Introduction
Neurodegenerative diseases are often caused by protein misfolding resulting in the accumulation of protein deposits, such as amyloid fibrils [
1]. Alzheimer disease (AD), the most common form of dementia, is characterized by two types of pathological protein deposits, extracellular amyloid plaques consisting of Amyloid-β (Aβ) peptide and intracellular neurofibrillary tangles (NFTs) consisting of Tau [
2]. Tau is a microtubule (MT)-binding protein that promotes and stabilizes the assembly of MTs and the regulation of axonal transport [
3,
4]. Binding of Tau to MT is regulated by post-translational modifications, especially by phosphorylation [
5]. Tau phosphorylation negatively regulates the binding of Tau to MT; as a result, MT stabilization and axonal transport are compromised and Tau aggregates into insoluble fibrils.
The assembly of Tau protein into paired helical filaments (PHFs) depends on two short hexapeptide sequence motifs,
306VQIVYK
311 (PHF6) and
275VQIINK
280 (PHF6*), which are located at the beginning of the third and second repeat regions, respectively [
6‐
9]. These motifs are important for filament assembly as they form a β-sheet structure [
7,
10,
11]. Accordingly, the repeat domain (RD) of Tau is sufficient for forming PHFs which are thought to contribute to AD pathology [
12‐
17]. In addition, there is evidence that Tau-induced neuronal toxicity is predominantly caused by smaller soluble oligomeric species formed in the Tau aggregation pathway rather than large protein deposits [
18‐
22].
In recent years, a large number of potential therapeutic substances have been developed for the prevention of Aβ-based pathology in AD, such as Aβ production inhibitors, Aβ aggregation inhibitors, or Aβ antibodies [
23‐
26]. Most of them failed in clinical trials due to side effects and lack of therapeutic success [
27,
28]. Recently, only one Aβ drug candidate (aducanumab) obtained preliminary approval from the US Food and Drug Administration (FDA), while its effectiveness will still have to be proven [
29‐
31]. In contrast to Aβ pathology, pathological changes in Tau correlate well with cognitive decline [
32,
33]. A potential approach to developing Tau-directed therapies against dementia could involve targeting the beginning of the Tau fibrilization cascade, thereby preventing the formation of toxic oligomeric species which are hypothesized to propagate from cell to cell in a prion-like manner [
22,
34].
A large number of Tau aggregation inhibitors have already been described as potential therapeutic agents [
35‐
39]. In particular, D-amino acid peptides are emerging as promising drug candidates [
40‐
42]. At least some D-peptides can be administered orally [
43,
44] and are able to cross the blood-brain barrier in combination with high bioavailability [
41,
43‐
48]. A promising D-peptide designated RD2, a derivate of D3 selected against D-Aβ-peptide using mirror image phage display, was shown to reduce plaque formation and inflammatory reactions and led to a significant improvement in the cognitive abilities of transgenic mice [
43,
46‐
49]. The RD2 peptide has successfully completed phase 1 clinical trials. Several Tau-directed D-peptides have also been characterized in pre-clinical studies [
8,
9,
50,
51]. While the D-peptides TLKIVW [
9] and TD28 [
50] were developed to bind PHF6, MMD3 [
52] was selected against the hexapeptide sequence motif PHF6*.
In the present study, we selected a peptide against the wild type full-length Tau (TauFL) protein in order to develop potential inhibitors acting on the early stages of a pathological fibrillization cascade. First, we selected a novel L-peptide ISAL1 using a phage display selection procedure with TauFL as a target and synthesized its D-amino acid counterpart, ISAD1, and its retro inversed version, ISAD1rev. We found that ISAD1 and its reversed form inhibit not only fibrillization of TauFL, but also of several disease-associated mutant Tau variants. Furthermore, our novel D-peptides penetrate neuronal cells and prevent cytotoxicity induced by externally added pro-aggregant repeat domain Tau mutant ΔK280 (TauRDΔK) fibril preparations as well as of internally expressed TauRDΔK. Thus, our data suggest that our novel peptide ISAD1 has an improved potential for treatment of AD, whereas ISAD1rev inhibited Tau fibrillization only moderately.
