Abnormally hyperphosphorylated tau is a defining pathological feature of tauopathies, such as Alzheimer’s disease (AD), and accumulating evidence suggests a role for iron in mediating tau pathology that may lead to cognitive decline in these conditions. The metal chelator deferiprone (DFP), which has a high affinity for iron, is currently in clinical trials for AD and Parkinson’s disease. However, the effect of DFP on tau pathology remains underexplored.
We aimed to investigate the impact of chronic DFP treatment on tau pathology using a well-characterized mouse model of tauopathy (rTg(tauP301L)4510).
Animals were treated daily with DFP (100 mg/kg) via oral gavage for 16 weeks. After 14 weeks, mice were tested in the Y-maze, open field, Morris water maze, and rotorod. At the end of the study, brain tissue was collected to examine metal levels (using inductively coupled plasma-mass spectrometry) and for western blot analysis of DFP on tau and iron associated pathways.
DFP significantly reduced anxiety-like behavior, and revealed a trend toward improved cognitive function. This was accompanied by a decrease in brain iron levels and sarkosyl-insoluble tau. Our data also showed downregulation of the tau kinases glycogen synthase kinase 3β and cyclin dependent kinase-5 in DFP treated mice and an increase in the methylation of the catalytic subunit of protein phosphatase 2A.
These data support the hypothesis that suggests that iron plays a neurotoxic role in tauopathies and may be a potential therapeutic target for this class of disorders.
Tauopathies are a broad class of neurodegenerative disorders that includes Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). While the clinical manifestation of tauopathies is quite heterogenous, they are all characterized by intracellular inclusions composed of the microtubule-associated protein tau. In the human brain, tau is encoded by the MAPT gene on chromosome 17q21 and is predominately localized in the axons of neurons and to a lesser extent in dendrites and glial cells . Tau is involved in several different cellular processes such as microtubule regulation and stabilization, iron metabolism , regulating axonal transport , and neurotransmission . In tauopathies, tau is abnormally hyperphosphorylated, which is speculated to lead to a disruption in tau function and subsequently to its aggregation into paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) . This is counterintuitive to its physiological nature, as tau is natively unfolded in solution and has a low tendency for aggregation [6, 7]. While the etiology of tau-mediated neurodegeneration is still unknown, a number of studies report that the aberrant activation of tau kinases and MAPT mutations appear to be the most probable reasons for tau dysregulation [8– 11]. However, numerous tau/tau kinase-targeting therapies have been developed with little to moderate efficacy in clinical trials , which may suggest that other pathways may be involved in these disorders. Indeed, the metal hypothesis is an emerging area of research in this field which suggests that transition metals (in particular iron) may play a mechanistic and therapeutic role in neurodegenerative disorders.
Dysregulation of cerebral iron in neurodegenerative tauopathies is well established and several studies have validated a putative link between iron and tau [13– 17]. In postmortem brains of patients with AD and PSP, iron is highly concentrated with tau in NFTs [15, 16] and in Lewy bodies in Parkinson’s disease (PD) . Iron is reported to influence the pathways associated with NFT formation, such as promoting tau hyperphosphorylation by regulating the activity of the tau kinases glycogen kinases 3β (GSK3β) and cyclin dependent kinase-5 (CDK5) [18, 19], which are both linked to the development of tauopathies. These kinases are found to be associated with early tau deposits and tangles in brain samples from PSP, Pick’s disease, and AD [9, 20]. Furthermore, iron is reported to bind to tau and promote conformational changes in the protein that may subsequently facilitate its aggregation [15, 21]. While, the causes of abnormal brain iron elevations in tauopathies is not well understood, it is speculated that loss/disruption of tau function may impair the ferroportin-mediated iron export pathway. Ferroportin is the only iron export protein and is stabilized by the amyloid-β protein precursor (AβPP), which itself is trafficked to the neuronal surface by tau . Loss of tau function therefore prevents the trafficking of AβPP and subsequently leads to cellular iron accumulation [2, 22, 23]. Elevations of iron in a dysregulated system can cause an increase in the labile iron pool— comprised of chelatable and redox-active iron — that may lead to the generation of unregulated reactive oxygen species (ROS) and inevitably oxidative damage and cell death. There is a high incidence of oxidative damage evident in AD, Huntington’s disease, and PSP [25– 27]. Furthermore, interactions between iron and pathological tau are reported to act as a source of ROS in neurons . As such, targeting iron is currently under investigation as a potential therapeutic avenue for tauopathies, with one of the first clinical studies undertaken more than two decades ago.
