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Published as doi: 10.1096/fj.08-113795.
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(The FASEB Journal. 2009;23:739-750.)
© 2009 FASEB

Minocycline reduces the development of abnormal tau species in models of Alzheimer’s disease

Wendy Noble*,1, Claire Garwood*, John Stephenson*, Anna M. Kinsey{dagger}, Diane P. Hanger* and Brian H. Anderton*

* Department of Neuroscience and

{dagger} Division of Old Age Psychiatry, MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry, London, UK

1Correspondence: MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry, Department of Neuroscience, PO37, De Crespigny Park, London, SE5 8AF, UK. E-mail: wendy.noble{at}iop.kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer’s disease (AD) is characterized by the presence of neurofibrillary tangles of hyperphosphorylated, aggregated tau protein and extracellular deposits of β-amyloid peptide. Increased β-amyloid levels are thought to precede tangle formation, but tau pathology is more closely related to neuronal death. Minocycline, a tetracycline derivative, has potent antiinflammatory, antiapoptotic, and neuroprotective effects in several models of neurodegenerative disease, including models of AD with amyloid pathology. We have used both in vitro and in vivo models of AD to determine whether minocycline may have therapeutic efficacy against tau pathology. In primary cortical neurons, minocycline prevents β-amyloid-induced neuronal death, reduces caspase-3 activation, and lowers generation of caspase-3-cleaved tau fragments. Treatment of tangle-forming transgenic mice (htau line) with minocycline results in reduced levels of tau phosphorylation and insoluble tau aggregates. The in vivo effects of minocycline are also associated with reduced caspase-3 activation and lowered tau cleavage by caspase-3. In tau mice, we find that conformational changes in tau are susceptible to minocycline treatment, but are not directly associated with the amount of tau fragments produced, highlighting a dissociation between the development of these pathological tau species. These results suggest a possible novel therapeutic role for minocycline in the treatment of AD and related tauopathies.—Noble, W., Garwood, C., Stephenson, J., Kinsey, A. M., Hanger, D. P., Anderton, B. H. Minocycline reduces the development of abnormal tau species in models of Alzheimer’s disease.


Key Words: caspase-3 • therapeutic • neurodegeneration • tauopathy • β-amyloid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALZHEIMER’S DISEASE (AD) IS CHARACTERIZED by deposits of β-amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated tau, synaptic dysfunction, and neuronal death. In AD, abnormal tau phosphorylation and/or processing are thought to occur as a result of elevated brain Aβ (1) . While the presence and density of NFTs correlates with cognitive decline in AD (2 , 3) , recent evidence indicates that NFTs are not neurotoxic, but that an earlier, abnormal tau species induces neurodegeneration (4 , 5) . C-terminal truncation of tau as a result of caspase-3-mediated cleavage is an early event in AD pathogenesis (6) , and tau fragments generated by cleavage have a propensity to aggregate and display toxic properties (7 , 8) , suggesting that caspase cleavage of tau may be a key early pathogenic process during neurodegeneration.

Caspases are a family of cysteine proteases closely associated with induction of the apoptotic cascade (9) . The specific role of caspases in AD remains unclear; however, in AD a number of caspases are activated in the brain, where they have been shown to cleave amyloid protein precursor (APP), presenilin, actin, fodrin, and tau (10 , 11) . Aβ activates caspase-3, which cleaves tau at aspartate (D) 451 to generate fragments with proapoptotic properties in vitro (12 , 13) , with tau cleavage thought to precede its hyperphosphorylation (11 , 14) . Furthermore, increased amounts of cleaved tau fragments ({Delta}tau) correlate with the clinical progression of AD (14) . Thus, inhibition of caspase-mediated tau cleavage may be a relevant target for therapeutic intervention.

Minocycline, a tetracycline derivative, has potent antiinflammatory, antiapoptotic, and neuroprotective properties. Minocycline readily crosses the blood-brain barrier and effectively delays disease progression and reduces neuronal death in mouse models of amyotrophic lateral sclerosis (15) , Huntington’s disease (16) , and Parkinson’s disease (17) . In many cases the neuroprotective properties of minocycline have been attributed to inhibition of caspases.

Recently, minocycline was shown to protect against Aβ-induced cell death and prevent fibrillization of Aβ in vitro (18) , prevent Aβ deposition and cognitive decline in APP transgenic mice (19) , and inhibit neuronal death and attenuate learning and memory deficits following administration of Aβ to rats (20 , 21) . However, as yet the effects on tau pathology remain unstudied.

Here we have investigated the neuroprotective effects of minocycline in tangle-forming tau transgenic mice (htau), in addition to further investigating the in vitro effects of minocycline. Htau mice progressively develop hyperphosphorylated tau and NFTs in the cortex and hippocampus, and widespread neuronal loss is apparent in aged mice (22) . We find that minocycline reduces Aβ-induced neuronal death in primary neuronal cultures and that this is associated with reduced caspase-3 activation and caspase-3-mediated tau cleavage. These effects are associated with an inhibitory effect of minocycline on cytochrome c release from mitochondria. Treatment of 2 groups of htau mice with minocycline results in inhibition of insoluble tau aggregate levels and tau phosphorylation. The reduction in tau pathology correlates with caspase-3 inhibition and reduced caspase-3 cleavage of tau. Importantly, minocycline reduced the amount of abnormal tau species in both young and aged mice. These results suggest that minocycline may have therapeutic benefit for the treatment of AD and related tauopathies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All reagents were obtained from Sigma (Poole, Dorset, UK) unless otherwise stated.

