damaged neurons. Michaelis and colleagues found that paclitaxel inhibited amyloid-β (Aβ)-related tau hyperphosphorylation thereby mitigating tau aggregation [6]. Findings from Brunden and colleagues suggest that microtubule stabilization may be effective in treating AD; however, this work has primarily focused on compounds with better brain uptake, such as Epothilone D [1 , 7–9]. Other research has reported improved outcome after paclitaxel administration in a variety of acute CNS injury models (e.g., spinal cord, optic nerve crush, needle stick) [10 –13]. Recently we administered paclitaxel via the intranasal (IN) route following closed-skull repetitive impact mild traumatic brain injury (TBI) and found that TBI mice treated with paclitaxel showed improved spatial memory performance compared to vehicle-treated TBI mice [14]. Neuropathological examination revealed that paclitaxel prevented TBI-induced axonal injury and synaptic loss in the hippocampus. Taken together, these studies suggest that paclitaxel may have therapeutic effects to counter neuronal injury and neurodegenerative disease. While these findings are encouraging, the potential mechanisms by which paclitaxel prevents neurodegenerative pathology remain to be elucidated.

In addition to neurotherapeutic effects, research has indicated that paclitaxel may alter specific immune processes associated with neuronal injuries. Paclitaxel has been used to treat spinal cord injury [15] and optic nerve crush injury where it reduced injury-induced microglia/macrophage activation in the brain [12]. A transient reduction (at 1 day) was found in GFAP stain (astrocyte reactivity) with paclitaxel [11, 12]. In addition, impaired Smad-dependent transforming growth factor-β (TGF-β) signaling processes including reduction in extracellular matrix secretion and cell migration were found [11]. There is growing evidence that neuroinflammatory processes play important roles in AD pathogenesis. These seemingly divergent theories are summarized in recent review articles [16 –21]. Once neurodegeneration has commenced, many processes including aberrant neuroinflammatory responses are observed [16, 18]. These findings led us to hypothesize that treatment with paclitaxel may mitigate pathology-induced neuroinflammation in 3XTg-AD mice.

Many MT stabilizers including paclitaxel have limited blood-brain barrier (BBB) permeability, which can hamper application to CNS disorders. To address this limitation, we administered paclitaxel via the IN route. More than two decades ago, Frey and colleagues investigated intranasal administration for CNS drug delivery [22, 23]. Also, as indicated above, we previously administered paclitaxel via the intranasal route to treat TBI in a mouse model [14]. In addition to providing a non-invasive route to circumvent the BBB for relatively non-permeable compounds, intranasal administration limits systemic distribution, thereby minimizing uptake in non-target organs [24 –28].

Here, we report data supporting our hypothesis that intranasal paclitaxel alters the phenotypic progression of AD in 3xTg-AD mice, while maintaining axonal transport and reducing the basal neuroinflammatory response. Alterations in phenotypic features were indicated when drug was administered to 8–10-week-old 3xTg-AD mice prior to onset of AD pathology and, even more surprisingly, when administration was initiated in older 3xTg-AD mice (>11 months) at an age when pathology was likely well-established [29].

MATERIALS AND METHODS

In this study, we investigated the effect of treatment by paclitaxel to alter the development of tau-related pathology and prevent deficits in axonal transport as well as changes in astrocyte morphology related to neuroinflammation by treating 3xTg-AD mice from 2 months to 6.5 months of age. We then investigated if treatment by paclitaxel could improve cognitive performance and reduce anxiety in 3xTg-AD mice, which were treated from 11 months to 17 months of age.

Subjects

All procedures were conducted in accordance with the animal care guidelines issued by the National Institutes of Health and by the Institutional Animal Care and Use Committee. Subjects were “triple transgenic” 3xTg-AD mice (the K670N/M671L mutation in amyloid precursor protein (APP), the presenilin mutation PS1 (M146V) and the human four-repeat Tau harboring the P301L mutation) [30] and wild-type (WT) fully back-crossed controls (young: n = 17 AD, n = 15 WT, male, age = 75±10 days and aged: n = 8 AD, n = 5 WT, male, age = 11 months) (Jackson Labs, Bar Harbor, ME, USA). Mice were kept on a 12-h light/12-h dark cycle with ad libitum access to water and food before and during experimental procedures and were randomly assigned to receive drug or vehicle.

Paclitaxel treatment

First, we wished to investigate if treatment by paclitaxel would alter the development of AD pathology in 3XTg-AD mice. Mice, 8–10-week-old 3XTg-AD and WT controls, were anesthetized with isoflurane (5%in flowing O₂ at 2L/min until sedated) and administered paclitaxel (Hospira, Inc., Lake Forest, IL), 0.0175μmol in 5μl volume) or 5μl saline (vehicle) via intranasal lavage with a micropipette. Mice received six treatments at intervals of 14±0.2 days until age = 24.6±2.28 weeks. At 30.4±1.07 weeks, mice were perfused and brains removed for neuropathological assessment. Previous research indicates that the 3xTg-AD model develops early neocortical Aβ pathology at approximately 12 weeks and abnormal tau after 6 months initially in the CA1 region of hippocampus [30]. Although neurofibrillary tangles are not apparent at this age, phosphorylated tau accumulation is indicated in neurons.

For the second part of this study, we tested if paclitaxel treatment could “rescue” cognitive deficits in 3XTg-AD mice. Transgenic 3XTg-AD mice are cognitively normal at birth but show subtle deficits of memory retention in the Morris Water maze by 6 months of age [31]. In treatment naïve 3XTg-AD mice (44.8±0.54 weeks old), intranasal paclitaxel (n = 4) or saline vehicle (n = 4) was administered at the same dose and method described above. A total of 14 treatments were administered every 2 weeks for 7 months (final age = 72 weeks).