In 1991, Crapper McLachlan and colleagues reported on the potential benefits of targeting metals in neurodegeneration with the metal chelator deferoxamine . Although there were a number of caveats associated with the clinical trial, deferoxamine was reported to reduce the rate of symptomatic decline in AD patients compared to control groups. Deferoxamine, however, was actually being used to target aluminum in this study, despite the fact that its affinity is six times greater for iron compared to aluminum and it is a compound commonly used for the treatment of iron-overload disorders . The outcomes that were observed in this trial, therefore, may have been driven more by an effect of chelating iron rather than aluminum as originally postulated. More recently, the metal chelator deferiprone (DFP) has commenced phase II clinical trials for PD (FAIR PARK II; ClinicalTrials.gov identifier: NCT02655315)  and AD (Deferiprone to Delay Dementia (The 3D Study); ClinicalTrials.gov Identifier: NCT03234686). DFP is a metal chelator that is commonly used to treat iron-overload disorders, such as hemosiderosis  and Friedreich’s ataxia . Animal models have provided some insight into the potential effect of DFP on tau, with reports of reduced phosphorylation of the protein and downregulation of GSK3β [34, 35]. A more recent study in a mouse model of PD reported improved motor function following DFP treatment . Importantly, in the FAIRPARK trial, DFP improved clinical symptoms and reduced iron deposits in the substantia nigra and putamen in PD . While Lewy bodies, which are primarily composed of aggregates of alpha-synuclein, are the pathological hallmark of PD, tau pathology has also been evidenced in PD . Interestingly, mutations in the gene encoding alpha-synuclein (SNCA) are reported to lead to the development of tau pathology and dementia [38, 39]. Furthermore, a genome-wide association study revealed that the MAPT H1 haplotype is closely linked to sporadic PD  Importantly, the genetic ablation of tau is reported to elevate brain iron levels and induce parkinsonism with dementia in mice . Cumulatively, these data suggest an involvement of tau in PD. While pre-clinical and clinical trials have demonstrated the potential efficacy of DFP in AD and PD, it is important to note that these disorders are characterized by the co-occurrence of tau and amyloid-β or α-synuclein pathology, which are both mediated by and interact with iron (and other metals) and tau in neurodegeneration [41– 43]. Therefore, to further our understanding of the role of iron in tauopathies we examined the therapeutic efficacy of DFP treatment in the rTg(tauP301L)4510 mouse model which overexpresses P301L mutant human tau (referred to as rTg4510) . We hypothesized that a “prevention” paradigm undertaken in young (4-month-old) mice would limit the accumulation of tau pathology and slow disease progression. Indeed, our results revealed that DFP reduced hyperactive behavior and promoted a trend toward improved cognitive function. This was accompanied by a decrease in brain iron levels and alterations in tau phosphorylation pathways. Taken together, these data support the targeting of iron as a tractable therapeutic approach for tauopathies.
MATERIALS AND METHODS
All animal experimental procedures were approved by The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee and conducted in accordance with the Prevention of Cruelty to Animals Act and the NH&MRC Code of Practice for the Use of Animals for Scientific Purposes. This study used the rTg(tauP301L)4510 mouse model of tauopathy (referred to as rTg4510), that is associated with heredity tauopathies [44, 45]. Drug treatment commenced at 4 months and concluded at the end of 7 months of age. This age was chosen to begin treatment as our previous work demonstrated a significant age-related increase in brain iron levels from 4 months to 7 months of age in rTg4510 compared to age-matched WT mice . Mice were housed in Techniplast IVC cages with free access to mouse chow and water. The cages were lined with a bed of sawdust and mice were given a solid enclosure as enrichment and tissue paper for nesting. Animals were weighed daily throughout the experimental part of the study in order to determine drug dose and to monitor adverse reactions to treatment. The study was performed in a mixed gender cohort with a total of 8 SSV treated mice (n = 4 males, n = 4 females) and 10 DFP treated mice (n = 6 males and n = 4 females).
Drug preparation and treatment
DFP (3-Hydroxy-1,2-dimethyl-4(1H)-pyridone, Sigma) was dissolved by probe sonication in standard suspension vehicle (SSV; (NaCl 0.9% w/v, carboxy methyl cellulose 0.05% w/v, Benzyl alcohol 0.05% v/v, Tween 80, 0.04 % v/v)). Sonication was carried out at room temperature in 2– 3 rounds of 15 s at an amplitude of 40% until DFP was completely dissolved. Mice were treated for 4 months (∼17 weeks) with 100 mg/kg of DFP (n = 10), delivered at a rate of 4×body weight by oral gavage using a 23-gauge gavage needle. Vehicle treated mice (n = 7) were gavaged an equivalent volume of SSV (relative to body weight).
Behavioral tests were performed 2 weeks prior the end of the trial duration. Animals were tested in a randomized order.
Morris water maze
The Morris water maze (MWM) performed in this study has been adapted from the original study . The experiment was performed in a 1.4 m diameter circular pool filled with water, made opaque with non-toxic paint and maintained at 23– 25°C. The maze was divided into four quadrants: northeast, northwest, southeast, and southwest. Data was collected using Ethovision automated tracking system (USA). Geometric patterns and lamps were used as extra-maze visual cues. The MWM was conducted over 7 consecutive days; task acquisition training occurred on days 1 through 6 and the probe trial occurred on day 7. Mice were acclimated by allowing them to explore the water maze for 60 s on the day before training commenced. On training days, the platform was submerged 1 cm under the water surface in the center of the target quadrant. The mouse was placed in the water maze facing the wall. Each mouse completed four 90 s trials per day, with a randomized start quadrant for each trial. If the mouse failed to find the platform within the allotted timeframe it was gently guided to it. On probe day, the platform was removed, and each mouse was placed in the quadrant opposite the target quadrant and allowed to search for the platform for 60 s.
The three identical arms of the Y-maze were randomly designated as start, novel, and other arm, with different visual cues at the end of each arm. The y-maze arena was covered in 2 cm of sawdust and each mouse was randomly assigned a start and novel arm. The Y-maze consisted of two trials separated by 1 h inter-trial interval. The first trial (training) was for 10 min, which allowed the mouse to explore 2 arms (start and other; the third arm was blocked off) freely. The retention trial commenced 1 h after training and the mouse could freely explore all three arms of the maze for 5 min. Data was collected using the Ethovision automated tracking system using a ceiling mounted CCD camera.