Primary neuronal cultures and cell treatments
Cortical neurons were obtained from embryonic day 18 (E18) rat embryos and cultured as described previously (23) . Neurons were pretreated with 10 µM minocycline for 24 h prior to the addition of 0.5 µM staurosporine, 100 µM glutamate, 10 µM soluble oligomeric Aβ1-42 (California Peptide Co., Napa, CA, USA), or 0.5 µM ionomycin. Predominantly oligomeric, soluble Aβ1-42 was prepared and stored prior to use according to the method described in Town et al. (24) , a preparation which reliably induces caspase activation.

Preparation of RIPA-extracted neuronal lysates
After cell treatments, the medium was removed and retained while cells were washed in ice-cold PBS followed by lysis in modified RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% (w/v) Nonidet P-40, 0.25% (w/v) sodium deoxycholate, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulphonylfluoride (PMSF), 1 mM sodium orthovanadate, 1 mM sodium fluoride, and protease inhibitor cocktail for mammalian tissues] and centrifugation at 25,000 g for 20 min at 4°C. The protein concentration of supernatants was measured using a BCA protein assay kit (Pierce Endogen, Rockford, IL, USA) and standardized. Protein (5–20 µg) was loaded for Western blot analysis. Each experiment was repeated 3 times with triplicate samples. Representative blots are shown, while bar charts give average data from all 3 experiments.

Toxicity assays
Cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) release in 50 µl collected culture medium using Cytotox 96 assay kits (Promega, Madison, WI, USA) according to the manufacturer’s directions. Each experiment was repeated 3 times (6 replicate wells/experiment); representative data are shown. Optical density was measured at 492 nm with a spectrophotometer. For each experiment, LDH release from untreated wells was measured and designated as time point 0. Samples of culture medium from vehicle-treated neurons served as a control at each time point. Values shown represent LDH release from cells into the medium (arbitrary photometric units).

Cytochrome c release assay
Cytochrome c release from mitochondria was measured using a cytochrome c release apoptosis assay kit (QIA87, EMD Biosciences Inc., San Diego, CA, USA). Primary cortical neurons were prepared and treated as described above. Neurons (9.5x106/treatment condition) were collected in ice-cold PBS by centrifugation at 600 gav for 5 min. Following removal of the supernatant, neurons were resuspended in 0.1 ml cytosol extraction buffer and incubated on ice for 10 min. Neurons were homogenized, followed by centrifugation at 700 gav for 10 min. The supernatant was collected and further centrifuged at 10,000 gav for 30 min. All centrifugation steps were performed at 4°C. The resulting pellet was resuspended in mitochondrial extraction buffer and run on a 12% SDS-PAGE gel, as described below. Tissue from wild-type and transgenic mice was homogenized at 200 mg/ml (w/v) in cytosol extraction buffer and processed as detailed above. The final mitochondria-containing pellet was resuspended in 200 µl mitochondrial extraction buffer. All blots were probed with an anti-cytochrome c antibody (mouse IgG2b; EMD Biosciences).

Treatment of htau transgenic mice with minocycline
Tau transgenic mice (htau line) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Two groups of htau mice were examined: 1) 3–4 months old, n = 5 (vehicle), n = 8 (minocycline); and 2) 12 months old, n = 8 (vehicle), n = 9 (minocycline). In addition, 2 age-matched control groups of wild-type mice were used (n=8). All mice received intraperitoneal injections of 10 mg/kg minocycline hydrochloride in PBS daily for 14 days. According to the methods of the Friedlander group (15 , 25 , 26) , minocycline was freshly prepared each day. Control animals were administered sterile PBS. Mice were sacrificed by cervical dislocation 2 h after the final injection. Brain regions were dissected and immediately snap-frozen on dry ice.

Preparation of RIPA-extracted brain proteins
Frozen hippocampal tissue was homogenized in modified RIPA buffer using a mechanical homogenizer (Polytron; Fisher Scientific Ltd., Loughborough, UK) at 100 mg/ml (w/v). Homogenates were centrifuged at 20,000 gav for 20 min at 4°C, and supernatants were collected. Protein concentrations of the final supernatants were determined using a BCA protein assay kit (Pierce Endogen), and RIPA-extracted proteins (5–20 µg protein) were analyzed on Western blots, as described below.

Isolation of aggregated tau
Sarkosyl extractions were performed on cortical tissue using a method adapted from that of Greenberg and Davies (27) , resulting in the generation of 3 fractions: S1, the low speed supernatant prior to addition of sarkosyl; S2, the sarkosyl-soluble tau; and P3, the sarkosyl-insoluble tau fraction as described by Kelleher et al. (28) . For quantification, the amount of tau present in the S1 fraction was standardized to β-actin and the amount of P3 tau standardized against that in the S1 fraction.