Behavioral assessment

Spatial memory and anxiety were tested in WT and 3XTg-AD mice using the radial water tread (RWT) maze at 13 months (after four paclitaxel treatments) and in the elevated-plus maze at 17 months of age (after 14 treatments). We did not test the mouse cohorts treated from 2–6 months in the maze because cognitive deficits in 3XTg-AD mice are subtle before 6 months of age [31]. The RTW maze protocol and application to a mouse model of TBI has been published previously by our group [32] and in an aging mouse model by other investigators [33]. This test requires no swimming and capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the edges of an apparatus (thigmotaxis). In brief, the RWT maze consists of a 32-inch galvanized steel tub with nine holes placed at equal intervals around the device, roughly 1 1/2 inch above the apparatus floor. Of these, eight terminate after roughly 1 inch (decoy exits), and one leads to a safety box hidden behind a 90-degree angle bend to prevent visual confirmation of escape route. The safety box consists of a dark plastic container (30” L×15” W×15” H) heated externally via electronic heating pad. Five large unique visual cues line the sides of the apparatus for spatial orientation. Testing was conducted in a brightly lit room. Before testing, the maze was sanitized with 70%ethanol and filled with 1 inch of 12–14°C water. Water was changed between mice, but not between trials, and the temperature was monitored to ensure the desired range. The mouse was placed in the center and allowed 180 s to find the escape hole. If the mouse was unable to escape within the time allowed, the mouse was guided to the exit and the trial was recorded as 180 s. If a mouse attempted to enter a decoy hole and did not voluntarily re-enter the maze after 5 s, they were returned to the center of the maze by hand. Once inside the safety box, the mouse was given a food treat and allowed to remain in the box for 60 s. The safety box and maze were sprayed with 70%ethanol between trials to prevent olfactory cues. Mice were given three trials a day for 4 days (acquisition period), followed by a short-term memory test consisting of three trials on day five. Mice were unexposed to the maze until day 12, at which time they underwent three trials (long-term memory test).

Mice were tested via elevated-plus-maze (EPM) at 70 weeks of age, following 14 bi-monthly intranasal injections of either sterile saline (n = 3) or paclitaxel (n = 4). One saline-treated 3XTg-AD mouse was found dead in the cage before EPM testing could be performed. The elevated-plus maze measures anxiety, exploration and activity levels in mice by taking advantage of their innate tendency to avoid open and elevated areas. The maze is elevated 45 cm above the floor and consists of a central square (5×5 cm), from which radiate four arms (5×30 cm). Two of the arms have transparent plexiglass walls (15 cm high) around the edge (closed arms), whereas the other two arms do not have walls (open arms), but do have a 0.25 cm-high edge to prevent the mice from falling. A videotracking system (Noldus Ethovision, Noldus IT, Wageningen, The Netherlands) was used to measure entries and duration in the center, open arm, and closed arm zones. The test was performed in 100 lux white lighting, and mice were given a full hour to acclimate before testing commenced. During testing, mice were individually placed in the central square of the apparatus, facing an open arm, and allowed to explore the apparatus for 5 min while data were collected.

Immunostaining

Mice were euthanized using intraperitoneal ketamine/xylazine until respiratory arrest and then transcardially perfused with 4%formaldehyde. Brains were removed and postfixed for two hours in 4%paraformaldehyde in PBS at 4°C, followed by 24 h in 20%sucrose in PBS, and then 24 h in 30%sucrose. Coronal sections were cut at 50μm thickness from bisected brains, embedded in OCT (Tissue-Tek, Torrance, CA) with a CM1850UV cryostat (Leica, Buffalo Grove, IL). Free-floating sections were permeabilized (1 h, room temperature, 0.2%Triton X-100 and 0.01%sodium azide in PBS) followed by three 5 min washes in PBS. Antigen retrieval was performed by heating at 80°C for 30 min in 50 mM sodium citrate (pH 8.5–9.0). Sections were then cooled to room temperature, washed in PBS (3 times for 5 min each) and blocked in PBS with 10%normal goat serum (NGS) for 2 h at room temperature with gentle agitation on an orbital shaker (10 rpm). Sections were incubated in primary antibody solution (PBS, 5%NGS; 200μl per section) at 4°C overnight with glial fibrillary acidic protein (GFAP, Dako, Carpinteria, CA), a reactive astroglial marker, or Iba-1 (Wako, Richmond, VA), a microglial marker. Tau phosphorylation: sections were incubated with the AT8 antibody (Ser202; ThermoFisher, Grand Island, NY). Goat secondary antibodies labeled with Alexa 488, Alexa 647, and Cy3 were obtained from Jackson Immunoresearch (West Grove, PA). Nuclei were stained with Hechst fluorescent dye (ThermoFisher, Grand Island, NY) using the manufacturer’s recommended conditions. Floating tissue sections were coverslipped with a drop of Prolong gold antifade solution without DAPI (Life Technologies, Grand Island, NY). Laser scanning confocal imaging was conducted using a Leica TCS SP2 confocal/multiphoton hybrid microscope with tunable emission gating and using sequential, between stack, single photon excitation at 488, 543, and 633. Z-plane images acquired the full volume thickness of each slice imaged (i.e., 50μm for floating sections and 5μm for paraffin-embedded sections) using system optimized stepping. Within an experiment, all directly compared sections/slides were identically and simultaneously prepared.

The P-tau immunostained images were evaluated by a rater blind to treatment. The P-tau neurons were localized to the region of the dentate gyrus, which encompassed an area of 70μm×254.5μm for all the sections. The rater counted the number of visible phospho-tau containing neurons in each image and the total number was averaged and compared between PTX and SAL treated groups. The 3xTg-AD mice at 7 months do not accumulate neurofibrillary tangles; however, phosphorylated tau accumulation is apparent.

