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Neurodegenerative tauopathies, such as Alzheimer’s disease (AD), are characterized by insoluble deposits of hyperphosphorylated tau protein within brain neurons. Increased phosphorylation and decreased solubility has been proposed to diminish normal tau stabilization of microtubules (MTs), thereby leading to neuronal dysfunction. Earlier studies have provided evidence that small molecule MT-stabilizing drugs that are used in the treatment of cancer may have utility in the treatment of tauopathies. However, it has not been established whether treatment with a small molecule MT-stabilizing compound will provide benefit in a Tg model with pre-existing tau pathology, as would be seen in human patients with clinical symptoms. Accordingly, we describe here an interventional study of the brain-penetrant MT-stabilizing agent, epothilone D (EpoD), in aged PS19 mice with existing tau pathology and related behavioral deficits. EpoD treatment reduced axonal dystrophy and increased axonal MT density in the aged PS19 mice, which led to improved fast axonal transport and cognitive performance. Moreover, the EpoD-treated PS19 mice had less forebrain tau pathology and increased hippocampal neuronal integrity, with no dose-limiting side effects. These data reveal that brain-penetrant MT-stabilizing drugs hold promise for the treatment of AD and related tauopathies, and that EpoD could be a candidate for clinical testing.
Insoluble fibrillar inclusions comprised of hyperphosphorylated tau proteins are found in the brains of patients with Alzheimer’s disease (AD) (Brunden et al., 2009; Ballatore et al., 2007; Lee et al., 2001b). The occurrence of tau aggregates in AD, frontotemporal lobar degeneration (FTLD), and several other “tauopathies” suggests that neurodegeneration is mechanistically linked to pathological tau. This theory is supported both by the discoveries that tau gene mutations cause inherited FTLD (Hutton et al., 1998; Hong et al., 1998) and by the correlation between tau pathology and the degree of AD dementia (Arriagada et al., 1992; Wilcock and Esiri, 1982; Gomez-Isla et al., 1997).
In healthy neurons, tau is enriched in axons and promotes MT stabilization (Cleveland et al., 1977b; Cleveland et al., 1977a). The sequestration of tau into insoluble inclusions could result in disruption of normal tau function (Lee et al., 1994), which could alter MT stability and FAT. An approach to compensate for tau loss-of-function would be to utilize molecules that stabilize MTs (Lee et al., 2001b; Brunden et al., 2009; Lee et al., 1994). This strategy was first tested (Zhang et al., 2005) when the MT-stabilizing drug, paclitaxel, was administered to transgenic (Tg) mice that develop primarily brain stem and spinal cord tau pathology (Ishihara et al., 1999). The tau Tg mice had improved MT density, FAT, and motor performance after paclitaxel absorption at neuromuscular junctions. However, paclitaxel is not suitable for human tauopathies because of poor blood-brain barrier (BBB) permeability (Brunden et al., 2011; Fellner et al., 2002).
Subsequent studies demonstrated that intranasal delivery of the MT-stabilizing octapeptide, NAP, reduced tau and Aβ in the brains of 3X Tg mice (Matsuoka et al., 2007; Matsuoka et al., 2008). This molecule (AL-108) is undergoing evaluation in patients with progressive supranuclear palsy (Gravitz, 2011). We more recently identified epothilone D (EpoD) as a preferred MT-stabilizing compound for the potential treatment of tauopathies (Brunden et al., 2010; Brunden et al., 2011). In a preventative study, EpoD was administered weekly for 3 months to young PS19 tau Tg mice that initially lacked significant tau pathology. EpoD largely prevented the axonal MT loss and dystrophy, as well as spatial learning deficits, that manifested as these mice developed forebrain tau pathology with age (Yoshiyama et al., 2007; Brunden et al., 2010).
Left unresolved in the prophylactic study of EpoD was the important question of whether this agent would be efficacious in Tg mice with established tau pathology, which would more accurately model the human clinical setting where tauopathy patients are identified after the onset of cognitive deficits and tau pathology (Jack et al., 2010). We have now evaluated EpoD in aged PS19 mice with pre-existing tau pathology. EpoD treatment resulted in significant improvements of MT density, axonal integrity, FAT and cognitive performance, without the onset of side-effects. Moreover, EpoD treatment reduced hippocampal neuron and axon loss. Thus, EpoD safely corrected axonal defects in an interventional mouse model of AD-like tauopathy, suggesting that this agent has potential for the treatment of human tauopathies.
EpoD was prepared as previously described (Lee et al., 2001a; Rivkin et al., 2004). The spectroscopic properties of EpoD were identical to those reported in the literature. Compound purity was >95% as demonstrated by LC-MS.