Materials and methods
Tau protein expression and purification
The gene of the human Tau
FL isoform, encoding 441 amino acids (Tau 2N4R, Uniprot P10636-F), and the pro-aggregant mutant Tau
RDΔK were commercially synthesized and cloned into a pET28A(+) vector (Novagen, San Francisco, USA). Tau protein expression and purification was carried out according to Margittai et al. and Barghorn et al. with some modifications [
53,
54]. The purity of the protein was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined by the bicinchoninic acid (BCA) method.
Preparation of TauFL fibrils for ELISA
The fibrillization was started by incubating 10 μM TauFL in 20 mM HEPES buffer, pH 6.8 with 2.5 μM heparin (16000 daltons (Da), H16K) at room temperature (RT) for 24 h. Fibril formation was verified using the Thioflavin-T (ThT) assay. For ThT fluorescence measurements, 20 μL of the sample with 10 μM ThT was pipetted into a black 96-well half area clear flat-bottom plate. TauFL without addition of heparin was used as a negative control. The fluorescence measurement was performed using a photometer POLARstar optima (BMG-Labtechnologies, Ortenberg, Germany), and excitation/emission wavelengths were set at 440/490 nm.
Phage display selection
Selection of novel peptides binding to recombinant TauFL was performed by a phage display selection method. The target protein TauFL was prepared in 50 μg/ml concentration in coating buffer (0.1 M NaHCO3, pH 8.6) and immobilized on 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) overnight at 4 °C. The next day, the coating solution was discarded and each well was completely filled with blocking buffer (0.1 M NaHCO3, pH 8.6, 5 mg/ml bovine serum albumin (BSA)). After blocking for 1 h, the wells were washed 6 times with tris-buffered saline with Tween20 (TBST: TBS + 0.1 % [v/v] Tween-20). One hundred microliters of a 100-fold dilution of the phage library, displaying 12-mer random peptides (Ph.D.-12, New England Biolabs, Frankfurt a.M., Germany), was incubated for 1 h with agitation. To remove unbound phages, the wells were again washed 10 times with TBST. Bound phages were then eluted using 0.2 M Glycine-HCl (pH 2.2) with 1 mg/ml BSA. The phages were then amplified according to the manufacturer’s instructions (New England Biolabs, Frankfurt a.M., Germany) and used for the following 3 panning rounds.
Single phage clone ELISA
A single clone binding assay was performed by enzyme-linked immunosorbent assay (ELISA) with the supernatant of amplified phage clones from selection round three and four to identify the single phages showing the strongest binding to TauFL. Therefore, TauFL (50 μg/ml) diluted in coating buffer was immobilized on polystyrene 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) overnight at 4 °C. The control wells contained only buffer without target protein. The next day, the coating solution was discarded and incubated with 100 μl of blocking buffer (1 % BSA in 50 mM Tris, 150 mM NaCl, pH 7.6) for 1 h. To avoid the selection of possible BSA-binding phages, the supernatant of third and fourth selection round was mixed with blocking buffer in a ratio of 1:1 and pre-incubated for 20 min at RT with gentle agitation. After blocking, the plate was washed 6 times with TBST and 100 μl of the pre-incubated diluted samples was transferred into the appropriate wells. Subsequently, the plates were incubated for 1 h at RT with gentle agitation, followed by a washing step with TBST (6 times with 100 μl). Afterwards, 150 μl of the anti-M13 antibody dilution horseradish peroxidase (HRP)/anti-M13 (Monoclonal Conjugate; GE-Healthcare, Freiburg, Germany) was added to the adequate wells for 1 h at RT, followed by 6 times washing with TBST. The anti-M13 antibody was diluted 1:5000 in blocking buffer. Detection was conducted by measuring the conversion of the substrate tetramethylbenzidine (TMB) by HRP. One hundred microliters of the substrate solution was transferred to the respective sample wells. The enzyme reaction was stopped by adding 100 μl of 20 % [v/v] H2SO4. The absorption of the reaction product was measured at 450 nm in a Multiscan Go (Thermo Fisher Scientific, Darmstadt, Germany) microplate reader.
Positive phages from ELISA were selected for DNA isolation. DNA sequencing was performed at LGC Genomics (Berlin, Germany). The DNA sequences were translated into 12-mer amino acids, aligned using CLUSTAL Omega program (
http://www.ebi.ac.uk/Tools/msa/clustalo/), and analyzed using the SAROTUP Database (an abbreviation of “Scanner And Reporter Of Target-Unrelated Peptides”) [
55].