Imaging and image analysis

We performed manganese-enhanced magnetic resonance imaging (MEMRI) to assess axonal transport in vivo using an ultra-high resolution 14T MRI (Avance III, vertical bore, Bruker BioSpin Corp, Billerica, MA). In vivo estimates of axonal transport were assessed by MEMRI in 3XTg-AD mice at 3 and 6 months of age. Briefly, the method takes advantage of the neurophysiological properties of manganese ion (Mn^2 +), which is taken up into neurons through voltage-gated Ca^2 + channels, packaged into vesicles, and transported within neurons on the microtubule framework. The transport also occurs transsynaptically, so the method can be used to estimate bulk flow of transport in vivo. Manganese is paramagnetic and results in a T1 shortening effect on MR images (increased signal enhancement). Using an imaging protocol modified from our previous research [34], T1-weighted structural imaging was performed using a 3D Modified Driven Equilibrium Fourier Transform (MDEFT) sequence with a voxel size of 0.14×0.14×0.25 mm³, 64 slices, flip angle/repetition time/echo time (FA/TR/TE): 12°/5000 ms/1.9 ms. A pre-enhancement T1-weighted image was acquired. On a subsequent day, 2 days after treatment with paclitaxel or saline, subjects were administered 1M MnCl₂ (Sigma-Aldrich, Inc., St. Louis MO.) in 5μl saline via intranasal lavage to the right nostril. Eleven T1-weighted images were acquired at 100 min (early) and again from 280–350 min (late) post MnCl₂. For analysis and rate estimation, image sets were coregistered and normalized to the mouse atlas [35]. Time-intensity curves were constructed for the olfactory nucleus using manual ROIs (ImageJ, NIH) placed in the olfactory bulb (uptake) and tract and a regression line was used to estimate averaged, group-wise relative transport rates. This methodology was modified from our previous method, which used tracer kinetic analysis of a dispersion model. However, due to constraints on imaging time and subject anesthesia [34] we modified the protocol to a single session as described above. Single factor ANOVA was used to determine significant differences between 3- and 6-month treatment groups.

In vitro assessment of paclitaxel on axonal transport deficits

Hippocampal cell culture and expression of transgenes

As confirmation of the effect of paclitaxel on axonal transport, we used primary hippocampal neuronal cultures from E18 embryonic rats (Charles River, USA) of either sex, which were prepared as described by Kaech and Banker [36] and kept in PNGM primary neuron growth media (Lonza, Basel, Switzerland). The glial feeder layer was derived from murine neural stem cells as described by [37]. At 10 days in vitro (DIV), cells were co-transfected with pβ-actin-BDNF-mRFP and pmUβa–enhanced blue fluorescent protein (BFP; from Gary Banker, Oregon Health and Sciences University, Portland, OR) using Lipofectamine 2000 (Invitrogen). Cells expressed the plasmids for 24–36 h before live imaging. All experiments with animals were approved by and followed the guidelines set out by the Simon Fraser University Animal Care Committee; Protocol 943-B05.

AβO and paclitaxel treatments

Full-length, synthetic Aβ 1–42 peptides (AβOs; American Peptide) were prepared exactly according to the method of Lambert et al. [38] and applied to 11–13 DIV cells at a final concentration of 500 nM for 18 h. We use synthetic AβOs in our studies for the following reasons. First, they mimic the toxic properties of natural oligomers (brain or cell derived) as described previously [39, 40]. Second, unlike natural oligomers, synthetic AβOs can be detected by immunocytochemistry. Confirmation of AβO binding is crucial in our experiments because it varies considerably between neurons. Although they may not be identical to natural oligomers, synthetic AβOs are a tractable tool for investigating mechanisms of AD pathogenesis [41]. Prior to AβO or vehicle exposure, cells were incubated with paclitaxel or equivalent volumes of vehicle (ethanol). Cell viability assays were performed using the LIVE/DEAD Viability Kit per manufacturer’s recommendations (ThermoFisher Scientific).

Live imaging and analysis of BDNF-mRFP transport

Red fluorescence protein-labeled brain-derived neurotrophic factor (BDNF-mRFP) transport was analyzed using a standard wide-field fluorescence microscope equipped with a cooled charge-coupled device camera and controlled by MetaMorph (Molecular Devices, Sunnyvale, CA) as described previously [42]. All imaging, typically 100 frames, was recorded by the “stream acquisition module” in MetaMorph. Briefly, cells were sealed in a heated imaging chamber, and recordings were acquired from double transfectants at an exposure time of 250 ms for 25 s. This captured dozens of transport events per cell in 100-μm segments of the axon. Axons were initially identified based on morphology and confirmed retrospectively by immunostaining against MAP2, a dendritic cytoskeletal protein. Soluble BFP detection was necessary to determine the orientation of the cell body relative to the axon and thus to distinguish between anterograde and retrograde transport events. Vesicle flux, velocity, and run lengths were obtained through tracing kymographs in MetaMorph. Flux was defined as the total distance traveled by vesicles standardized by the length and duration of each movie (in micron-minutes), $(\sum_{i = 1}^{n} d_{i}) / t$ , where d_i are the individual DCV run lengths, l is the length of axon imaged, and t is the duration of the imaging session. A vesicle was defined as undergoing a directed run if it traveled a distance of≥2μm. This distance was determined as a safe estimate of the limit of diffusion based on the assumption that root-mean-squared displacement equals 2Dt, where D is the diffusion coefficient (D = 0.01μm²/s for DCVs) and t is the duration of the imaging period (t = 50 s) [43]. A run was defined as terminating if the vesicle remained in the same position for at least four consecutive frames. In healthy control neurons, a significant fraction of vesicles typically pause while navigating microtubule track intersections and maneuvering around obstacles, or when captured at synaptic sites of release [44, 45]. Percentage flux represents the flux in treated neurons normalized to controls (100%). Statistical analyses were performed using Microsoft Excel or GraphPad Prism. Data are presented as mean +/–SEM. Significant differences between treatments were analyzed by Student’s t-tests with equal or unequal variance at a 95%confidence interval. For live imaging experiments, a minimum of 15 cells from 3 independent cultures (n = 3) were analyzed.