Semiquantitative tau ELISA
Tissue was homogenized in 50 mM TBS, pH 7.4, containing 2 mM EGTA, 1 mM PMSF, 10 mM sodium fluoride, and 1 mM sodium orthovanadate at 100 mg/ml (w/v). Homogenates were centrifuged at 20,000 gav for 20 min at 4°C, and the amount of tau in the resulting supernatants was determined by ELISA, as described previously (29) . The amount of tau in abnormal conformations (MC1) was calculated following normalization to total tau (TG5).

Immunoblotting
Proteins were separated on 10% (w/v) SDS-PAGE gels and electrophoretically transferred to nitrocellulose membrane. After blocking with 5% (w/v) dry milk for 1 h, membranes were probed with primary antibodies, detected using fluorophore-coupled secondary antibodies, and visualized, then quantified, using the Odyssey infrared imaging system (Li-Cor Biosciences, Cambridge, UK).

Antibodies
The following primary antibodies were used, with specificity, isotype, and source given in parentheses: Tau, nonphosphorylation dependent tau, Dako (rabbit polyclonal; Dako Ltd., Ely, UK); Tau-1 (dephosphorylated tau at Ser-199/202/205, mouse monoclonal; Chemicon, Millipore, Watford, UK); Tau-C3 (truncated at Asp421, mouse IgG1; Chemicon). Caspases used were cleaved caspase-3 (Asp175, rabbit IgG; New England Biolabs, Ipswich, MA, USA); cleaved caspase-8 (clone IIG10, Asp384, mouse IgG; Calbiochem, Merck, Darmstadt, Germany); cleaved caspase-9 (clone 96-222, mouse IgG; Abcam, Cambridge, MA, USA). The following tau antibodies were kindly gifted by Peter Davies (Albert Einstein College of Medicine, Bronx, NY, USA): TG5 (residues 220-240, mouse IgG1); CP13 (phospho-Ser-202; mouse IgG1); PHF-1 (phospho-Ser-396/404; mouse IgG1); MC1 (recognizes conformational change around residues 5-15 and 312-322, mouse IgG1); and TG3 (recognizes conformational change and phosphorylation around residue Thr231, mouse IgM).

Statistical analysis
Paired analyses were performed between littermates in vehicle- and minocycline-treated groups. Changes in tau phosphorylation, aggregation, and conformation were analyzed by ANOVA and multiple comparison procedures of the Tukey test. Correlation coefficients and significance were established by nonparametric, 2-tailed, Spearman {rho} tests using SPSS (v15.0; SPSS Inc., Chicago, IL, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Minocycline protects against neurotoxicity in vitro
Minocycline can protect against caspase-induced neurotoxicity in vitro (15) . We began by testing its effectiveness in our model system using staurosporine to activate caspase-dependent apoptotic pathways (30) . Rat cortical neurons were treated with staurosporine (0.5 µM) for 2 min to 24 h, resulting in significant neuronal death from 1 h onwards (P<0.05), as indicated by the increased amount of LDH released into culture medium. Pretreatment of cultures with minocycline (10 µM) for 24 h blocked staurosporine-induced neuronal death at all time points (Fig. 1A ), supporting the idea that minocycline inhibits caspase-induced neurotoxicity.


Figure 1
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  		Figure 1. Minocycline prevents caspase-mediated neuronal death. LDH release was measured in rat primary neuronal cultures exposed to 0.5 µM staurosporine (A), 100 µM glutamate (B), 10 µM Aβ1-42 (C), or 0.5 µM ionomycin (D), with or without pretreatment with 10 µM minocycline for 24 h. LDH release from untreated wells was measured for each experiment (time point 0). Samples of culture medium from vehicle-treated neurons served as a control at each time point. Values represent LDH release from cells into the medium (arbitrary photometric units). *P < 0.05 vs. control (vehicle treated); {diamondsuit}P < 0.05, protective effect of minocycline. Error bars = SEM. Six replicate wells/experiment; n = 3.

In AD, increased glutamate and/or Aβ levels are proposed as primary triggers for neurodegeneration (31) . Since both glutamate and Aβ activate caspases and induce cell death in vitro (32) we sought to determine whether minocycline could also prevent glutamate- and Aβ-induced toxicity. Treatment of neurons with glutamate (200 µM) resulted in significant neuronal death after 20 and 60 min (P<0.05 and P<0.01, respectively), while neurons pretreated with minocycline were resistant to glutamate-induced neurotoxicity (Fig. 1B ; P<0.05 at 20 min, P<0.001 at 60 min). Similar results were obtained following treatment of neurons with 10 µM Aβ1-42. A significant degree of neurotoxicity was apparent following incubation of neurons with Aβ1-42 for 48 and 72 h (P<0.05 for 48 h, P<0.001 for 72 h). Toxicity was suppressed in cultures pretreated with minocycline (Fig. 1C ; P<0.05 for 48 h, P<0.001 for 72 h). These results demonstrate that minocycline can prevent glutamate- and Aβ-induced neuronal death in vitro.