Mice were first placed on the rotarod for 3×2 min sessions, set at an acceleration of 4 rpm to acclimate to the equipment. The rotarod task was performed over 2 days (training and test day) and consisted of 3 inter-trial intervals separated by 1 h. Mice were placed on the rod at a starting speed of 4 rpm, which increased by 1 rpm every 8 s for a total of 5 min. On day 2 (test day), the time spent on the rotarod for each mouse was recorded and averaged over the 3 inter-trials
Locomotor (Open field test)
Mice were placed in clear perspex tracking chambers, equipped with a grid of infrared beams (Coulburn TruScan, U.S.A), for 60 min. The tracking software recorded the total movements in the floor plane by the interruption of the beams.
Animals were euthanized (with sodium pentobarbitone, 80 mg/kg, via intraperitoneal injection) followed by transcardial perfusion (0.1 M phosphate buffer saline; PBS). From the left hemisphere, the cerebellum, hippocampus, and cortex were rapidly dissected and each brain sample along with the remaining tissue from the left hemisphere (referred to as whole tissue) was stored at – 80°C until analysis.
Metal quantification was performed in whole tissue: samples were weighed and homogenized by probe sonication (2– 3 rounds of sonication for 15 s on ice, 40% amplitude) in 1 ml of homogenization buffer (Dulbecco’s PBS with EDTA free protease inhibitor and phosphatase inhibitor cocktails 2 and 3; 1 : 500; Roche). For metal analysis, 100– 200μL of total homogenate was centrifuged at 100,000×g for 30 min at 4°C. The supernatant was collected, and both the pellet and supernatant were stored at – 80°C until further use.
All measurements were performed on an Agilent 7700 Series ICP-MS (Agilent Technologies, CA, USA) with a glass MicroMist nebulizer (Agilent Technologies). Helium was used as a collision gas (1.7 mL min–1) for analysis of the elements 56Fe, 63Cu, 66Zn, and 89Y and Hydrogen was used as a collision gas (6 mL min–1) for analysis of the elements 78Se and 89Y. The instrument was calibrated with 0, 0.05, 0.1, 1, 5, 10, 50, 100, and 500 ppb of multi-element standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3, and ICP-MS-CAL-4, AccuStandard, CT, USA). An internal standard solution containing 200 ppb of Y89 was used as an internal standard. Samples were all thawed at room temperature (RT) on the day of analysis. The soluble material and was diluted 1 : 20 with 1% nitric acid and metal content was analyzed. For the insoluble material, the samples were lyophilized. The dry material was digested with 50μL of concentrated nitric acid (65%) overnight. The samples were heated to 90°C for 15 min and then 50μL of hydrogen peroxide was added. The samples were heated at 70°C for 15 min and then diluted 1 : 20 with 1% nitric acid and metal content was analyzed. Metal content in the soluble fraction (supernatant) was normalized to protein concentration determined by BCA Protein Assay Kit (ThermoFisher Scientific) as per manufacturer’s instructions. Metal content within the insoluble fraction was normalized to wet tissue weight.
Size exclusion chromatography-ICP-MS
Size exclusion chromatography-ICP-MS (SEC-ICP-MS) analysis was performed using the previously described method for injection of 100μg of protein . Samples were chromatographically separated using a BioSEC-3 column (4.6×300 mm) 150A with 200 mM ammonium nitrate containing internal standard (133Cs, 121Sb; 10μg L–1 each), pH 7.5, at a flow rate of 0.4 ml/min. The HPLC was directly connected to MicroMist nebulizer (Glass Expansion, Australia) fitted to an Agilent Technologies 7700× ICP-MS. Helium was used as the collision gas (3 mL min–1) to minimize polyatomic interferences with all elements. The following elements were analysed: 56Fe, 63Cu, 66Zn, 121Sb, and 133Cs.
Hippocami and cortical samples were weighed and homogenized in homogenization buffer at a ratio of 1 : 10 (w/v) by probe sonication (as described above). For immunoblotting, 20μL of total homogenate was centrifuged at 100,000×g for 30 min at 4°C. The supernatant was collected and the pellet was resuspended in homogenization buffer. Protein concentration was determined by BCA protein assay.
|Total tau||1 : 1000||DAKO||A0024|
|p-tauSer396||1 : 1000||Invitrogen||35– 5300|
|p-tauSer202/205||1 : 1000||Invitrogen||MN1020|
|AT100||1 : 1000||Invitrogen||MN1060|
|p-tauThr231||1 : 1000||Invitrogen||35– 5200|
|GSK3β||1 : 1000||Cell Signalling||9315S|
|p-GSK3β Ser9||1 : 1000||Cell Signalling||9336|
|CDK5||1 : 200||Santa Cruz Biotechnology||sc-6247|
|p-CDK5 Tyr15||1 : 200||Santa Cruz Biotechnology||sc-377558|
|Ferroportin/SLC40A1||1 : 500||Novus Biologicals||NBP1-21502|
|Ferritin||1 : 1000||Abcam||Ab75973|
|PP2A subunit A||1 : 1000||Cell Signalling||2041S|
|PP2A subunit B||1 : 1000||Cell Signalling||2290S|
|PP2A subunit C||1 : 1000||Cell Signalling||2259|
|Pin-1||1 : 1000||Cell Signalling||3722S|
|PME-1||1 : 1000||Thermo Fisher Scientific||PIEPA5-27754|
Sarkosyl tau extraction
A modified method by Greenberg and Davis  was used to perform sarkoysl tau extraction in the cortex. Briefly, 15μL of total homogenate was centrifuged at 27,200×g for 20 min at 4°C. The supernatant was collected (S1) and the pellet was resuspended in cold H buffer (10 mM Tris-HCl, 1 mM EGTA; 0.8 M NaCl, 10% surcrose, pH 7.4) in the sample initial volume. The samples were centrifuged again and the supernatant was collected (S2) and combined with S1. The combined supernatant was then incubated with 1% sarkosyl (N-Lauroylsarcosine sodium salt; Sigma Aldrich) for 90 min with shaking at 37°C. After incubation, samples were centrifuged at 150,000×g for 35 min at 20°C. The supernatant was collected and the pellet was resuspended in 10μL of 50 mM Tris.