Immunocytochemistry and fixed cell imaging

For wide-field fluorescence imaging, neurons were fixed in 4%paraformaldehyde and blocked with 0.5%fish-skin gelatin [46]. Following live cell imaging to confirm AβO binding to dendrites and verify qualitatively that AβOs remained oligomeric after 18 h in culture, cells were stained with an Aβ oligomer–specific antibody (NU-4, 1:1000; from W. L. Klein, Northwestern University, Evanston, IL) or 6E10 (1:1000; Covance, Berkeley, CA) and anti-MAP2 (1:2000; Millipore, Billerica, MA). Neurons were subsequently incubated with compatible secondary antibodies.

To qualitatively assess microtubule composition by super-resolution microscopy, neurons were fixed and stained as described in [47]. Neurons were washed with 1 mL of pre-warmed (37°C) phosphate buffered saline (PBS), then fixed and permeabilized using 0.3%glutaraldehyde and 0.25%Triton X-100 in cytoskeleton buffer (CB; 10 mM MES pH 6.1, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, and 5 mM MgCl₂) for 1–2 min. This was followed by a second fixation step of 2%glutaraldehyde in CB for 10 min. Neurons were then treated with 0.1%NaBH₄ for 7 min to reduce background fluorescence and rinsed with PBS 3X for 10 min. Coverslips were incubated in blocking buffer then with primary antibody in blocking buffer. Cells were briefly rinsed 3X in PBS and then 3 X for 5 min in 0.05%Triton/PBS. After incubation with the secondary antibody, coverslips were washed 6X for 10 min each with gentle rocking in 0.05%Triton in PBS. A final fixation to crosslink the primary and secondary antibodies was performed in 4%paraformaldehyde/4%sucrose for 15 min at 37°C and rinsed 3×5 min in PBS.

Super-resolution images were acquired on a Zeiss LSM 710 with AiryScan detector through a 63x1.4NA PlanApo objective. Pinhole diameter and pixel array were set according to the ZEN software suggestions for optimal resolution results. Acquisition settings were unchanged across all samples, and raw data files were processed using identical filtering parameters.

Statistical assessment

Imaging statistical analyses are described in the imaging methods section. For behavioral studies, one-factor between-group analysis of variance (ANOVA) or Student t-tests were used to compare paclitaxel-3XTg-AD, saline-3XTg-AD, and WT groups. All reported p values were based on two-tailed critical values (p≤0.05). Radial water tread maze data were analyzed by ANOVA with training or test days as a repeated measures within-subjects factor and treatment (drug/no drug) as a between-subjects factor. Following the ANOVA we tested the a priori hypothesis that saline-treated 3XTg-AD mice would be significantly different from both WT and paclitaxel-treated 3XTg-AD mice was performed using a Helmert individual comparisons test [48, 49]. Statistical analyses were carried out using SPSS software (IBM, Armonk, NY, USA). In vitro values were compiled for analysis in Microsoft Excel and SPSS (IBM, North Castle, NY, USA), and significance difference between treatments was analyzed by Student’s t-test with equal or unequal variances at a 95%confidence interval.

RESULTS

Intranasal paclitaxel improves spatial memory and reduces anxiety in 3xTg-AD mice

Using a radial water tread maze to test memory performance and an elevated-plus maze to evaluate anxiety, we found that paclitaxel treatment rescued cognitive impairment, which has been demonstrated previously in 3xTg-AD mice starting at 6 months of age [31]. Paclitaxel treatment also reduced anxiety-related behaviors in the elevated-plus maze. In our study, starting at age of 11 months. Three groups of mice were assessed: 1) 3xTg-AD treated with intranasal paclitaxel (4 bi-monthly treatments at 48, 50, 52, and 54 weeks of age); 2) 3xTg-AD vehicle treatment (Saline); and 3) non-treated non-transgenic mice (WT). At 13 months of age the mice were evaluated using a radial water tread (RWT) maze, an established spatial memory task tailored specifically for mice that requires no swimming. This avoids species-specific stress-induced motor performance confounds in mice that can occur when using standard Morris water maze methods, which were originally optimized for use in rats [14 , 51]. Animals were also tested on an elevated-plus maze at 17 months of age after 14 bi-monthly treatments.

As shown in Fig. 1A, an overall two-way ANOVA comparing WT, 3xTg-AD paclitaxel-treated, and 3xTg-AD saline-treated mice on days 1-12 revealed a statistically significant difference between groups (F[2,10, 2,10] = 5.440, p≤0.025), indicating that paclitaxel treatment significantly improved performance in the water tread maze task in 13-month-old 3xTg-AD mice. In keeping with this, a Helmert a priori individual comparisons test [49] further confirmed that saline-treated 3xTg-AD mice performed significantly worse than the paclitaxel-treated and WT (p≤0.021) and that the paclitaxel-treated group performance was not significantly different from WT controls (p≤0.125, n.s.).

Fig. 1

Intranasal paclitaxel improves spatial memory and reduces anxiety in 3xTg-AD mice. A) Radial Water Tread (RWT) maze tests spatial learning and memory. Graph indicates learning/acquisition phase (Days 1–4), short-term (Day 5), and long-term (Day 12) memory of three 13-month-old groups of mice: non-treated wild-type (dashed line/triangles) and 3xTg-AD mice that received either 4 paclitaxel treatments (solid/squares) or 4 saline treatments (solid/circles). A two factor ANOVA indicated the paclitaxel-treated animals displayed significantly better performance in this task compared to saline-treated 3xTg-AD mice (p≤0.025) and not significantly different from WT animals. B) Anxiety was tested using an elevated-plus maze. Paclitaxel-treated 3x-Tg-AD mice spent significantly less time in the center (p≤0.02) and more time exploring the open arms (p≤0.04). compared to saline-treated 3xTg-AD mice. Error bars indicate standard error of the mean (S.E.M.).