A transgene encoding the human T34 tau isoform (1N4R) containing the P301S mutation found in FTDP-17 (Goedert and Jakes, 2005) and driven by the mouse PrP promoter was used to create tau Tg mice on a B6C3/F1 background as described (Yoshiyama et al., 2007). Groups (n=12–13) of 9-month old male PS19 tau Tg mice or 9-month old non-Tg littermates were administered weekly IP injections of 1 mg/kg EpoD or 0.3 mg/kg of EpoD, or vehicle (DMSO), for a total of 3 months. Male PS19 mice were chosen for these studies as they develop tau pathology more consistently than do females. Animals were monitored for signs of abnormal behavior or distress, and were weighed weekly. After 11 weeks of dosing, the mice underwent behavioral testing as described below. A subset of mice (n=3) from each study were utilized for FAT studies (below). The remaining animals were euthanized at study completion, with blood collected for complete blood cell counts and brain and optic nerve (ON) recovered for immunohistochemical and biochemical analyses. The mice from each group also underwent necropsy evaluation with organ weights recorded.
Transmission EM was performed on cross-sections of ONs from vehicle- and drug-treated PS19 mice or non-Tg littermates. The ONs were removed and post-fixed in 2% glutaraldehyde and 2% paraformaldehyde, and then processed for standard EM as described (Zhang et al., 2005). Ultrathin sections from ON blocks were cut and mounted on EM grids and examined using a JEM1010 electron microscope (Jeol, Peabody, MA) at 80 kV. Cross sections of the entire ON were systematically sampled at 10,000x and 50,000x magnification, with dystrophic axons and MTs counted from multiple fields of 128 μm2 and 0.035 μm2, respectively, from at least 25 separate photos. The assessors were masked to the treatment group.
Three mice from each treatment group underwent bilateral intravitreal injections (2 μl) of 0.5 mCi of 35S-methionine (PerkinElmer, Boston, MA) with a 33 gauge needle connected to a 5 μl Hamilton syringe over a period of 2 min while under deep anesthesia (Brady et al., 1999; Kirkpatrick et al., 2001), in accordance with protocols approved by the University of Pennsylvania. Mice were sacrificed 3 h after injection, with removal of ONs and dissection into seven consecutive 1-mm long segments. Matched pairs of segments from each individual mouse were homogenized in 66 μl of BUST buffer (0.5% SDS, 8 M urea, 2% β-mercaptoethanol, 0.1% proteinase inhibitor cocktail, and 0.1 M Tris HCl, pH 6.8) at room temperature overnight, and then sonicated with 20 pulses of a tip-sonicator followed by centrifugation at 85,000 × g for 40 min at 30° C as previously described (Zhang et al., 2005). The extent of FAT was examined by comparing the proximal-to-distal distribution of a single 35S-labeled protein within each ON segment after SDS-PAGE. Aliquots of the ON segment homogenates containing equal volumes were resolved on 7.5% SDS-PAGE gels and transferred onto nitrocellulose membranes. The radioactive bands were detected and quantified with a Phosphor Imager (Molecular Dynamics/Amersham, Piscataway, NJ) after 48 hours exposure on Phosphor Imager plates. A mixed-effect model was used to examine the rate of proximal-to-distal decline of the % of the densitometric value of the protein band in the ON segments (Laird and Ware, 1982). The intercept and the regression coefficient for the distance were treated as random effects such that each sample has a unique intercept and regression coefficient for the distance. This statistical procedure accounts for the correlations that are due to the repeated measurements of outcomes in the same animals.
Mice were perfused with 20 ml of PBS after being deeply anesthetized by an i.p. injection of ketamine hydrochloride (1 mg/10 g) and xylazine (0.1 mg/10 g), in accordance with protocols approved by the University of Pennsylvania. The brains of mice were then removed and processed as described (Brunden et al., 2010). Paraffin-embedded tissue blocks were cut into 6-μm thick sections, and were stained with antibodies that recognize: tau phosphorylated at ser202/205 (AT8, 1:7500 dilution) (Goedert et al., 1995);, misfolded tau (MC1, 1:2000 dilution; kind gift from Dr. Peter Davies) (Jicha et al., 1997);, neurons (NeuN, 1:500 dilution; Millipore, Billerica, MA;); synapses (synaptophysin, 1:500; Millipore); and axons (neurofilament light chain (NFL), 1:9000) (Tohyama et al., 1993). Semi-quantitative analysis of tau pathology was performed using the AT8 and MC1 antibodies, utilzing a previously published criteria adapted from the Braak staging system for AD that assesses both the intensity and spatial distribution of tau pathology in Tg mice (Hurtado et al., 2010). The area occupied by NeuN-positive neurons in each mouse was assessed from matched brain sections using 20X images of a defined portion of the hippocampal CA3 region (see Figure 7). The NeuN-positive area was quantified using ImageJ software (National Institutes of Health). Synaptic density was assessed by determining the area of synaptophysin immunoreactivity using matched 20X images within the same CA3 area used for NeuN quantification, with free-hand demarcation of the synaptophysin-positive area as depicted in Figures 8A & 8B. Quantification was with ImageJ software. Finally, hippocampal axonal integrity was evaluated in matched 20X images by determining the area occupied by mossy fibers after free-hand demarcation based on NFL immunostaining, as depicted in Figures 8D and 8E. Quantification was with ImageJ software. All immunostained slides were assessed by a rater who was masked to the treatment group.