Samples were prepared for SDS-PAGE by the addition of 4× NuPage LDS sample buffer (Life Technologies) and 10× NuPage reducing agent (Life Technologies; both to a final 1× concentration) to 5μg of protein. Samples were heated to 90°C for 5 min and loaded on 26-well NuPage Novex 4– 12% Bis-Tris gel (Life Technologies) alongside Odyssey One-Color protein molecular weight marker (Millennium Science). Gels were run at 140 V for 80 min in 1× MES buffer (Life Technologies) and then transferred to iBlot PVDF transfer stacks (Life Technologies) using the iBlot dry transfer system with the following settings: step 1) 15 V for 6 min, step 2) 20 V for 5 min, and step 3) 25 V for 2 min. Membranes were blocked for 30 min in 1× Tris buffered saline with Tween 20 (TBST; 10 mM Tris, 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk powder and 1% BSA at room temperature (RT) then incubated with primary antibody (Table 1) diluted in 3% BSA in TBST overnight at 4°C or 1 h at RT. Blots were rinsed in TBST (3×5 min washes) and incubated with IRDye secondary antibody (Millennium Science) diluted in 0.01% SDS in TBST for 30 min at RT. Blots were washed again in TBST, followed by 3 quick washes in PBS and imaged using a LI-COR Odyssey Imaging system (LI-COR Biosciences, USA). Blots were analyzed using Image Studio Lite software and samples were normalized to β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. The relative levels of phospho-proteins were obtained by dividing the normalized relative florescence of the phosphorylated protein by the normalized relative florescence of the total protein.
Statistical analysis was carried out using GraphPad Prism 8 software. Two-way ANOVA with Tukey’s post hoc test for multiple comparisons was used to analyze MWM. Unpaired t-test was used to compare differences between treatment groups for Y-maze, rotarod, open field and biochemical tests. The primary comparisons of interest in this study were within the rTg4510 groups ± DFP. As such. the tissue experiments were set up to maximize to rigour of these comparisons. Western blots were performed in the WT groups, but these were run separately to examine any drug effects in the WT animals, not for comparisons between genotypes.
Treatment with DFP improved behavior
The rTg4510 mice have previously been reported to develop deficits in spatial memory and locomotor activities [44, 45, 50]. To investigate if DFP had an effect on these behavioral deficits, this study employed MWM, Y-maze, rotarod and open field (locomotor) tests. As the focus of this work was on the rTg4510 mice and the effect of DFP, then for optimal experimental design (in terms of minimizing any variations that may arise from protracted behavioral testing in larger groups of mice across different parts of the diurnal cycle) the rTg4510 and WT mice were assessed separately. As such, the WT data was not included in the statistical analysis and is presented in the Supplementary Material.
Trend toward improved MWM performance in DFP treated rTg4510
Two-way ANOVA with repeated measures revealed no differences in spatial acquisition (time taken to learn the task) between rTg4510 treatment groups (Fig. 1a). While post-hoc analysis revealed a significant difference between groups at day 4 (p < 0.05), by day 6 no difference in the duration to locate the hidden platform was observed between groups. In the probe trial (recall task), an indication of whether the animal has learnt the task can be measured by the duration spent in the target quadrant. Vehicle treated rTg4510 mice spent an average of 34.7% in the target quadrant and DFP treated rTg4510 mice spent an average of 43.1%. Unpaired t-test revealed a trend towards improved performed in the probe trial (Fig. 1b; p = 0.076) in DFP treated rTg4510 mice. In WT mice, DFP did not influence performance in spatial acquisition (Supplementary Figure 1a) or in the probe trial (Supplementary Figure b). In the probe trial, vehicle control WT mice spent 30% of the total duration within the target quadrant and DFP treated WT mice spent a duration of 33%.
No effect of DFP in rTg4510 mice in Y-maze performance
In the Y-maze an indication of a willingness to explore a new environment is measured by the duration spent in the novel arm. No difference between rTg4510 groups were observed in the duration spent in the novel arm (Fig. 1c; vehicle treated spent 45.3% and DFP averaged 41.2% in the novel arm). Based on historical data, the durations spent in the novel arm suggest there are no impairments in behavior in this task in this cohort. In WT mice, DFP did not affect Y-maze performance; the duration spent in the novel arm was 46% and 47% for vehicle treated and DFP treated WT mice respectively (Supplementary Figure 1c).