The elevated-plus maze has been used previously to demonstrate anxiety-related behavior in 3xTg-AD [52]. At 17 months of age, we found that paclitaxel (after 14 bi-monthly treatments) resulted in an 84%increase in time exploring the open arms (129.1±46.3 versus 21.0±18.1, mn±sem compared to saline-treated 3xTg-AD animals respectively, p≤0.04) and 75%decreased time in the center zone (14.9±6.4 versus 59.9±13.9, mn±sem for paclitaxel and saline respectively, p≤0.02). Time spent in closed arms in mice treated with paclitaxel, was not significantly different (p≤0.21 n.s.) (Fig. 1B).

Paclitaxel reduces aberrant phospho-tau levels and decreases reactive gliosis in 3xTg-AD mice

In 3xTg-AD mice after intranasal paclitaxel (or vehicle) treatment from age of 10 to 24 weeks (mice were 7 months at time of perfusion), we assessed the neuropathology in the CA1 region of the hippocampus. Figure 2 shows representative examples from confocal microscopic examination of 4 saline and 5 paclitaxel-treated 3xTg-AD mice. The top row shows microglia (iba-1) of 3xTg-AD mice that appear less ramified, consistent with a state of chronic partial activation in 3xTg-AD, which was ameliorated by paclitaxel treatment (Fig. 2A, B). The middle row shows sections immunostained with antibodies recognizing GFAP in astrocytes and indicates that paclitaxel reduced GFAP expression levels (Fig. 2C, D). These findings are consistent with the idea that paclitaxel may influence the basal inflammatory state of the CNS in 3xTg-AD mice. The bottom row shows double-label confocal microscopic images, where red indicates phospho-tau that was immunostained with an antibody recognizing phospho-epitopes at tau Thr-181. DAPI was used to counter-stain nuclei in the CA1 hippocampal pyramidal cells in blue. The representative mages indicate that paclitaxel treatment reduced the number of phospho-tau (Thr-181) positive neurons (Fig. 2E, F). To confirm this observation, p-tau immunostained images from all 5 paclitaxel- and 4 saline-treated 3xTg-AD mice were evaluated by a rater blinded to the treatment status. This analysis identified significantly fewer phospho-tau containing neurons/imaged field (5.25±3.8 versus 8.33±2.5, t-test, p < 0.04) for the paclitaxel- versus saline-treated, respectively.

Fig. 2

Paclitaxel reduces phosphorylated tau and reactive gliosis in 3xTg-AD mice. Representative examples from confocal microscopy of 4 saline (SAL) and 5 PTX-treated 3X-Tg-AD mice (7mo). Microglia (iba-1) in the CA1 are less ramified, indicating a state of chronic activation in 3X-Tg-AD, which was ameliorated by PTX (A,B). Staining for GFAP in astrocytes, showed that PTX reduced GFAP expression, indicating that PTX may influence the basal inflammatory state of the CNS (C,D). Double-labeling, of CA1 pyramidal cells, where red indicates phospho-tau that was immunostained for phospho-epitopes at tau Thr-181 (arrowheads) (E,F) and blue indicates Dapi counter-stained nuclei. PTX reduced the number of phospho-tau positive neurons. P-tau immunostained images from 5 PTX and 4 SAL evaluated by a rater blind to treatment, identified significantly less phospho-tau containing neurons/imaged field (5.25±3.8 versus 8.33±2.5, p < 0.04). Scale bars = 50μm.

Intranasal paclitaxel increases axonal transport in vivo

A primary rationale for this therapeutic strategy was that stabilizing microtubules would improve deficits in axonal transport in 3xTg-AD mice. Our previous research used MEMRI to assess transport rates in the olfactory tract during aging and with AD pathology in vivo [34, 53]. Paramagnetic, MnCl₂ 1M solution is administered intranasally. Mn^2 +, a calcium analog, is taken up by neurons and transported via axonal transport processes. Dynamic T1-weighted MR imaging detects signal enhancement and post-processing algorithms calculate a relative rate of transport in the olfactory tract. Details of the imaging technology were described previously [34]. In this study, the MEMRI was obtained at 3 and 6 months in 3xTg-AD mice before and after treatment with paclitaxel (or vehicle). No significant differences in relative Mn^2 + transport were found in saline treated mice from baseline, however paclitaxel intranasal administration increased transport significantly (0.9±0.2%at baseline versus 1.0±0.3%and 2.7±0.7%, for saline and paclitaxel respectively, F[2,18, 2,18] = 3.55, p≤0.003, Fig. 3).

Fig. 3

Intranasal paclitaxel increases axonal transport in the olfactory tract of 3xTg-AD mice on MEMRI. Manganese-enhanced MRI was obtained at 3 and 6 months in 3xTg-AD mice before and after treatment with paclitaxel versus saline. Compared to saline-treated animals intranasal PTX significantly increased manganese transports (0.9±0.2%at baseline versus 1.0±0.3%and 2.7±0.7%, mean±standard error for saline and paclitaxel respectively, F[2,18, 2,18] = 3.55, p≤0.003)