Sequentially extracted brain homogenates were prepared as previously described (Yoshiyama et al., 2007). Whole-brain hemispheres were homogenized in 5 μl/mg tissue of cold high-salt (HS) buffer (0.1M MES, 1 mM EGTA, 0.5 mM MgSO4, 0.75M NaCl, 0.02 M NaF, 1 mM PMSF and 0.1% protease inhibitor cocktail), followed by centrifugation at 40,000 × g for 40 min at 4°C. Protein concentration within the supernatant fraction (high-salt soluble) was determined using a BCA assay. The HS-insoluble pellet were homogenized in HS buffer with 1 M sucrose and centrifuged at 85,000 × g for 20 min at 4°C to remove myelin and lipids. The resulting pellets were resuspended in a volume of RIPA (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40 and 5 mM EDTA) that was equal to the original HS buffer amount, and again centrifuged at 85,000 × g for 20 min at 4°C. The supernatants were removed and kept as the RIPA-soluble fraction, and the pellets were treated with 70% formic acid (FA) at a volume equal to 50% of the original HS buffer extract. The FA samples were sonicated and then centrifuged at 13,000 × g for 20 min at 4°C. The FA supernatant fractions were dried in a SpeedVac, and resuspended in RIPA containing 1% SDS at a volume equal to 50% of the original HS buffer extract. For ELISA measurements of total insoluble tau, equal fractions of the RIPA and FA samples from each brain homogenate were combined, and these were identically diluted over 1000-fold in EC buffer (0.02M sodium phosphate pH 7.0, 2 mM EDTA, 0.4 M NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% BlockAce (AbD Serotec, Oxford, UK) and 0.05% NaN3) for analysis in a tau ELISA.
Measurements of tau were made with a sandwich ELISA that is similar to that which has recently been described (Yamada et al., 2011). 384-well plates were coated with Tau5 antibody (5 μg/ml) in 30 μl of cold 0.1 M bicarbonate buffer. After overnight incubation at 4°C, the plates were washed with cold PBS containing 0.05% Tween (PBST), followed by the addition of 100 μl of Block-Ace solution and overnight incubation at 4°C. The blocking solution was removed and the wells were washed with cold PBST. Aliquots (30 μl) of Tau40 (Goedert et al., 1989) standards (37.5–1200 pg/ml) diluted in EC buffer or diluted brain homogenate RIPA + FA samples were added to the wells, and were allowed to incubate overnight at 4°C. The plates were subsequently aspirated and washed with cold PBST, followed by addition of 30 μl/well of reporting antibodies BT2 and HT7 (Thermo Scientific, Rockford, IL) diluted to 125 ng/ml in C buffer (0.02 M sodium phosphate, pH 7.0, 2 mM EDTA, 0.4 M NaCl, 1% bovine serum albumin and 0.05% thimerisol). The plates were incubated overnight at 4°C, followed by washing with cold PBST and addition of 30 μl/well of Streptavidin-HRP (Thermo Fisher, Rockford, IL) that was diluted 1:8000 in C buffer. After a 1 h incubation at 25°C, the plates were washed with PBST, followed by addition of peroxidase substrate solution (30 μl). The reaction was quenched after 5–10 minutes by addition of 30 μl of 10% phosphoric acid. Plates were read on a SpectraMax M5 plate reader at 450 nm. The amount of insoluble tau in the RIPA + FA samples was determined from the tau40 standard curve, and normalized to the average amount of insoluble tau found in the samples from the vehicle-treated PS19 mice.
Blood from PS19 or non-Tg mice was analyzed for complete blood cell counts as previously described (Brunden et al., 2010).