Because these toxic agents are all reported to increase intracellular calcium (Fig. 1A-C ), we then determined whether the neuroprotective effects of minocycline are mediated by inhibiting calcium-induced toxicity. Treatment of neurons with the calcium ionophore, ionomycin (0.5 µM), resulted in significant neuronal death 60 min after application (Fig. 1D ). However, ionomycin-induced neurotoxicity was unaltered in the presence of minocycline. This indicates that minocycline-mediated neuroprotection involves specific targets, including caspases, rather than preventing toxicity arising from general increases in intracellular calcium concentration.

Minocycline-mediated neuroprotection against Aβ toxicity is associated with decreased caspase activation and reduced caspase cleavage of tau
To investigate whether minocycline protects against Aβ-induced neurotoxicity by inhibiting caspase activation we analyzed cell lysates from Aβ-treated neurons on Western blots probed with antibodies to tau, {alpha}-spectrin, cleaved caspase-3, and caspase-3- cleaved tau (Tau C-3; Fig. 2 ). Neurons were treated with 10 µM Aβ1-42 in he presence or absence of 10 µM minocycline for up to 72 h.


Figure 2
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  		Figure 2. Minocycline reduces Aβ-induced caspase-3 activation and tau cleavage. Western blots of cell lysates from rat primary cortical neurons exposed to 10 µM Aβ for various times up to 72 h ± 10 µM minocycline. A, B) Blots were probed with antibodies against β-actin as a loading control and tau (Dako) (A), and with antibodies against {alpha}-spectrin, caspase-3, and caspase-3-cleaved tau (Tau C-3) (B). C) Following treatment of primary neurons with 10 µM Aβ for 48 h, amount of cytochrome c present in mitochondrial and cytoplasmic fractions was quantified by Western blotting. D) Bar chart illustrates effect of Aβ on cleaved caspase-3, showing increased activation of caspase-3 with time and partial prevention after preincubation with minocycline. E) Bar chart illustrates increased caspase-3-cleaved tau amounts with Aβ incubation and minocycline-mediated reductions in cleaved fragments. F) Bar chart illustrates amount of cytochrome c protein present in mitochondria and cytoplasm of treated neurons. Aβ treatment significantly increases cytochrome c release from mitochondria; minocycline reduces this effect. Data are representative from 3 experiments (3 replicates/experiment); bar charts present average ± SEM from all 3 experiments. *P < 0.05, **P < 0.01.

At 48 and 72 h, a small decrease in tau mobility on SDS-PAGE gels was apparent when blots were probed with the Dako antibody. β-Actin was used to ensure equal protein loading on gels (Fig. 2A ).

Caspase cleavage of {alpha}-spectrin generates fragments of 120 and 150 kDa, while calpain cleavage results in a 150-kDa {alpha}-spectrin fragment. Aβ1-42 treatment for 48 and 72 h induced increased amounts of 120- and 150-kDa {alpha}-spectrin fragments, concomitant with a decrease in the full-length protein (240 kDa), indicating increased caspase (and possibly calpain) activation. In cultures pretreated with minocycline, reduced cleavage of {alpha}-spectrin was apparent at both 48 and 72 h (Fig. 2B ). Caspase-3 is activated by cleavage, generating fragments of ~19 and 17 kDa. In untreated primary rat cortical cultures, we found fragments of ~19, 17, and 15 kDa. Significantly increased amounts of both the 19- and 17-kDa cleavage products were apparent from 24 h in Aβ1-42-treated neurons, indicating increased caspase-3 activation (Fig. 2D ). These results demonstrate that caspase-3 activation precedes Aβ-induced neuronal death. In cultures pretreated with minocycline, there was a significant reduction in the amount of cleaved caspase-3 (Fig, 2B, D ), strongly suggesting that minocycline inhibits Aβ-mediated caspase-3 activation and that this may be the mechanism by which minocycline prevents Aβ-induced neurotoxicity.

Tau is cleaved by caspase-3 to generate proaggregation, toxic fragments (13) . We used an antibody specifically recognizing tau cleaved by caspase-3 at D421 (Tau C-3), and in control cultures detected a small amount of caspase-cleaved tau of ~50 kDa (Fig. 2B ). From 24 h after incubation with Aβ, the amount of tau fragments detected increased, with highly significant elevations apparent from 48 h onwards. Neurons pretreated with minocycline contained significantly reduced amounts of caspase-3-cleaved tau fragments after 24, 48, and 72 h (Fig. 2B, E ; P<0.05, P<0.01, and P<0.05, respectively). These results indicate that minocycline-mediated caspase-3 inhibition is sufficient to reduce Aβ-induced caspase-3 cleavage of tau.

Minocycline has previously been shown to inhibit caspase-3 activity by preventing cytochrome c release from mitochondria. Following 48 h treatment with Aβ, we detected a significant decrease in the amount of cytochrome c detected in mitochondria, suggesting that Aβ increases the release of cytochrome c from mitochondria into the cytoplasm. Pretreatment of neurons with minocycline increased the retention of cytochrome c in mitochondria (Fig. 2C, F ). Similarly, increased amounts of cytochrome c were detected in the cytoplasmic fraction of neurons following treatment with Aβ. This was reduced in the presence of minocycline (Fig. 2C, F ). This supports published data that inhibition of cytochrome c release is a primary target of minocycline and that this is sufficient to reduce activation of caspases downstream in the apoptotic cascade (15) .