Paclitaxel improves AβO-induced vesicle transport deficits in vitro

To further support the in vivo results from MEMRI that paclitaxel treatment can improve axonal transport rates, we employed a well-established in vitro neuron model of dense core vesicle protein cargo axon transport by monitoring red fluorescence protein-labeled brain-derived neurotrophic factor (BDNF-mRFP) in primary rodent neurons [54, 55]. Using this approach, we tested whether oligomeric Aβ 1–42 (AβO)-induced BDNF transport impairment could be prevented with PTX. Neurons were imaged 18–24 h after exposure to 500 nM AβOs. AβO binding to dendrites was confirmed retrospectively by immunocytochemistry (Fig. 4A) using an oligomer-specific antibody (NU-4) [56]. Representative kymographs illustrate differences between BDNF transport in control (vehicle-treated), AβO-treated neurons, and PTX-AβO-treated neurons (Fig. 4B). Total axonal BDNF flux was markedly reduced by AβOs (∼68%decrease compared to control), yet significantly restored in neurons treated with 10 nM PTX (Fig. 4B, Table 1) in both the anterograde and retrograde directions. As previously reported [55, 57], AβOs did not significantly decrease average velocities or run lengths (Table 1). Notably, AβOs do not lead to concomitant reduction in cell viability or structural alterations of the microtubule network (also see [55 , 58]. To confirm the integrity of the microtubules in this study, using immunocytochemistry to detect tubulin and super-resolution microscopy we determined that bundled, parallel arrays of microtubules were present in all cells treated with AβOs (n = 6) and/or PTX (n = 6; Fig. 3C). Cell viability in the presence of 10 nM PTX (92.1%± 8.4%viable) was unchanged from control neurons (94%± 12%); however, 100 nM and 1 mM PTX reduced viability significantly (61.4%± 18.3%; 48.2%± 20.8%; p < 0.001 compared to control). Results show that AβOs perturb BDNF transport, yet can be prevented in the presence of PTX, and that treatments do not obviously alter microtubule structure.

Fig. 4

Low dose paclitaxel prevents AβO-induced BDNF transport deficits in vitro. Expression of soluble BFP and BDNF-RFP in an AβO-treated neuron (A). Immunocytochemistry shows that AβOs remain oligomeric after 18 h in culture. The inset (see dashed box in BDNF-mRFP) is an example of BDNF-filled vesicles in the axon (far right panel). Arrows indicate axon; arrowheads indicate dendrites. Scale bars = 25μm and 5μm. Transport rates of BDNF-RFP assessed in vitro for AβO-treated versus control with 10 nM PTX-treated hippocampal neurons (B). Representative kymographs comparing BDNF-RFP transport in control and treated neurons. Lines with a positive slope represent anterograde transport; lines with a negative slope represent retrograde transport. There is a significant increase in vesicle flux (min^–1) with AβO versus AβO with 10 nM PTX for all events. Bar graphs display means +/–SEM; Significant differences between treatments were analyzed by t-tests with equal or unequal variance at a 95%confidence interval, ^***p < 0.001 when compared with vehicle control; ^# # #p < 0.001 when compared with AβO-treated cells; nonsignificant relations are not indicated. Complete transport statistics are reported in Table 1. Super-resolution images demonstrate that microtubule organization is preserved after treatment with paclitaxel and AβOs (C). Scale bars = 25μm and 500 nm.

Table 1

BDNF Transport Defects Prevented By Application of 10 nM PTX

	BDNF Transport
	Traffic			%
	All Events	Anterograde	Retrograde	%All Events
Flux (min^-1)
Control	11.35±0.74	5.57±0.54	5.78±0.39	100±6.53
1 nM PTX	11.25±1.4	5.35±1.01	5.9±0.67	99.13±12.28
10 nM PTX	12.48±1.2	5.19±0.57	7.3±0.84	110.02±10.55
100 nM PTX	2.98±0.8^***	1.39±0.39^***	1.6±0.49^***	26.27±7.05
ABO	3.74±0.38^***	1.78±0.27^***	1.97±0.22^***	32.94±3.34
ABO + 1 nM PTX	4.61±0.61	2.16±0.35	2.46±0.37	40.59±5.37
ABO + 10 nM PTX	11.35±1.24^# # #	6.62±0.92^# # #	6.47±0.83^# # #	99.99±10.9
ABO + 100 nM PTX	2.81±0.87	1.37±0.5	1.45±0.47	24.77±7.63
Run Length (μm)
Control	6.06±0.37	6.35±0.57	5.67±0.3	100±6.01
1 nM PTX	6.79±0.49	7±0.72	6.54±0.51	112.16±7.98
10 nM PTX	6.67±0.31	6.55±0.41	6.57±0.35	110.07±5.11
100 nM PTX	5.71±0.8	5.79±1.37	4.77±0.39	94.24±13.07
ABO	5.72±0.42	5.5±0.4	6.62±1.17	94.46±6.85
ABO + 1 nM PTX	5.12±0.36	5.3±0.47	4.79±0.4	84.53±5.95
ABO + 10 nM PTX	6.65±0.24	7.05±0.44	6.28±0.29	109.72±3.88
ABO + 100 nM PTX	4.8±0.48	4.12±0.81	4.59±0.47	79.26±7.87
Velocity (μm/s)
Control	1.32±0.05	1.3±0.07	1.3±0.05	100±3.76
1 nM PTX	1.65±0.08^**	1.67±0.1^**	1.61±0.07^**	124.61±5.52
10 nM PTX	1.49±0.05^*	1.44±0.06	1.49±0.05^**	112.78±3.08
100 nM PTX	1.18±0.1	0.99±0.13^*	1.21±0.13	88.94±7.23
ABO	1.19±0.07	1.16±0.08	1.23±0.09	88.94±5.21
ABO + 1 nM PTX	1.45±0.1#	1.46±0.13^#	1.36±0.08	109.69±7.34
ABO + 10 nM PTX	1.63±0.07^# # #	1.64±0.09^# # #	1.62±0.08#	123.02±5.07
ABO + 100 nM PTX	1.04±0.13	0.95±0.19	0.98±0.1	78.72±9.4

Control n = 39 kymographs (39 cells, 2238 vesicles), 1 nM PTX n = 14 kymographs (14 cells, 705 vesicles), 10 nM PTX n = 30 kymographs (30 cells, 1699 vesicles), 100 nM PTX n = 10 kymographs (10 cells, 159 vesicles), ABO n = 30 kymographs (30 cells, 577 vesicles), ABO + 1 nM PTX n = 23 kymographs (23 cells, 594 vesicles), ABO + 10 nM PTX n = 20 kymographs (20 cells, 722 vesicles), ABO + 100 nM PTX n = 9 kymographs (9 cells, 148 vesicles). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, when compared with vehicle control (from each vehicle control column). ^#p < 0.05, ^# #p < 0.01, ^# # #p < 0.001, when compared with ABO control (from each ABO control column).