All mice were tested for working memory using spontaneous alternation behavior (SAB) in a Y-maze, as previously described (Rosario et al., 2006), one week prior to sacrifice. Briefly, each animal was placed in the maze and allowed to explore freely for 8 min. SAB score was calculated as the proportion of alternations (an arm choice differing from the previous 2 choices) to the total number of alternation opportunities (total arm entries-2) (King and Arendash, 2002). To assess spatial learning and memory, mice were tested on the Barnes maze (San Diego Instruments; model #7001-0235) essentially as previously described (Brunden et al., 2010). Mice were randomly assigned to different escape compartments and were allowed to habituate to the testing room 1 h prior to testing, and to the maze for 30 sec before each trial in a start compartment. On 5 consecutive testing days, mice were exposed to the Barnes maze for three 2.5 min trials that were 15 min apart. Performance was not scored during the first trial and mice who did not find the hidden escape compartment were gently guided there and allowed to remain for 30 sec. Mice were tested for their ability to learn and remember the fixed position of this escape compartment during trials two and three, and their performance was scored for success rate and the time taken before finding the correct compartment (Harrison et al., 2009; Patil et al., 2009). The maze was cleaned with 70% ethanol in between each animal to minimize odor cues. Statistical analyses of Barnes maze data was as previously described (Brunden et al., 2010).
To assess potential treatment effects on peripheral sensory nerve function, mice were evaluated for tactile allodynia by the Von Frey Test (Pitcher et al., 1999; Smith et al., 2004). Von Frey filaments of increasing diameters ranging from 0.008 to 300 g of force (Bioseb) were placed underneath the hind paw of mice standing on a wire mesh grid to determine the minimum pressure needed to elicit a withdrawal response. Values were determined from the average of five trials.
Prior pharmacokinetic studies revealed that EpoD has a significantly longer residence time in the brain than in plasma of mice (Brunden et al., 2010; Brunden et al., 2011), with the compound being detectable in brain for at least one week after a single 3 mg/kg administration. Moreover, a measurable pharmacodynamic effect, as evidenced by increased levels of a marker of stable MTs, acetyl-tubulin (Black et al., 1989; Laferriere et al., 1997), was apparent one week after a single 3 mg/kg dose of EpoD to normal mice (Brunden et al., 2011). These characteristics led to the selection of EpoD as a preferred MT-stabilizing agent for testing in Tg mouse models of tauopathy (Brunden et al., 2010). Here we have assessed EpoD in an interventional study to address the important question of whether EpoD might improve efficacy outcomes in aged PS19 mice with pre-existing tau pathology, a paradigm that more closely approximates the clinical setting in which AD and other tauopathy patients will first display cognitive symptoms after the onset of tau pathology. In this study, groups of 9-month old male PS19 mice with existing tau pathology (Brunden et al., 2010; Hurtado et al., 2010; Yoshiyama et al., 2007) were administered once-weekly intraperitoneal (IP) doses of either vehicle, 0.3 mg/kg of EpoD or 1.0 mg/kg of EpoD for 3 months, followed by the evaluation of a series of efficacy and safety endpoints. In addition, vehicle-treated 9-month old non-Tg WT littermates were included in the study as a control group.
PS19 mice show an age-dependent increase in axonal dystrophy that is detectable at 3 months of age and becomes very apparent by 6 months (Brunden et al., 2010). In agreement with these previous results, a comparison of the axonal dystrophy within the optic nerves (ONs) of vehicle-treated WT and PS19 mice revealed a significant increase in dystrophic axons in the tau Tg mice (Figure 1). Not surprisingly, the number of dystrophic axons per unit area was increased in the 12-month old PS19 mice relative to the value previously reported for PS19 mice at 6 months of age (Brunden et al., 2010). Interestingly, the incidence of abnormal axons in the 12-month old WT mice also appeared to be increased relative to that measured in 6-month old WT mice (Brunden et al., 2010), suggesting an age-related deterioration. Notably, the 12-month old PS19 mice treated with either dose of EpoD showed a significant decrease in axonal dystrophy, such that the density of abnormal axons was similar to that observed in the vehicle-treated WT mice (Figure 1B). This decrease in the number of degenerating axons presumably resulted from EpoD-mediated effects on MTs, and an evaluation of MT density in the ON (Figure 2) revealed an increase of MTs in the higher dose EpoD-treated PS19 mice that was significantly greater than vehicle-treated PS19 mice (Figure 2C). Although the average MT density in the vehicle-treated 12-month old PS19 mice was reduced relative to that in WT mice (Figure 2C), this difference did not reach statistical significance. The MT density of the 12-month old vehicle-treated PS19 mice is nearly identical to that reported for 6-month old PS19 mice (Brunden et al., 2010); however, the MT number per area in the 12-month old WT mice is somewhat diminished relative to what was observed in 6-month old WT mice. This age-dependent reduction of MT density in the WT mice may cause the increase in axonal dystrophy that was observed when comparing 12- and 6-month old WT mice, and may explain why the difference in MT density between the vehicle-treated PS19 and WT mice did not reach statistical significance.