Minocycline reduces the generation of caspase-cleaved tau fragments in vivo
Since our in vitro results demonstrate that minocycline can prevent disease-associated changes in tau, we have examined its effects in wild-type and tangle-forming htau mice.

Minocycline has previously been shown to inhibit caspase-3 activity by preventing cytochrome c release from mitochondria and thus preventing activation of caspase-9, upstream of caspase-3 in the apoptotic caspase cascade. Caspase-8 is upstream of cytochrome c and is not a reported target of minocycline. Therefore, we first measured the amount of cleaved (active) caspase-3, -8, and -9 in lysates prepared from the hippocampus of minocycline- and vehicle-treated mice (Fig. 3 ). In all groups, we found a significant reduction in the amount of cleaved caspase-3 following minocycline treatment (Fig. 3A, B ), indicating that minocycline reduces caspase-3 activation both in vitro and in vivo. We also found reduced caspase-9 cleavage in minocycline treated mice, whereas caspase-8 activity was unchanged (Fig. 3A, B ).


Figure 3
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  		Figure 3. Minocycline prevents caspase-3 activation and tau cleavage in vivo. Amount of cleaved (active) caspases and degree of caspase-3-mediated cleavage of tau were examined in cortex of htau mice following minocycline treatment. A) Western blotting of cortical protein extracts from 3- to 4-month old htau (n=5 vehicle, n=8 minocycline) and wild-type mice (n=8) using antibodies specific for cleaved caspase-8, -9, and -3 and for caspase-cleaved tau (Tau C-3). Bar chart shows reduction in amounts of both the 50-kDa doublet and 23- to 28-kDa tau fragments after minocycline treatment of htau mice, as well as reduced cleaved (active) caspase-3. B) Western blot shows caspase activation and tau cleavage in 12-month-old htau (n=8 vehicle, n=9 minocycline) and wild-type mice (n=8). Bar chart shows amounts of cleaved caspase-3 and tau fragments present, illustrating that caspase cleavage of tau is reduced by minocycline. C) Amount of cytochrome c present in wild-type and htau mice was assessed by Western blotting following protein fractionation. Minocycline treatment increased retention of cytochrome c in mitochondria of all groups. Error bars = SEM. *P < 0.05.

We next examined the amount of caspase-cleaved tau in htau mice, and observed bands at ~23 and 28 kDa, together with a doublet of ~50 kDa in hippocampal lysates (Fig. 3A, B ). In both age groups, we detected significant decreases in all tau fragments following minocycline treatment. These results support our in vitro data and suggest that, by reducing caspase-3 activity, minocycline can effectively reduce the generation of caspase-3-cleaved tau fragments. Wild-type mice were found to contain very low levels of caspase-3-cleaved tau, and these results were not quantified.

We then extracted cytoplasmic and mitochondrial proteins from the amygdala of 12-month-old wild-type mice and both ages of htau mice and assessed the amount of cytochrome c present in these fractions. The total amount of cytochrome c measured showed no significant difference between wild-type and htau mice (Fig. 3C ). Minocycline treated mice showed greater amounts of cytochrome c in the mitochondrial fraction, with a corresponding decrease in cytoplasmic cytochrome c (P<0.05; Fig. 3C ). These results confirm those found in primary neuronal cultures, suggesting that cytochrome c is a target for minocycline.

Minocycline reduces tau phosphorylation and aggregation in young (3–4 month) htau mice
We then examined amount of total tau and phosphorylated tau in cortical lysates from wild-type and htau mice using specific antibodies against total tau (Dako) and tau phosphorylated at Ser-202 (CP13), Ser-396/404 (PHF1), and dephosphorylated tau at Ser-199/202 (Tau-1). Htau mice overexpress human tau, and this is reflected in the increased amount of tau detected by Western blotting. Furthermore, the tau in htau mice becomes progressively more hyperphosphorylated with age, and this is also indicated by the increased levels of phosphorylated tau detected in 12-month-old htau mice compared with 3- to 4-month-old mice, particularly when comparing htau and wild-type mice (Fig. 4A ).


Figure 4
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  		Figure 4. Minocycline reduces phosphorylation of tau in young htau mice. A) Western blotting was used to demonstrate differences in total tau protein and phosphorylated tau in wild-type, 3- to 4-month-old htau, and 12-month-old htau mice. Total tau protein was detected with the Dako antibody, while tau phosphorylation was assessed using antibodies recognizing phosphorylation of tau at epitopes Ser-202 (CP13), Ser-396/404 (PHF1), and dephosphorylated tau at Ser-199/202 (Tau-1). B) Western blotting shows that minocycline treatment has no effect on total tau amounts in hippocampal lysates from wild-type and 3- to 4-month-old htau mice. β-Actin was used as a protein loading control. C) Phosphorylation of tau was examined using phosphorylation-specific tau antibodies in both 3- to 4-month-old htau and wild-type mice. D) Bar chart shows amounts of phosphorylated tau present in 3- to 4-month-old htau mice after standardization to total tau amounts. Minocycline treatment resulted in reduced phosphorylation at Ser-202 and Ser-396/404 (n=5 vehicle, n=8 minocycline for 3–4 month htau; n=8 for wild-type). Error bars = SEM. *P < 0.05.