DFP altered locomotor activity in rTg4510 mice
Hyperactivity and anxiety-like behavior can be measured by the open field test, which can be indicated by resting time, total distance travelled and walking speed over the 60 min test period. There was a trend toward a decrease hyperactivity in DFP treated rTg4510 mice, as indicated by increased resting times (Fig. 1e; p = 0.080) and a decrease in distance travelled (Fig. 1f; 0.060). Walking speed and time in DFP rTg4510 treated mice was 35% slower in the open field test (Fig. 1g, h; p < 0.05) compared to vehicle treated rTg4510 mice. While this may suggest DFP may affect motor coordination, no difference in rotarod performance was observed between treatment groups (Fig. 1d), suggesting that a slower walking speed may indicate improved exploratory behavior. In WT mice, DFP treatment appears to increase hyperactivity as indicated by a decrease in resting times (Supplementary Figure 1e, – 10%, p < 0.05), an increase in distance travelled (Supplementary Figure 1f, +50%, p < 0.05), walking speed (Supplementary Figure 1g, +83%, p < 0.05), and walking time (Supplementary Figure 1h, +52%, p = 0.01) compared to vehicle treated WT.
Brain iron is decreased in DFP treated mice in whole tissue
The effect of DFP on brain iron levels was measured using ICP-MS within the soluble and insoluble fractions in whole tissue. Iron was increased by 21% in rTg4510 mice compared to WT vehicle treated mice (Fig. 2a; p < 0.001) and decreased by 25% in DFP treated rTg4510 (compared to vehicle treated rTg4510, p < 0.001). No difference was observed between WT treatment groups in brain iron levels (p > 0.01). In the insoluble fraction (Fig. 2b), iron was increased by 40% in rTg4510 mice compared to WT vehicle treated mice (p < 0.001) and decreased by 24% in rTg4510 DFP treated mice (compared to vehicle treated rTg4510, p < 0.05). No difference in iron levels in the insoluble fraction were evident between WT treatment groups (p > 0.05).
Iron-associated with ferritin-like proteins is decreased in DFP treated rTg4510
To further assess the effect of DFP on iron, SEC-ICP-MS was employed to measure iron bound to metalloproteins such as ferritin. The chromatogram revealed 4 peaks (Supplementary Figure 2); peak 1 may be associated with ferritin-like proteins as this protein-complex elutes with the ferritin standard, and peak 2, 3, and 4 may be associated with metallothioneins . The area under the curve for each peak was used to quantify the amount of iron associated with each metalloprotein complex respectively. There was a 15% decrease in iron bound to ferritin-like proteins (ferritin-iron) in DFP treated rTg4510 mice compared to vehicle treated rTg4510 (Fig. 2c; p < 0.05). In WT mice, DFP decreased ferritin iron by 20% compared to vehicle treated WT (p < 0.05). No difference was observed between vehicle treated rTg4510 and WT mice. Iron associated with proteins within peak 3 and 4 (Fig. 2e, f) were significantly decreased in rTg4510 mice by 13% (p < 0.05) and 23% (p < 0.05) respectively compared to vehicle treated WT mice, which was not altered following DFP treatment in rTg4510 mice. Interestingly, DFP treatment in WT mice resulted in a similar decrease in peak 3 (14%; p < 0.05) and 4 (27%; p < 0.05) compared to vehicle treated WT.
Ferritin protein was decreased in the hippocampus in DFP treated rTg4510 mice
To examine if DFP altered ferritin protein levels, western blotting was employed to investigate the steady-state level of the protein within the hippocampus and cortex. Steady states levels of ferritin were increased by 68% in rTg4510 mice compared to WT vehicle treated mice in the hippocampus (Fig. 3a, p < 0.05), which was decreased by 92% in DFP treated rTg4510 mice (p < 0.05 compared to vehicle treated rTg4510). There was no change in ferritin between WT treatment groups. In the cortex, ferritin was increased by 22% in rTg4510 mice compared to vehicle treated WT mice, though this was not statistically significant (Fig. 3b, p > 0.1). There was a trend towards a decrease in ferritin in DFP treated rTg4510 mice in the cortex (42%; p = 0.06, compared to vehicle treated rTg4510). In WT mice, DFP increased ferritin by 18%, though this was not significant (Fig. 3b, p > 0.1).
Steady-state levels of ferroportin are decreased in DFP treated mice
The decrease in brain iron levels prompted investigation of the effect of DFP on the iron export protein ferroportin. In the hippocampus, ferroportin was decreased by 26% in vehicle treated rTg4510 mice compared to WT (Fig. 3a; p < 0.05). There was a 32% increase in ferroportin in DFP treated rTg4510 mice compared to vehicle treated rTg4510 (p < 0.05). No change in ferroportin was observed between WT treatment groups in the hippocampus. In the cortex, there was a 57% decrease in ferroportin in vehicle treated rTg4510 mice compared to WT (Fig. 3b; p < 0.05). This was increased by 37% in DFP treated rTg4510 mice (p < 0.05; compared to vehicle treated rTg4510 mice. Between WT treated groups, there was a 28% decrease in ferroportin in DFP treated WT mice, which was not statistically significant (p > 0.1).