Peptides
The selected peptide sequences obtained from the phage display selection were first synthesized as L-amino acid peptides. ISAL1 to 4 and ISAL9 (Table
1) were purchased from JPT Peptide Technologies (Berlin, Germany). L-peptides ISAL5 to ISAL8 and ISAL1sam were synthesized in the lab of Prof. Eichler as described in the supplement. Later, unlabeled and fluorescein amidites (FAM)-labeled peptides ISAD1 and ISAD1rev (all amino acids of both peptides are D-enantiomers) with > 95% purity were purchased from JPT Peptide Technologies as well. The FAM label is attached to the C-terminus of the peptides with an additional lysine residue in between. PHF6 and PHF6* were purchased as N-terminally acetylated hexapeptides to allow self-aggregation.
Table 1
Selected L-peptides from phage display selection against TauFL
1 | SVFKLSLTDAAS
| 1/80 | + | ISAL1 |
2 | NHDMDLLVWWMN
| 1/80 | +/- | ISAL2 |
3 | NWSMPGMTQGFL
| 13/80 | - | ISAL3 |
4 | DFHQRDDDSQQA
| 1/80 | - | ISAL4 |
5 | AMYQFSRNPHLP
| 3/80 | - | ISAL5 |
6 | VSPAWDARTRSA
| 2/80 | - | ISAL6 |
7 | MTPHGNSKTPSG
| 1/80 | - | ISAL7 |
8 | HDWYRSPRMGLF
| 1/80 | - | ISAL8 |
9 | DLSHGQDLMHHH
| 1/80 | - | ISAL9 |
10 | SASVTSKFDALL
| - | - | ISAL1sam |
Detection of peptide binding to Tau conformers using ELISA
A 96-well microtiter plate (Greiner Bio-One GmbH, Frickenhausen, Germany) was coated with 5 μg/ml TauFL or fibrils in coating buffer for incubation overnight at 4 °C. For investigation of the binding properties to the hexapeptide, 5 μM PHF6 was coated overnight. After three times washing with 300 μl phosphate-buffered saline with Tween20 (PBST: PBS with 0.1 % [v/v] Tween20), the plate was blocked with 3 % [w/v] BSA in PBS for 1 h at RT, followed by further washing steps. Subsequently, 100 μl FAM-labeled peptides was added at a final concentration of 0.1 to 20 μg/ml in PBST and incubated for 1 h at RT. The plate was washed three times with PBST before 100 μl sheep anti-fluorescein isothiocyanate (FITC) HRP-conjugate (1:5000 dilution in PBST; AbD Serotec, Puchheim, Germany) was added and incubated for 1 h at RT with gentle agitation. Again, the plate was washed for three times, followed by addition of the TMB substrate. The reaction was stopped with 20 % [v/v] H2SO4 and absorbance was measured at 450 nm.
In silico modeling of binding mode of ISAD1 to PHF6 fibrils
Modeling of the ISAD1 peptide complex with PHF6 was guided by previous models of PHF6 with D-peptides TLKIVW [
9] and TD28 [
50]. ISAD1 was modeled in the same extended geometry and the same binding register as the TLKIVW and TD28 D-peptides. The binding register was chosen according to the position of a conserved Φ+Φ (Φ hydrophobic residue; + central positively charged residue) sequence motif present in all these PHF6-binding peptides. Modeling was performed with Sybyl 7.3 (Tripos Inc., St. Louis, USA) and UCSF Chimera [
56]. Structural analysis of the complexes between the PHF6 oligomers and the docked peptides was performed with VMD [
57].
Fibrillization of TauFL monitored by ThT assay
Tau aggregation assays were performed under reducing conditions. Before the addition of heparin and peptides, a final concentration of 1 mM dithiothreitol (DTT) was added to the Tau protein solution and heated at 95 °C for 10 min. For TauFL inhibition assays, 5 μM TauFL were incubated in HEPES buffer (pH 6.7) in the presence of 1.25 μM heparin at 37 °C for 48 h with or without novel D-peptides (ISAD1 and ISAD1rev) at different concentrations (1 nM to 200 μM). Final concentration of 10 μM ThT was used for monitoring fibrillization. In case of the two hexapeptides, 5 μM PHF6 and 5 μM PHF6*, respectively, without addition of the aggregation inducer heparin (16000 Da) were used for monitoring the fibrillization process. The assays were performed with 50-μl sample volume per well in a 96-well half area microtiter plate (Greiner Bio-One GmbH, Frickenhausen, Germany). The fibrillization of TauFL was monitored by ThT, and the relative fluorescence intensity of ThT was read out at 440 excitation/521 emission nm in a BMG microplate reader (BMG Labtech, Ortenberg, Germany).