The increase of MT stabilization and the decrement in dystrophic axons observed in the EpoD-treated PS19 mice suggest that axonal function might also be improved. To examine this, an assessment of FAT was made in ON samples from mice in each treatment group. Briefly, [35S]-methionine was injected into the vitreous of each eye, and each ON was harvested 3 hours later. The extent of FAT was determined by quantifying the proximal-to-distal distribution of a single chosen 35S-labeled protein as quantified by densitometric analysis after SDS-PAGE and autoradiography [see (Zhang et al., 2005) and Figure 3A]. As depicted in Figure 3B, a greater percentage of the total 35S-labeled protein was found in the most proximal segment, and a correspondingly smaller fraction had progressed to the more distal segments, in the vehicle-treated PS19 mice compared to the other cohorts. This reveals an impairment of FAT in the vehicle-treated PS19 mice relative to the WT mice. Importantly, the proximal-to-distal distribution in the PS19 mice treated with either dose of EpoD was nearly indistinguishable from the WT vehicle-treated mice. Thus, the reduction in ON axon damage observed upon EpoD treatment of PS19 mice translated into an improvement of axonal function.
PS19 mice show deficits in spatial learning by 6-months of age (Brunden et al., 2010), and thus the 9-month old PS19 mice that first received EpoD treatment in this study are cognitively impaired. It was therefore of interest to determine whether the behavioral performance of the aged PS19 mice would be affected by 3-months of EpoD treatment. Working memory was analyzed using a Y-maze test (Hughes, 2004; Carroll et al., 2007) in which performance was assessed by determining whether the mice remembered which of the three arms of the maze they previously explored. The vehicle-treated PS19 mice performed more poorly in this task, demonstrating lower spontaneous alterations, than did the corresponding WT mice (Figure 4A). The PS19 mice that received EpoD had improved performance relative to those that received vehicle, with the 1 mg/kg treatment group reaching statistical significance. To examine further the cognitive abilities of the treatment cohorts, the mice also underwent spatial learning analysis in the Barnes maze (Brunden et al., 2010; Harrison et al., 2009). Again, the vehicle-treated PS19 mice showed deficits compared to WT mice, both in their success at correctly identifying the target escape box, as well as in the time required to find the target (Figures 4B & C). The EpoD-treated PS19 mice showed improved spatial learning when compared to the vehicle-treated PS19 mice, with the 1 mg/kg dose group reaching significance in the success endpoint and both EpoD dose groups showing significant improvement in the latency measure (Figures 4B & C). The Y-maze and Barnes maze data reveal that administration of EpoD to aged PS19 mice with existing cognitive deficits can lead to improvements in working and spatial memory, such that overall performance approaches that of comparably-aged WT mice.
The enhancement of MT density and axonal function observed upon EpoD treatment of the aged PS19 mice would not be expected, a priori, to affect tau misfolding and aggregation unless tau pathology is exacerbated by axonal dysfunction. A modest decrease in forebrain phospho-tau (pTau) immunostaining was previously observed when young PS19 mice were treated with EpoD, although this change was not statistically significant (Brunden et al., 2010). An analysis of pTau (pS202/pT205) in the aged PS19 mice, using an immunohistochemical rating scale to monitor both the intensity and distribution of immunostaining (Hurtado et al., 2010), revealed a decrease of pTau in EpoD-treated PS19 mice relative to those receiving vehicle (Figure 5A), which reached significance at the higher EpoD dose (Figure 5C). To confirm that there was a reduction of pathological tau in the EpoD-treated PS19 mice, immunohistochemistry was also performed with the MC1 antibody that recognizes misfolded tau (Jicha et al., 1997). Again, both EpoD groups demonstrated less staining than did the vehicle group (Figure 5B), with the higher dose of EpoD reaching statistical significance (Figure 5D). Finally, the amount of pathological tau was also evaluated in the vehicle- and EpoD-treated PS19 mice by ELISA measurement of high salt buffer-insoluble tau in hemi-brain homogenates. In accordance with the immunohistochemistry results, the amount of high salt buffer-insoluble tau that was detected with the ELISA, which measures both phosphorylated and non-phosphorylated tau, was found to be decreased in the PS19 mice receiving EpoD, with the 1 mg/kg treatment result being statistically significant (Figure 5E). The correspondence between the immunohistochemical and biochemical analyses of pathologic tau was further confirmed by comparing for each mouse the semi-quantitative scores assigned from the immunostaining methodologies with the relative amount of insoluble tau as determined by ELISA. As depicted in Figure 6, there was a strong and highly significant correlation between the immunohistochemical ratings and the insoluble tau level for each animal. Thus, these data indicate that EpoD treatment of the PS19 mice resulted in diminished tau pathology.