Decrease in tau phosphorylation in DFP treated mice
In the hippocampus, total soluble tau levels were increased by 29% in DFP treated rTg4510 mice compared to vehicle treated mice (Fig. 4a; p < 0.05). Relative to total tau levels, tau phosphorylated at Ser202/Thr205 and Ser396 were decreased by 63% and 49%, respectively, in DFP treated mice (Fig. 4a, p < 0.05). Relative to β-actin levels, Ser202/Thr205 and Ser396 were significantly decreased by 47% and 27% in DFP treated mice (p < 0.05; data not shown). There was a trend toward a decrease in tau phosphorylated at Thr231 (relative to total tau levels, p = 0.076) and a trend toward deceased p-tauThr231/β-actin (p = 0.065). Within the insoluble fraction of hippocampi samples (Fig. 4b), DFP did not alter total tau levels. However, tau phosphorylated at Ser396 and Ser202/Thr205 were decreased significantly by 63% (p > 0.05) and 42% (p < 0.05), respectively.
In the cortex, total soluble tau levels were not changed between treatment groups (Fig. 4d). There was a trend toward an increase in soluble Ser202/Thr205/Total tau in DFP treated rTg4510 mice (Fig. 4d, p = 0.078). There were no significant differences in Ser396/Total tau and Thr231/Total tau (Fig. 4d) between treatment groups in the cortex. Relative to β-actin no statistical differences were observed in the tau phosphorylated at Ser202/Thr205, Ser396, and Thr231 . Tau aggregates are insoluble in the presence of sarkosyl (also known as sodium lauroyl sarcosinate, an anionic surfactant) and is a standardized protocol for investigating insoluble tau aggregates. In the cortex, DFP treatment reduced sarkosyl-insoluble tau by 32% (Fig. 4e; p < 0.05). Due to lack of tissue, sarkosyl tau extractions were not performed in hippocampal tissue. In WT mice, DFP treatment did not significantly alter total tau levels (+8%, p > 0.1), or the relative ratios of phosphorylated tau/total tau (Ser396/Total tau: – 3%, p > 0.1; Thr231/Total tau: – 14%, p > 0.1). No Ser202/Thr205 was detected in the soluble fraction in WT mice.
Tau kinases are downregulated in DFP treated mice
There are over 20 kinases involved in tau hyperphosphorylation in tauopathies, which may promote NFT formation. Here, we investigated the effect of DFP on the two key tau kinases GSK3β and CDK-5. Within the hippocampus, there was no effect of DFP on the levels of total GSK3β (Fig. 5a), the inhibitory phosphorylation site of GSK3β at ser9 (p-GSK3β) or p-GSK3β/Total GSK3β (Fig. 5a) between rTg4510 treatment groups. In contrast, while no changes in total CDK-5 levels (Fig. 5a) were evident between groups, the levels of CDK-5 phosphorylated at Tyr15 (p-CDK-5, which is associated with the upregulation of the kinase), were significantly decreased by 62% in DFP treated mice (p < 0.05) and decreased by 29% relative to total CDK-5 levels (p-CDK-5/Total CDK-5; p < 0.05).
In the cortex, no difference in total GSK3β was evident between rTg4510 treatment groups (Fig. 5b; p > 0.5). However, p-GSK3β/β-actin was significantly increased by 54% in DFP rTg4510 treated mice and 36% p-GSK3β/Total GSKβ (Fig. 5b; p < 0.05). Total CDK-5 and p-CDK-5 are decreased by 21% and 36%, respectively, in DFP rTg4510 treated mice compared to vehicle controls (Fig. 5b, p < 0.05 and p < 0.05 respectively). However, no significant difference was observed between groups of p-CDK-5 relative to total CDK-5 levels. In WT mice, DFP did not significantly change total GSK3β (– 7%, p > 0.1); p-GSK3β (+3%, p > 0.1); pGSK3β/Total GSK3β ratio (– 4%, p > 0.1) compared to vehicle treated WT. There was no difference in total CDK-5 between WT treatment groups (p > 0.1). However, the effect of DFP in WT mice on p-CDK-5 requires further investigation.
PP2A subunit A and PME-1 are altered in DFP treated rTg4510 mice
The tau phosphatase PP2A is a heterotrimeric protein, comprised of three subunits: a structural subunit (A), regulatory subunit (B), and catalytic subunit (C). Methylation of the subunit C (PME-1) is associated with the downregulation of PP2A and Pin1 is a phosphorylation dependent cis/trans isomerase that primes sites for PP2A activity. There was no difference in protein levels of the subunits B or C of PP2A, following DFP treatment in the hippocampus (Fig. 6a). However, there was an increase in the structural subunit (subunit A) of PP2A in DFP treated mice (Fig. 6a; 65%; p < 0.05). In addition, both regulatory proteins PME-1 and Pin1 were significantly decreased following DFP treatment (Fig. 6a; 49% and 6a; 54%; p < 0.05). In the cortex, the subunits of PP2A (subunit A, B, or C) and Pin1 were unaltered between treatment groups (Fig. 6b). However, levels of PME-1 were decreased by 15% in DFP treated mice (Fig. 6b; p < 0.05). In WT mice, DFP did not significantly affect PP2A B (+3%, p > 0.1), PP2A C (– 12%, p > 0.1), or Pin1(– 12%, p > 0.1) compared to vehicle treated WT. However, there was a significant increase in PP2A A (+66%, p < 0.01) and PME-1 (+26%, p < 0.05) in DFP treated WT mice.