Fibrillization of TauRDΔK and Tau mutants monitored by Thioflavin-S (ThS) assay
For Tau fibrillization inhibition assays, 10 μM Tau mutant protein (TauRDΔK, TauFLΔK, TauFL-A152T, TauFL-P301L) was incubated in BES buffer (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 7) in the presence of 2.5 μM heparin at 37 °C for 24 h with and without novel D-peptides (ISAD1 and ISAD1rev) at different concentrations (1 nM to 200 μM). Final concentration of 20 μM ThS was used for monitoring fibrillization. The assay was performed with 40-μl sample volume per well in a 384-well microtiter plate (Thermo LabSystems, Dreieich, Germany). The fibrillization of Tau was monitored by ThS and the relative fluorescence intensity of ThS was read out at 440 excitation/521 emission nm in a Tecan micro titer plate reader (Tecan, Männedorf, Switzerland).
Dynamic light scattering (DLS)
After the ThS assay (end time point, 24 h), the samples were used for DLS measurements. Twenty microliters of the sample was placed in a quartz batch cuvette (ZEN2112) and the measurement was performed at 25 °C in a Zetasizer Nano S instrument (Malvern Instruments, Herrenberg, Germany). The sample was thermally equilibrated at 25 °C for 2 min. The mean value of the intensities of an individual sample was determined over 3 measurements with 15 runs each. Analysis and averaging of the collected data were performed with the Zetasizer software 7.11 (Malvern Instruments, Herrenberg, Germany) and the result is represented as a volume graph. TauRDΔK fibrils (Tau+heparin) formed in the absence of D-peptides was used as a positive control.
Pelleting assay and western blot
After the ThS assay (end time point, 24 h), 70 μl of each sample (pooled together from 2 wells) was centrifuged in a Beckmann coulter (Optima Max Ultra Centrifuge, TLA 100.3 rotor) at 61,000 rpm for 60 min at 4 °C. After centrifugation, the supernatant was separated from the pellet. Then, the pellet was dissolved in BES buffer in an equal volume as the supernatant. For the following western blot, 12-μl samples were mixed with 3-μl SDS-sample buffer (5x), heated for 5 min at 95 °C, and loaded onto a 8–16% SDS tris-glycine-gel (BioRad, Feldkirchen, Germany). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After the transfer, the membrane was blocked in 5% non-fat dry milk. After washing the membrane three times for 10 min with TBST, the primary pan-Tau K9JA antibody (1:5000; Agilent, Waldbronn, Germany) was incubated for 1 h at RT with gentle agitation, followed by again 3-times washing with TBST. For detection on western blot, the secondary antibody (goat anti-rabbit HRP, Agilent, Waldbronn, Germany) was incubated in a 1:2000 dilution for 1 h at RT with shaking. After another washing step (3 times with TBST), imaging was done with chemiluminescence substrate (AmershamTM, ECL Prime Western Blotting Detection Reagents, GE Healthcare, Chicago, USA) and Image QuantTM LAS 4000 (GE Healthcare, Chicago, USA). The quantification of intensities was performed using ImageJ.
Cell culture
Cells of a Neuro-2a (N2a) Tau
RDΔK inducible cell line (N2a-Tau
RDΔK) [
58] were grown in minimal essential media (MEM, Sigma Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS), 5 ml non-essential amino acids (PAA, Pasching, Austria), and 1X penicillin and streptomycin antibiotics at 37 °C with 5% CO
2. The inducible N2a cell line expressing Tau
RDΔK require antibiotics geneticin G418 (300 μg/ml) and hygromycin (100 μg/ml). Tau
RDΔK expression was induced by incubating cells with 1 μg/ml doxycycline (Dox) in the studies on the detoxification of cellular Tau
RDΔK by the D-peptides; otherwise, these cells did not express the Tau
RDΔK protein.