The observed reduction of tau pathology in the PS19 mice receiving EpoD, coupled with the improvement of axonal function, suggested that these mice might have less hippocampal neuron loss than is normally seen in these mice (Yoshiyama et al., 2007). An example of this hippocampal neuron death can be observed by comparing NeuN-stained sections from 12-month old WT (Figure 7A) and PS19 mice (Figure 7B). Quantification of NeuN area in the CA3 region of vehicle-treated WT and PS19 mice revealed a significant reduction of neurons in the latter group (Figure 7C). The PS19 mice that received EpoD demonstrated a dose-dependent increase of CA3 neurons relative to the vehicle-treated group which reached statistical significance at the 1 mg/kg dose (Figure 7C). A similar comparison of CA3 synaptic density, as measured by synaptophysin immunostaining, revealed a trend toward a loss of synapses in the 12-month old PS19 mice (Figures 8A–C), and both doses of EpoD caused an significant increase of synaptic area in the PS19 mice when compared to those receiving vehicle (Figure 8C). Finally, hippocampal axonal density was assessed by determining the area within a defined CA3 region occupied by mossy fibers, as visualized by neurofilament light chain (NFL) immunostaining (Figures 8D & E). A loss of mossy fibers was observed in the vehicle-treated PS19 mice relative to WT littermates, and EpoD treatment prevented this axonal loss, with the higher compound dose reaching significance. Thus, EpoD treatment reduced several measures of neurotoxicity in the 12-month old PS19 mice.
The data described above reveal that EpoD can significantly improve functional outcomes, reduce tau pathology and prevent neurotoxicity in tau Tg mice when they are treated in an interventional setting. This suggests that EpoD, which has previously advanced to Phase II clinical testing for oncology, might have utility for the treatment of AD and related tauopathies. However, MT-stabilizing agents are known to induce side-effects when used at the doses required for the treatment of cancer. Hence, it is important to note that the aged PS19 mice in this study received EpoD doses that were 30- and 100-fold less, on a mg/m2 basis, than was previously utilized in human testing (Beer et al., 2007). Thus, the substantially lower EpoD doses used here could attenuate the adverse complications associated with MT-stabilizing drugs, such as immune cell depletion and the onset of peripheral neuropathy (Bedard et al., 2010; Cheng et al., 2008; Cortes and Baselga, 2007). The PS19 mice receiving EpoD in the present study had body weights at the end of the dosing period that were indistinguishable from vehicle-treated PS19 or WT mice (Figure 9A), indicating an absence of gross toxicities. Moreover, there were no significant differences in major organ weights among the study cohorts, including brain weight (Figure 9B). An evaluation of complete blood cell counts from the different treatment groups demonstrated that there was no reduction of total white blood cells (WBCs) in the EpoD-treated PS19 mice relative to either the PS19 or WT mice that received vehicle (Figure 9C). Similarly, there were no alterations in the neutrophil content among WBCs in the EpoD-treated mice (Figure 9C). Thus, there is no evidence of immune suppression or neutropenia in the mice that received either of the EpoD doses. Finally, peripheral nerve sensory function was evaluated using the von Frey test to measure the sensitivity of the mouse foot pad to sensory stimuli by determining the force required to elicit a withdrawal response (Smith et al., 2004). As summarized in Figure 9D, neither of the EpoD-treated cohorts showed any change in foot pad sensitivity relative to the vehicle-treated PS19 mice. Therefore, it appears that the low doses of EpoD used in this study, which were sufficient to improve efficacy outcomes, elicited none of the adverse events that are observed when higher doses of MT-stabilizing agents are employed for the treatment of cancer.