The generation of tau fragments by caspase cleavage is reported to precede further pathological changes in tau proteins, such as hyperphosphorylation and aggregation. To determine whether minocycline treatment results in reduced tau pathology in vivo, we examined changes in the amounts of tau protein and phosphorylation in htau mice and wild-type controls at 3 to 4 months of age.

Antibodies against both phosphorylated and nonphosphorylated tau (Dako) showed no change in the total amount of soluble hippocampal tau present in 3- to 4-month-old htau mice following minocycline treatment (Fig. 4B, D ). In addition to inhibiting caspase activation, minocycline is reported to inhibit protein kinases active on tau, including cdk5 (33) , Akt/GSK-3 (34) , and p38 (35) . Therefore, we assessed the degree of phosphorylation at the CP13, PHF1, and Tau-1 epitopes (Fig. 4C ). We detected reduced amounts of tau phosphorylation (P<0.05) in minocycline-treated mice at these sites (Fig. 4D ). These results demonstrate that, in addition to preventing the caspase cleavage of tau in vitro, minocycline also reduces abnormally phosphorylated tau at relevant sites in htau mice at an early stage of tauopathy development.

To determine whether minocycline reduces tau phosphorylation and aggregate levels via inhibition of proline-directed protein kinases, we used a panel of antibodies to examine the effect of minocycline treatment by immunoblotting. We were unable to detect any significant change in either the total amount or the activation state of any kinase examined in minocycline-treated animals compared to vehicle-treated controls (Supplemental Fig. S1).

As we have previously found a correlation between tau phosphorylation and aggregation in tau transgenic mice (36) , we next examined whether minocycline affects the amount of insoluble, aggregated tau present in 3- to 4-month-old htau mice. Three fractions were prepared from the cortex of minocycline- and vehicle-treated mice: a low-speed supernatant (S1), supernatant after mixing with sarkosyl and a high speed centrifugation (S2; data not shown), and a sarkosyl-insoluble pellet (P3). Immunoblot analysis was performed on all brain fractions, and results from the total lysate (S1) and sarkosyl-insoluble fraction (P3) are shown (Fig. 5 ). Quantitative analysis of the S1 fraction showed a slight, but not statistically significant decrease in the amount of total tau (Dako antibody) in the minocycline-treated mice. Immunoblotting using antibodies for tau phosphorylation at Ser-202 (CP13) and Ser-396/404 (PHF1) indicated decreased phosphorylation at these sites relative to total tau (Fig. 5A, B ).


Figure 5
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  		Figure 5. Minocycline reduces tau aggregation in young htau mice. Insoluble, aggregated tau was isolated from cortex of vehicle- or minocycline-treated htau mice and analyzed by Western blotting using antibodies against total (Dako) and tau phosphorylated at Ser-202 (CP13) and Ser-396/404 (PHF1). A) Western blots of extracts containing both soluble and insoluble tau (S1 fraction). B) Bar chart shows amount of total tau present and extent of its phosphorylation following standardization to total tau (Dako). C) Amount of total and phosphorylated tau found after sarkosyl extraction (P3 fraction). D) Bar chart shows that minocycline treatment resulted in significant reduction in amount of insoluble tau following standardization to tau in S1 fraction. Minocycline reduces phosphorylation of insoluble tau at Ser-202 (CP13) and Ser-396/404 (PHF1), following standardization to total amount of insoluble tau (n=5 vehicle, n=8 minocycline) Error bars = SEM. *P < 0.05.

Analysis of the sarkosyl-insoluble tau fraction (P3) using a phosphorylation independent tau antibody (Dako) revealed a statistically significant decrease in aggregated tau migrating at ~50 to 68 kDa in the cortex of treated mice (Fig. 5C, D ). Quantification of these results showed a significant decrease in phosphorylation at Ser-202 (CP13) and Ser-396/404 (PHF1) following minocycline treatment. In agreement with our previous work (29) , the CP13 antibody labeled predominantly larger tau species (64–68 kDa), whereas most of the aggregated tau recognized by PHF1 was detected at 55–60 kDa. These results indicate that minocycline treatment of htau mice can either prevent the formation or reduce existing levels of pathological tau species, including abnormally phosphorylated and aggregated tau.

Minocycline treatment reduces tau aggregate load, but not phosphorylation in 12-month-old htau mice
We next examined changes in tau phosphorylation and aggregation in 12-month-old htau mice. Htau mice at this age demonstrate significant levels of hyperphosphorylated tau in neuronal cell bodies, with mature NFTs detected in cortical and hippocampal regions (22) . Again, we found no change in the total amount of soluble hippocampal tau following minocycline treatment using an antibody against both phosphorylated and unphosphorylated tau, Dako (Fig. 6A ). We then determined the amount of phosphorylation at CP13, PHF1, and Tau-1 epitopes and, unlike in the younger age group, we were unable to find any significant change in tau phosphorylation in RIPA extracts (Fig. 6B ).