DISCUSSION

In this study, we show that intranasal administration of paclitaxel alters the phenotypic features of the 3xTg-AD mouse model of AD, which has not been reported previously. These alterations included reduction of the basal neuroinflammatory response and phosphorylated tau-containing neurons in the CA1 hippocampus and increased axonal transport in the olfactory tract of mice treated prior to the onset of amyloid and tau pathology. Most importantly, we found that spatial memory was improved and anxiety reduced in aged 3xTg-AD mice when paclitaxel was administered at a more advanced stage of the disease.

The pathologic processes that initiate late onset AD pathogenesis remain unknown. Amyloid appears to accumulate prior to, or concurrently with other well-described features of the pathophysiological cascade such as abnormal tau aggregation, neuronal loss, and eventually, cognitive decline [59]. However, whether amyloid is the driver or merely a consequence of other age-related processes such as inflammation, cardiovascular disease, metabolic disease, etc., is still under debate [60]. Currently, numerous AD clinical trials have ended in disappointment, particularly those focused on reducing or clearing Aβ in the brain [20, 21]. Reducing Aβ after clinical dementia has been shown to do little to slow disease progression. Researchers that remain faithful to the “amyloid hypothesis” of AD have suggested that anti-amyloid therapies should be given prior to the onset of dementia. However, even if early amyloid removal should prove effective in preventing AD, this strategy does not address the need of millions of people in the world currently suffering from clinical AD.

Successful interventions for clinically advanced AD might well require combination therapies, which address the neurodegenerative process at multiple levels and across various systems. Thus, there is great urgency to identify other cellular/molecular targets, which may be affected by AD and to assess their potential as therapeutic targets [61]. Several lines of evidence point to the importance of loss of microtubule integrity and axonal trafficking dysfunction in AD [62]. It has been suggested that microtubule stabilizers, including paclitaxel, may have therapeutic promise in treating AD [1 , 63]. One such drug, Epothilone D, which has good blood-brain barrier (BBB) penetration and is an inhibitor of the P-glycoprotein (Pgp) transporter, was shown to improve cognitive performance and reduce associated tau pathology in transgenic mouse models of tauopathies [1 , 65]. In another study with intranasal delivery to 3xTg-AD mice of the octapapeptide, NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln), which has microtubule stabilizing effects, indicated reduced hyperphosphorylated tau and Aβ_1–40 and Aβ_1–42. With the current study, it was our goal to show that microtubule stabilization with paclitaxel could alter disease progression and phenotypic features at both early and late stages of the disease in 3xTg-AD mice. Various studies have indicated that microtubules and microtubule dynamics are involved in many processes critical to homeostatic brain function including axonal transport, maintenance of neuronal architecture, synaptic structure and plasticity [62 , 67]. In addition to supporting the neuronal cytoskeleton, microtubule dynamics influence microglia proliferation and motility [68] as well as astrocyte morphological changes [69].

The results of this study indicated alterations in the basal inflammatory response typically observed in AD transgenic mice, which included decreased reactive astrocytes and the altered appearance of the microglia. Our findings indicate microglia of 3xTg-AD mice that appear less ramified, consistent with a state of chronic partial activation in 3xTg-AD, which was ameliorated by PTX treatment A report by Cherry et al. in AD mice shows the same changes that we observed of microglia ramification after treatment to induce neuroinflammation [70]. Despite the specificity of GFAP as a marker of astrocytes, the biological properties of GFAP somewhat complicate the interpretation, especially at chronic time points in both animals and humans. Increased GFAP can indicate: 1) a change in expression level; 2) a change in astrocyte morphology; and 3) a change in the expression subcellular pattern within the astrocytes. Thus changing where astrocytes express GFAP can appear as a change in cell number. It was beyond the scope of this study to distinguish between these possibilities.

In addition to the above-mentioned alterations in the basal inflammatory response, we found that paclitaxel treatment improved axonal transport using in vivo MEMRI and an in vitro model of AβO-induced transport deficits. Our previous study as well as other research using the MEMRI technique indicated axonal transport deficits in AD transgenic mice [53 , 72]. In brief, Mn^2 + is a calcium analog and, with some slight differences in kinetics, behaves similarly in vivo. However, since it is a metal, on an MR image, it has a shortening effect on the parameter “T1”, which is the longitudinal relaxation time. The presence of Mn^2 + in brain tissue is “bright” compared to no Mn^2 + and this effect is approximately linear for the concentration levels achieved with MEMRI [74]. Also, the change in signal enhancement is dependent upon the total amount of Mn^2 + which is taken up into the olfactory tract and system. AD Tg mice have increased calcium metabolism and therefore the total Mn^2 + uptake is much higher than WT [75]. Therefore, we could only compare the effect of PTX administration in the treated versus non-treated 3xTg-AD mice and not the overall effect compared to WT. However, due to this limitation in the current study, we performed the in vitro analysis to verify the effect of PTX on transport, particularly at very small concentrations.