A growing body of evidence, from pre-clinical studies through to clinical cases, has demonstrated that iron may play a critical role in tauopathies . Furthermore, studies suggest that iron may have an important role in mediating NFT formation, by altering tau phosphorylation patterns and inducing tau aggregation [21, 52, 53]. To examine the intersection between iron and tau in tauopathies, this study investigated the effects of the iron chelator DFP in young rTg4510 mice, which is a well-established mouse model of tauopathy. Our data showed that decreasing brain iron levels with DFP also resulted in the concurrent decrease in pathological tau. Furthermore, DFP was found to reduce hyperactivity in rTg4510 as measured by open field and promoted a trend towards improved performance in the MWM.
Animal models of tauopathy such as JNPLtauP301L  and rTg4510 , along with APP/PS1 (AD model) [19, 56] and mouse models of PD  have provided valuable insights in assessing the efficacy of targeting metals as a therapy for neurodegenerative diseases. For example, the iron chelator deferoxamine improved cognitive function in JNPLtauP301L and APP/PS1 [19, 54, 56]. However, at the time of behavioral testing, the rTg4510 mice in this study did not display any deficits in Y-maze (Fig. 1c), though cognitive impairments were observed in the MWM, as the rate of learning the task was slower in rTg4510 compared to WT mice. While there was only a trend towards improved performance in MWM (Fig. 1b), this may suggest that a more chronic dose of DFP may be required to improve memory function. Indeed, a higher dose of DFP (ranging between 125– 150 mg/kg), is reported to rescue memory deficits in animal models of iron-induced neurodegeneration, PD and AD [36, 58, 59]. In rTg4510, cognitive deficits and tau pathology are more aggressive in females and occurs earlier compared to males . This study used both males (n = 4 – SSV treated; n = 5 – DFP treated) and females (n = 4 both SSV and DFP treated). However, no differences between gender were observed in the behavioral tasks, possibly due to insufficient numbers. MWM performance may also suggest that cognitive function is not significantly impaired at 8 months in rTg4510 and may therefore obscure any therapeutic benefits of DFP in this behavioral task. Future studies may need to utilize larger group sizes and/or other behavioral tasks, such as object recognition, to further examine the effect of DFP on learning and memory.
In addition to memory loss, patients with AD and FTD experience behavioral symptoms such as agitation, anxiety, and hyperactivity [61, 62]. The rTg4510 mice exhibit high levels of locomotor hyperactivity and anxiety-like behavior . In this study, the open field test revealed slower walking speeds in DFP treated mice (Fig. 1g), which could be interpreted as reduced hyperactivity/anxiety and improved exploratory behavior. However, this type of behavioral test generally assesses the distance travelled as a measure of hyperactivity and exploratory behavior , which was not significantly different between rTg4510 treatment groups. The open field test only provides an indication of anxiety-like behavior and future studies should incorporate more direct tests to examine the anxiolytic effects of DFP such as elevated plus maze. The potential therapeutic benefits of iron chelation have been supported by several clinical trials. In AD patients, the metal chelator deferoxamine was reported to slow the clinical progression of dementia over a 24-month period . More recently, DFP has shown efficacy in phase two clinical trials in PD  and is currently in clinical trial for AD patients (Deferiprone to Delay Dementia (The 3D Study); ClinicalTrials.gov Identifier: NCT03234686).
There are several properties of DFP that make it a favorable candidate for iron chelation therapy, including its oral bioavailability and high affinity for iron (Kd ≈ 10–35 M) . While high affinity iron chelators can have severe side effects such as audiovisual impairments, hypersensitivity reactions, lethargy, nausea, and abdominal pain over long term treatments , DFP is reported to be safe and well tolerated over long durations with limited side effects . In addition, DFP is relatively small in size, crosses the blood-brain barrier  and can redistribute iron to apotransferrin  thereby preventing systemic iron loss. It has also been reported to target iron in a region-specific manner [31, 64]. For example, DFP treatment reduced iron levels in the substantia nigra in PD patients, assessed using magnetic resonance imaging (MRI), but not the caudate and pallidum (regions not associated with PD) . In Friedreich’s ataxia patients, DFP reduced iron levels within the dentate nucleus and did not affect iron levels within the thalamus and putamen . In line with these studies, our results revealed a significant decrease in brain iron in rTg4510 treated with DFP compared to vehicle treated mice (Fig. 2a,b). However, further investigation is required to examine the anatomical distribution of iron in rTg4510 following DFP treatment. Based on the chelating properties of DFP and our previous data that has demonstrated significant increases in cortical and hippocampal iron levels in rTg4510, we hypothesize that DFP may reduce iron within these regions, which are also primarily affected in AD and in rTg4510 mice. As a metal chelator, DFP shows affinity for other biologically relevant metals such as copper and zinc  and as such are currently under investigation.