Figure 6
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  		Figure 6. Minocycline reduces tau aggregation, but not phosphorylation, in old htau mice with existing NFT pathology. Phosphorylation of tau was examined in hippocampal lysates from vehicle- or minocycline-treated 12-month-old htau mice. A) Western blots show β-actin loading control and total tau amounts in these extracts following minocycline treatment. B) Phosphorylation was assessed by Western blotting using antibodies recognizing phosphorylation of tau epitopes at Ser-202 (CP13), Ser-396/404 (PHF1), and dephosphorylated tau at Ser-199/202 (Tau-1). Amount of tau phosphorylated at these epitopes is illustrated in bar charts after standardization to the total tau amounts. C) Sarkosyl-insoluble tau was also assessed by Western blotting and quantified following standardization to total tau amounts. Minocycline treatment resulted in reduced amounts of aggregated tau, and reduced phosphorylation of the aggregated tau. D) Conformation of tau was assessed by semiquantitative tau ELISA; bar chart illustrates amount of tau in abnormal MC1 and TG3 conformations following standardization to total tau amounts (TG5; n=8 vehicle, n=9 minocycline). Error bars = SEM. *P < 0.05.

Sarkosyl-insoluble tau was prepared from the cortex of 12-month htau mice, as described above. No significant changes in the amounts of either total tau or phosphorylation were found in the S1 fraction of minocycline-treated mice (data not shown). However, analysis of the P3 fraction showed a significant reduction in the amount of insoluble, aggregated tau after treatment (Fig. 6C ; P<0.05), with reduced amounts of CP13 and PHF1 present (Fig. 6C ; P<0.05 for both). These results demonstrate that minocycline treatment can reduce or prevent the formation of tau aggregates in mice with significant preexisting tau pathology.

We next used semiquantitative tau ELISAs to determine the amount of tau present in an abnormal conformation in aged htau mice. Specific conformational changes of tau unique to AD brain may take place at an early stage in AD, preceding NFT formation, and may represent a more disease-specific alteration than phosphorylation (37 , 38) . Conformational change creates epitopes recognized by the MC1 antibody (39) and a later conformational change recognized by antibody TG3 (40) . Following standardization to total tau amounts (TG5) we found a significant reduction in the amount of tau in the MC1 conformation following minocycline treatment (Fig. 6D ). Only small amounts of tau in the TG3 conformation were detected, and there was no significant change in treated mice.

Finally, to determine whether the amount of cleaved tau fragments correlate with development of other abnormal tau species, we performed correlation analyses and found a highly significant correlation between the amount of caspase-cleaved tau fragments and tau aggregate load (Fig. 7 ). No correlation was apparent between the amount of tau in the MC1 conformation and caspase-cleaved tau fragments. We have previously demonstrated a significant correlation between MC1-positive tau and tau aggregate load in these mice, with increasing amounts of tau in the MC1 conformation found in the insoluble tau fraction with aging (28) . Taken together, these results suggest that the reduced amount of conformationally altered tau found may be an artifact of minocycline reducing tau aggregate load.


Figure 7
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  		Figure 7. Amount of caspase-3-cleaved tau correlates with insoluble tau load, but not altered conformation. Correlation analysis was used to determine the association between pathological tau species. A) Chart shows a significant correlation between caspase-3-cleaved tau fragments and tau aggregate load. B) Chart shows a lack of correlation between caspase-3-cleaved tau fragments and amount of tau in the MC1 conformation. For correlations, data from both age groups of htau mice were pooled. Correlation coefficients and probabilities are shown for each chart.

Thus, we find that minocycline treatment of aged htau mice is effective at reducing the development of some pathological tau species. Importantly, minocycline treatment was effective in aged htau mice, suggesting that it may have therapeutic benefit for patients with late-stage disease.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have used minocycline in models of AD and find that in Aβ-treated neurons, minocycline effectively reduces neuronal death and that this is associated with inhibition of caspase-3 activation and reduced generation of caspase-3-cleaved tau fragments. In addition, we demonstrate here that minocycline partially suppresses the development of tau pathology in vivo and that this is associated with a decrease in caspase-cleaved tau. Importantly, minocycline was effective at reducing tau aggregate load not only in young mice in the early stages of NFT development, but also in old htau mice with mature tau pathology.

There is an increasing literature demonstrating that the generation of caspase-cleaved tau fragments in response to aberrant caspase activation may be an early pathogenic event in AD (8 , 14) . In neuronal cultures treated with Aβ, we found increases in caspase-3 activation and generation of caspase-3-cleaved tau and, in agreement with previous reports, caspase-3 cleavage of tau preceded neuronal death (12 , 13) . Mutant forms of tau that are resistant to caspase-3 cleavage (D421E) can prevent caspase-induced cell death (13) , suggesting that the generation of caspase-cleaved tau fragments is a primary neurotoxic event. Since Aβ-induced neurotoxicity is abolished in the absence of tau (41) , our results support the idea that minocycline-mediated neuroprotection against Aβ may result from decreased generation of caspase-cleaved tau following inhibition of caspase-3 activation.