Nanomolar levels of PTX increased transport as well as lowered hyperphosphorylated tau in vivo (Figs. 2 3), in addition to preventing BDNF transport disruption in AβO-treated primary neurons (Fig. 4). In the micromolar range, PTX is toxic; however, it can be protective in the nanomolar range as shown by our results as well as previous research [12, 76]. One result of low-dose PTX may be to elicit a homeostatic response via microtubule stabilization by suppressing what might otherwise be a pathogenic response to a stressor, i.e., Aβ. For example, one study found that “Taxol” prevented activation of the tau kinase CDK5 in Aβ treated neurons, reducing hyperphosphorylated tau and cell death [77]. Zempel et al. also demonstrated reduced Aβ induced toxicity where “Taxol” prevented microtubule dissolution and tau mis-sorting, but did not show a concomitant reduction of hyperphosphorylated tau or reduced activity of several tau kinases [78]. Further experimentation is required to determine the broader effects of low-dose paclitaxel. However, it is possible that stabilization of the microtubule network itself acts as a homeostatic sensor for cells [6]. Further evidence in support of microtubule stabilization and intracellular signaling homeostasis comes from studies on dual leucine zipper-bearing kinase (DLK) during neuronal development and in injury. DLK is activated when neuronal microtubules are disrupted, resulting in a broader stress response in axons in a DLK-dependent manner [79]. It is possible that signaling cascades dysregulated by Aβ that impinge on microtubule stability are ameliorated by PTX action. Taken together, we can speculate that microtubule stability is a critical sensor of cell health with wide ranging implications, e.g., reduction in hyperphosphorylated tau, and transport recovery, and warrants further investigation.

Paclitaxel has been well-characterized, over more than 20 years in treating a number of cancers. Thus, paclitaxel has the important advantage that pharmacokinetics, pharmacodynamics, effective therapeutic window, and side effects have been studied extensively. “Druggability” in terms of target characterization and the availability of biological assays is well established. Paclitaxel binds to β tubulin on the inner surface of the microtubule and counteracts the effects GTP hydrolysis thereby preventing depolymerization [4, 5]. A common argument against using paclitaxel to target CNS disorders is that it crosses the BBB poorly and possesses potential side effects in cancer treatment that include neuropathy. Importantly, the use of intranasal delivery circumvents these obstacles by efficiently delivering PTX to the brain. In a previously published study to treat repeated concussive brain injury, we assessed the pharmacokinetics and brain distribution of 3H-paclitaxel after a single intranasal administration of the same dose used in this present study [14]. IN administration of PTX indicated that overall, the concentration in the brain remained fairly stable over 10–60 min. However, among the 11 brain regions analyzed, the uptake differed. As expected, olfactory bulb uptake was highest and uptake was also comparatively high in the striatum and hypothalamus, which is unique for PTX. We also assessed distribution in peripheral organs, which indicated 0.3%Inj/ml was found in serum and 2%, 0.66%, 0.3%, and 0.02%injected dose/g-tissue was in the kidney, liver, serum, and urine, respectively). The uptake of 0.08–0.23%injection/g brain tissue converts to a tissue concentration of 14–40 pmol/g of brain tissue, which is consistent with previously published findings of IN administration of other small molecules [80, 81]. However, due to these rather low brain concentrations and the 6–10 h half-life of PTX in tissue [82], we did not assess the brain distribution past 1-h post-administration.

Previous studies indicate neurotherapeutic effects of microtubule stabilizers, including paclitaxel in very low concentrations and doses, which may be critically important when distinguishing from studies reporting neurotoxicity. Intracortical delivery of 853.9 ng paclitaxel after a needle stick brain injury found reduced axonopathy [10]. Sengottuvel et al. found 3 nM paclitaxel promoted axon growth in vitro and 4.27 ng promoted optic nerve regeneration in vivo [12]. Intrathecal administration of 256 ng/day for 30 days facilitated axon regeneration in acute spinal cord injury in rats [11]. Only 8.54 ng paclitaxel delivered directly to a dorsal root ganglion injury limited axonal retraction bulbs, and in the same study, only 10 nM applied to cultured neurons promoted axonal growth [76]. In this present study, 10 nM applied to cultured neurons prevented axonal transport deficits induced by oligomeric Aβ, while larger doses had no effect on transport rates (Fig. 4). Collectively, these findings along with those from the present study suggest that low dose paclitaxel can be neurotherapeutic rather than neurotoxic as seen with higher doses used for cancer treatment.

One advantage to intranasal delivery is that using a commercially available drug delivery device [26], a drug can be readily administered to patients. These devices are portable, easy to administer, and resemble other inhalant drug delivery systems. Some skepticism for intranasal drug delivery remains within the field of central nervous system drug development, particularly with respect to translation of results from mouse studies to human. Main objections include: the mouse brain is diminutive and diffusion distances are shorter than in humans, and the olfactory bulb of the mouse is large compared to that of the human, so it is not surprising that mice have drug uptake via the olfactory nerve tract. The distribution of substances into the brain after intranasal administration is not dependent on classic brain distribution mechanisms. Instead, distribution is very rapid, reaches steady state quickly, and is fairly stable over 1 h [26]. It has been suggested that transport after intranasal administration is within the perivascular space of the brain [27, 28]. Research to improve the intranasal administration of therapeutics is ongoing [24 –27].

CONCLUSIONS

Currently, options for treating AD are extremely limited. Thus, there is a critical need for therapeutic approaches that can produce even modest delays in cognitive decline. Here we showed that intranasal administration of low dose paclitaxel altered the phenotypic features in young 3xTg-AD mice including reduced tau pathology, decreased neuroinflammation, improved axonal transport, as well as normalized cognitive function in older mice.

Footnotes

ACKNOWLEDGMENTS

Support for this research was provided by a grant from the Alzheimer’s Association (NIRG-10-174269) and the UW Center on Human Development and Disability animal imaging and animal behavioral cores (NICHD - U54HD083091) (DJC) and VA Biomedical Laboratory Research and Development Service from the U.S. Department of Veterans Affairs I01BX002311(DGC). MAS was supported by the Natural Sciences and Engineering Research Council (RGPIN-2018-03945) and the Canadian Institutes of Health Research (90396). The authors would like to acknowledge Nathalie Martin, Dalia Murra, Bradley Neel, Aumbreen Akram, Stefanie Kaech, and Greg Garwin for their assistance with this project.

Authors’ disclosures available online ().

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