Elevations in brain iron are usually accompanied by an increase in ferritin— the main iron storage protein. Iron levels modulate the biosynthesis of ferritin which in turn regulates the availability of iron for cellular processing to prevent iron deficiency and iron overload . In tauopathies, such as PSP and Huntington’s disease, ferritin is significantly elevated compared to healthy controls, which may be a compensatory response to the disease-associated iron overload. In rTg4510 mice, ferritin is significantly increased compared to WT (Fig. 3a), which corresponds with increased brain iron levels in this mouse model (Fig. 2a,b). Reducing brain iron in rTg4510 with DFP resulted in decreased ferritin levels (Fig. 3a). Ferritin stores iron in its redox-inert state (Fe3 +; ferritin-iron) and ferritin-iron is suggested to facilitate tau aggregation and NFT formation [16, 52]. In this study DFP decreased ferritin-iron levels (Fig. 2c), and this was accompanied by a significant decrease in pathological tau in the form of sarkosyl insoluble tau (Fig. 4e). DFP is a strong Fe3 + chelator  and is reported to induce ferritin degradation and release ferritin iron . The upregulation of ferroportin is also reported to promote ferritin degradation in vitro . In rTg4510, DFP increased ferroportin protein levels (Fig. 3a,b), suggesting a possible pathway that may result in decreased ferritin and brain iron levels. Further work is required to understand how DFP effects other iron associated proteins such as APP in rTg4510.
Studies have demonstrated that the phosphorylation of tau at different sites has a different impact on the biological function of tau under normal and disease conditions . For example, phosphorylation of tau at Thr231 inhibits its binding to microtubules . This site along with Ser396 promotes self-aggregation of tau into filaments and phosphorylation of tau at Ser202/Thr205 is associated with PHF . There are several kinases involved in tau phosphorylation; candidate tau kinases include GSK3β and CDK-5, which are reported to phosphorylate the sites mentioned above and promote tau aggregation [75, 76]. Both GSK3β and CDK-5 are found to be associated with early tau deposits and tangles in brain samples from PSP, Pick’s disease, and AD [9, 20] and are reported to be upregulated in the presence of iron [53, 56, 77– 80]. Our results revealed a decrease in tau phosphorylated at Ser396 and Ser202/Thr205 in the soluble hippocampal fraction (Fig. 4a) following DFP treatment, which was accompanied by downregulation of CDK-5 (Fig. 5a). However, while no significant decrease in phosphorylated GSK3β was evident within the hippocampus (Fig. 5a), it has been reported that the phosphorylation of tau by CDK-5 primes tau for phosphorylation by GSK3β . Interestingly, in the cortical region, there was an increase in the inactive form of GSK3β (Fig. 5b), but no significant decrease in tau phosphorylated at sites Ser396 and Ser202/Thr205 (Fig. 4b). We next examined the tau phosphatase PP2A, which is a heterotrimeric Ser/Thr protein phosphatase and is downregulated in AD . Compared to age-matched WT vehicle treated mice, PP2A A (structural subunit) and B (regulatory subunit) are decreased in rTg4510 (data not shown). While there was an increase in PP2A A in DFP tau mice within the hippocampus (Fig. 6a), no changes were observed in PP2A B or the catalytic subunit (PP2A C). PP2A accounts for 70% of tau phosphatase activity in the brain and is a critical regulator of the phosphorylation status of tau . The structural subunit of PP2A plays an important role in escorting the catalytic subunit and mediates interactions with the regulatory subunit and other substrates (such as tau) . The stability of PP2A is regulated by its methylation (promotes assembly) and phosphorylation (inhibits assembly). In clinical cases of AD and PSP, methylated-PP2A is reportedly decreased and protein phosphatase methylesterase-1 (PME-1, which catalyzes the demethylation of PP2A) is found to be increased in these disorders compared to healthy controls . Furthermore, demethylated-PP2A interacts weakly with phosphorylated tau and may promote PHFs and NFT assembly in tauopathies . Our results revealed a decrease in PME-1, in both the hippocampus and cortex in DFP treated rTg4510 mice (Fig. 6a,b). While the methylation status of PP2A requires further investigation in this study, we hypothesized that the decrease in PME-1 may lead to an elevation in methylated-PP2A in DFP treated mice. This may indicate a pathway that DFP effects that leads to a decrease in tau phosphorylation (Fig. 6) in rTg4510 mice. Lastly, the peptidyl prolyl isomerase (Pin1) is suggested to regulate interactions between PP2A and tau and is downregulated in AD  and in rTg4510 mice (compared to age-matched WT mice, data not shown). It was hypothesized that there would be an increase in Pin1 protein levels; interestingly our results revealed a decrease in Pin1 in DFP treated rTg4510 (Fig. 6a). It is speculated that Pin1 interacts differently with mutant and normal functioning tau. Knock out/knock down of Pin1 in transgenic tauP301L mice (mixed 129/SvJ and C57BL/6 background) was reported to abolish tau pathology, while in transgenic mice expressing human WT tau, it lead to NFT formation . Together, this data suggests that regulating iron with DFP may mediate multiple pathways involved in tau phosphorylation and subsequently prevent tau aggregation.
This study provides valuable insight on the effects of DFP on tau in a mouse model of tauopathy. Our data demonstrates that early intervention with DFP prevents iron accumulation, reduces pathological tau, and that the compound may potentially have anxiolytic properties. Furthermore, this research opens possible avenues for clinical trials of DFP in tauopathies to investigate its effect of early intervention the role of iron in disease progression.
The Florey Institute of Neuroscience and Mental Health acknowledge the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant. The authors would like to thank A/Prof Blaine Roberts and the Neuroproteomics Facility (Florey) for the use of the SEC-ICPMS equipment and Irene Volitakis for generating the preliminary ICP-MS data. SSR would also like to thank the Core Animal Services staff and other members of the Florey and The Melbourne Dementia Research Centre for their assistance throughout the project and was supported by a Parkinson’s Victoria— Argyrou Family PhD Partner Scholarship.
Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/20-0551r1).
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