Caspase-cleaved tau is present in tauopathies lacking amyloid pathology (8) , suggesting that additional factors, other than Aβ, and intrinsic to neurodegeneration, can initiate caspase activation and tau cleavage. In htau mice we detected significant amounts of caspase-cleaved tau, that was absent from wild-type mice. These results are in agreement with data from human postmortem brain showing that caspase-cleaved tau is present in AD, where the number of caspase-cleaved tau containing neurons correlates positively with Braak staging and inversely with cognitive function, but is absent from control brain (8) .

Treatment with minocycline significantly reduced the amount of caspase-cleaved tau in htau mice, together with decreasing the tau aggregate load in both young and old mice. Immunohistochemical studies have demonstrated that the generation of caspase-cleaved tau is a relatively early event in NFT formation, preceding alterations in tau conformation, phosphorylation, and aggregation (6 , 14) . Following caspase cleavage, the conformation of tau is reported to be altered, rendering it more readily phosphorylated by kinases such as GSK-3 (14) . However, our results show no correlation between tau in the MC1 conformation and the amount of cleaved tau fragments. Caspase-cleaved tau also exhibits proaggregation properties (12) and enhances nucleation-dependent filament formation from full-length tau (8 , 14) . Although the precise sequence of these events is unclear, a combination of tau phosphorylation and cleavage is thought to result in elevated tau aggregation and increased amounts of sarkosyl-insoluble (filamentous) tau (42) . Since we were unable to detect any changes in activation of any of the protein kinases examined here, it seems likely that minocycline may reduce tau aggregation by suppressing caspase-3 activation and thereby inhibiting the primary event of caspase-mediated tau cleavage that occurs prior to further pathological changes in tau proteins. However, our results from the 12-month htau group suggest that there is a dissociation between tau phosphorylation and its altered conformation and aggregation. If an early, highly phosphorylated soluble form of tau is indeed the toxic species of tau, then it is possible that there is a critical period for therapeutic intervention using agents designed to prevent the generation of caspase-cleaved tau fragments, with more progressed and mature tauopathy resistant to this type of therapy.

Pleiotropic effects of minocycline in central nervous system (CNS) disorders have been described, including inhibition of glial activation (18 , 19 , 43) and suppression of cyclooxygenase-2 (20) . Interestingly, in tau transgenic mice, development of tau pathology is often accompanied by glial activation (44 , 45) . Indeed, we observed significant activation of astrocytes in htau mice, and reduced amounts of glial fibrillary acidic protein following minocycline treatment (Supplemental Fig. S2). A more detailed study of the effects of minocycline on neuroinflammation in mouse models of tauopathy is currently underway in our laboratory, and preliminary results suggest that many of the protective effects of minocycline are mediated via inhibition of glial responses. In addition, minocycline inhibits poly (ADP-ribose) polymerase-1 (46) , blocks the activation of protein kinases (33 34 35) , reduces oxidative stress (20) scavenges free radicals (47) , and reduces the expression of BclII (48) . We cannot rule out the possibility therefore that additional targets of minocycline might also contribute to the reduced tau pathology we observe following minocycline treatment.

In wild-type mice treated with minocycline, we observed a slight, nonsignificant increase in tau phosphorylation by Western blotting in contrast with results from htau mice. Seabrook et al. (19) reported differential effects of minocycline on plaque deposition depending on the age of the mice. It is likely that different targets of minocycline may be specifically affected depending on the dosing method (oral gavage, intraperitoneal, intracerebroventricular), concentration of drug used (11–100 mg/kg/day), and/or the up-regulation of specific pathways during disease progression. Indeed, the method of administration of minocycline appears to be particularly important, with widely different results found by different laboratories (49 50 51) . A comprehensive proteomic analysis is currently underway in our laboratory to dissect out the specific neuroprotective targets of minocycline. Finally, although numerous preclinical trials of minocycline in cell and animal models of disease have shown disease-modifying effects, many of these promising results appear to have been "lost in translation" during clinical trials (52) , although in some cases this may be a consequence of mistakes in trial design and interpretation. Recent results from a Phase III trial reported harmful effects of minocycline on patients with ALS (53) , however this report has been criticized for the high dose of minocycline used (double the well-tolerated dose) and the suitability of the functional tests (in patients susceptible to fatigue) (54) . Thus, although minocycline may be a potentially effective therapeutic agent for the treatment of a wide spectrum of CNS disease, caution should be used until its mechanism of action is fully understood.

In summary, we have shown that minocycline has therapeutic promise in both in vitro and in vivo models of Alzheimer’s disease. The beneficial effects of minocycline are associated with inhibition of caspase-3-mediated tau cleavage. Minocycline effectively reduces tau aggregate load in mice during both early and late tauopathy development, suggesting that minocycline may be of use for patients with both early- and late-stage disease. Taken together with studies showing a beneficial effect of minocycline in amyloid models of AD (18 19 20 21) , these results indicate that minocycline may represent a novel therapeutic agent for the treatment of AD and related neurodegenerative disorders.


   ACKNOWLEDGMENTS
 
The authors thank Professor Peter Davies (Albert Einstein College of Medicine, Bronx, NY, USA) for his generous gift of tau antibodies. This work was supported by the Medical Research Council, the Alzheimer’s Society UK, and the Alzheimer’s Research Trust.

Received for publication May 16, 2008. Accepted for publication October 16, 2008.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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