There is growing evidence that the hyperphosphorylation and misfolding of tau contributes to neurodegenerative processes in AD and other tauopathies through both gain-of-function and loss-of-function toxicities (Brunden et al., 2009; Ballatore et al., 2007; Lee et al., 2001b). Although there may be multiple mechanisms of tau-mediated neuron damage, there is evidence in AD patients of a decline of normal tau function. This includes data which demonstrate a reduction in the number and length of MTs in neurons within the AD brain (Cash et al., 2003), decreased levels of MT-binding competent tau in affected regions of AD brains (Bramblett et al., 1992), and diminished acetylated tubulin in AD neurons (Hempen and Brion, 1996). Moreover, decreased MT density has been observed in Tg mouse models of tauopathy (Ishihara et al., 1999; Zhang et al., 2005), including the PS19 mice that were utilized in the study presented here (Brunden et al., 2010). Tau Tg mouse models have proven to be valuable in testing the hypothesis that MT-stabilizing drugs that are used in the treatment of cancer can compensate for an alteration of tau-mediated stabilization of MTs. For example, paclitaxel improved MT density, axonal function and motor performance after absorption at neuromuscular junctions in another tau Tg mouse model that develops tau inclusions primarily within the brain stem and spinal cord (Zhang et al., 2005). Because paclitaxel does not effectively cross the BBB (Brunden et al., 2011; Fellner et al., 2002), it is not suitable for the treatment of human tauopathies where forebrain tau inclusions predominate. More recently, we demonstrated that prophylactic administration of the brain-penetrant MT-stabilizing agent, EpoD, alleviated the onset of MT deficits and axonal dystrophy in young PS19 mice, resulting in improvements of cognitive performance (Brunden et al., 2010).
Here, we have confirmed and substantially extended these previous studies by examining the safety and efficacy of EpoD in an interventional treatment paradigm, using aged PS19 mice with existing tau pathology. This study design was critical for demonstrating that a BBB-permeable MT-stabilizing agent could decrease disease in a tauopathy model that more closely approximates the symptomatic patients who are candidates for therapeutic treatment. Although significant advances are being made in the ability to diagnose patients before the onset of advanced disease, it is likely that those at the prodromal stages of disease progression (i.e., mild cognitive impairment in AD patients) already have substantial tau pathology (Jack et al., 2010). Therefore, the determination that very low EpoD doses can safely and effectively mitigate axonal deficits and improve cognitive performance in aged PS19 mice with pre-existing tau pathology provides critical evidence that such a therapeutic strategy might ameliorate disease in human tauopathy patients.
Although EpoD was demonstrated to increase MT density in aged PS19 mice, the possibility exists that the drug-induced improvements of FAT, cognitive performance, tau pathology and neuronal health also resulted from effects that are not directly attributable to tau loss-of-function. For example, tau over-expression in cultured neurons has been shown to reduce the engagement of the kinesin motor proteins required for anterograde FAT (Stamer et al., 2002; Vershinin et al., 2007; Dixit et al., 2008). Since there is evidence that tau binding to MTs is decreased by paclitaxel (Kar et al., 2003; Samsonov et al., 2004), and because EpoD shares a common MT-binding site with paclitaxel (Bollag et al., 1995), it is conceivable that EpoD may increase FAT in the PS19 mice by decreasing tau binding to MTs and thus enhancing kinesin interaction with MTs. It is also possible that tau over-expression in the PS19 mice could result in MT over-stabilization, with EpoD normalizing MT stability by decreasing tau binding to MTs. However, it should be noted that the modest decrease in MT density observed in the PS19 mice relative to WT mice [(Brunden et al., 2010) and present study] would argue that the extent of tau bound to MTs in the Tg mice is below normal levels, and that compensation for tau loss-of-function is the most parsimonious explanation for the beneficial effects of EpoD.
A somewhat unexpected finding from our study was the reduction of tau pathology after EpoD treatment, as evidenced by decreased immunohistochemical staining with antibodies that recognize a major phospho-tau epitope (pS202/pT205) and misfolded tau, as well as by a reduction of insoluble tau. How a change of MT stabilization or an enhancement of FAT might reduce the amount of hyperphosphorylated and misfolded tau in the PS19 mice is unclear, although a similar reduction of tau pathology was observed after intranasal administration of the pleiotropic peptidic MT-stabilizing agent, NAP (Matsuoka et al., 2007; Matsuoka et al., 2008). We have examined whether EpoD has a direct effect on tau fibrillization, using a well-characterized in vitro assay (Crowe et al., 2009), and find that the compound has essentially no effect on tau assembly (data not shown). Although it is impossible to definitively exclude the possibility of off-target effects of EpoD that might lead to increased degradation of misfolded tau, it is interesting to note that kinesin-1 deficient mice develop dystrophic axons with increased amounts of hyperphosphorylated tau (Falzone et al., 2009). Moreover, JNPL3 tau Tg mice crossed to kinesin light chain knockout mice have an exacerbation of tau pathology (Falzone et al., 2010). Thus, it appears that impaired FAT can lead to an enhancement of tau pathology, perhaps through JNK pathway activation (Falzone et al., 2009). These findings suggest that a vicious cycle may develop in tauopathies, whereby tau loss-of-function results both in MT destabilization and FAT deficits that can then further enhance tau pathology. This may explain why EpoD treatment prevents the development of additional tau pathology in the aged PS19 mice, as the compound improves FAT, and thus attenuates this possible feed-forward mechanism.
In addition to reducing tau pathology, EpoD treatment also prevented the loss of hippocampal neurons, axons and synapses in the PS19 mice. The improvements in neuronal measures, along with increased axonal transport, are consistent with the enhanced cognitive performance of the EpoD-treated PS19 mice. It is difficult to separate the absolute contributions of improved axonal transport, increased synaptic density and greater neuronal survival to the observed learning and memory improvements in the EpoD-treated mice, as all of these parameters may be interrelated.
The efficacious properties of EpoD in this interventional study suggest that this compound, which progressed to Phase II clinical testing in cancer (Beer et al., 2007), might be a possible clinical candidate for the treatment of tauopathies. However, MT-stabilizing agents have been plagued by side-effects in oncology settings (Bedard et al., 2010), and there is a concern that such drugs would not be tolerated when dosed on a chronic basis, as would likely be required for neurodegenerative diseases. For this reason, we have carefully monitored the mice in this study for potential drug-related adverse events. Notably, we did not observe any side-effects after 3-months of weekly EpoD administration to aged PS19 mice, with the compound-treated mice showing normal body weight gain and no signs of organ toxicity. Moreover, the mice receiving EpoD did not show alterations of neutrophil counts or evidence of peripheral neuropathy, complications that are typical of this class of drug in cancer patients (Bedard et al., 2010; Cortes and Baselga, 2007; Cheng et al., 2008). The absence of EpoD-related side-effects in this study presumably relates to the low doses that were utilized, which are 30–100X less than used in cancer patients (Beer et al., 2007). To the extent that the PS19 mice accurately model human tauopathy, our data would suggest that much lower concentrations of EpoD are required to overcome tau loss-of-function than are needed to inhibit the growth of rapidly dividing cancer cells. We should also note that the unique pharmacokinetic and tissue distribution properties of EpoD likely contribute to the favorable efficacy-to-safety margin (i.e., therapeutic index). In particular, the retention of EpoD in the brain long after the compound has been cleared from the blood (Brunden et al., 2010; Brunden et al., 2011) appears to provide for prolonged MT stabilization in the brain while minimizing peripheral side-effects. As a comparison, we have also examined in PS19 mice the drug ixabepilone (Ixempra™), which is another member of the epothilone family of MT-stabilizing drugs that is currently approved for the treatment of metastatic breast cancer (Hunt, 2009). Although brain-penetrant, the plasma levels of ixabepilone are higher than those of EpoD at the same drug dose (Brunden et al., 2011). A subset of PS19 mice receiving a weekly 4 mg/kg dose of this drug began to show significant body weight loss after 6–8 weeks of treatment, with some animals dying (data not shown). In contrast, we previously demonstrated that a weekly 3 mg/kg EpoD dose administered over a 12-week period was well-tolerated (Brunden et al., 2010).
The combined safety and efficacy of EpoD in a mouse model with pre-existing tau pathology suggests that this compound merits consideration as a candidate for clinical testing in tauopathy patients. The results obtained with EpoD also provide impetus to identify additional BBB-permeable MT-stabilizing agents that might have an even greater therapeutic index. Finally, EpoD and related drug candidates might also have utility in other neurodegenerative conditions, particularly in Parkinson’s disease (PD) where tau pathology can be found (Kalaitzakis and Pearce, 2009) and where genome-wide association studies have identified a significant association between PD and the tau gene locus (Simon-Sanchez et al., 2009; Edwards et al., 2010).
The authors thank Biao Zuo of the Biomedical Imaging Core Facility of the University of Pennsylvania for his assistance. These studies were supported by NIH grant U01AG029213, and in part by the Marian S. Ware Alzheimer Program, the Karen Cohen Segal and Christopher S. Segal Alzheimer Drug Discovery Initiative Fund, the Paula C. Schmerler Fund for Alzheimer’s Research, the Barrist Neurodegenerative Disease Research Fund, the Eleanor Margaret Kurtz Endowed Fund, the Mary Rasmus Endowed Fund for Alzheimer’s Research, Mrs. Gloria J. Miller and Arthur Peck, M.D. Drs. Brunden, Lee and Trojanowski collaborate with Bristol-Myers Squibb, which has certain intellectual property rights to epothilone D, on unrelated research studies.