Cell viability assays
Cell viability was analyzed in accordance with the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany; cell proliferation kit II (MTT)). This assay is based on the cleavage of the yellow tetrazolium salt MTT into purple formazan dye by metabolic active cells. The color changes only in viable cells and can be directly quantified using a scanning multiwell spectrophotometer. In all experiments, the cells were grown as described previously. The cells (25,000 cells/well or 80% confluence) were plated on poly D-lysine-coated 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) for stronger attachment from overnight to 24 h at 37 °C. Fibrillized TauRDΔK was generated by incubating 200 μM TauRDΔK in BES buffer at 37 °C for 24 h in the presence of 2 mM D-peptides (TauRDΔK:peptide = 1:10). Successful fibrillization was verified using ThS (5 μM TauRDΔK, 10 μM ThS) measurement. The aggregated TauRDΔK (10 μM final concentration), TauRDΔK (10 μM) + peptide (100 μM) samples (ISAD1 and ISAD1rev), buffer only (negative control, set to 100% cell viability), and TritonX-100 (2%, cytotoxic agent, positive control) were incubated on N2a-TauRDΔK cells (100 μl solution) for another 24 h. The cell viability was measured in accordance with the manufacturer’s protocol.
Measurement of lactate dehydrogenase (LDH) release
N2a cells expressing TauRDΔK were plated on poly D-lysine-coated 96-well plates with a density of 25,000 cells/well. At 70 to 80% confluence, the cells were treated for 24 h with various concentrations of D-peptides (25, 50, 100, and 250 μM) or TauRDΔK (10 μM final concentration) in the presence of ISAD1 and ISAD1rev (100 μM final concentration). The ability of D-peptides to neutralize the toxicity of TauRDΔK oligomers/fibrils was investigated by measuring the amount of released LDH (Roche Diagnostics, Mannheim, Germany). Therefore, 50 μL of each well was transferred to a fresh 96-well plate and 50 μL of reagent was added followed by a 30-min incubation period at RT. Finally, 50 μL of stop solution (1 N HCl) was added and absorbance was recorded at 492 nm (TECAN spectrofluorometer, Männedorf, Switzerland). Absorbance values were corrected by background values and the percentage of LDH release was calculated.
Reactive oxygen species (ROS) measurements
Toxic Tau oligomers and fibrils can induce the production of superoxides and peroxy radicals in cells which can be measured with fluorescent dye dichlorodihydrofluorescein (DCF). N2a-TauRDΔK cells were plated on D-lysine-coated 96-well plates. At 70 to 80% confluence, the cells were washed once with warm PBS and then incubated with 20 μM of DCF (Abcam, Cambridge, UK) diluted in 1X dilution buffer for 30 min at 37 °C. After 30 min, the cells were washed once with 1X PBS. After washing, the cells were incubated with desired concentrations of different samples (10 μM oligomers/fibrils ± treated with 100 μM D-peptides or controls) for 30 min. The cytotoxic agent TBHP (tert-butylhydroperoxide) was used as the positive control. The fluorescence intensity was measured using a spectrofluorometer (Tecan, Männedorf, Switzerland; excitation at 485 nm and emission at 535 nm).
Effect of ISAD1 and ISAD1rev on cellular TauRDΔK aggregation
After the N2a-TauRDΔK cells reached the desired confluence (25,000 cells/well or 80%), intracellular TauRDΔK expression was induced by the addition of 1 μg/ml Dox. The cells were plated on poly D-lysine-coated 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and treated with ISAD1 and ISAD1rev in a concentration range of 25 to 250 μM. TritonX-100 (2%) was used as a positive control. The incubation and TauRDΔK expression time was 72 h. Following incubation, cell viability was studied by measuring MTT and LDH release in accordance with the manufacturer’s protocol.
Discussion
Even through Alois Alzheimer characterized AD more than 100 years ago, there is currently no curative treatment available
. These circumstances and the predicted steady increase in the number of AD patients highlight the urgency of developing a causal AD therapy. Recently, the Aβ antibody aducanumab obtained preliminary approval from the FDA. Nevertheless, the approval is controversial and the efficacy of aducanumab has to be proven in further studies [
29‐
31]. A number of potential therapeutic substances targeting the Aβ pathology appeared successful in pre-clinical models, but failed in clinical trials [
64‐
66].
Apart from the amyloid peptide Aβ, another compelling target for AD therapy is Tau. A large body of evidence suggests that Tau pathology has a strong correlation with the disease progression and clinical symptoms [
32], which has led to increased research into Tau-associated compounds [
35,
67,
68]. Tau pathology not only occurs in AD but also in other neurological diseases, called tauopathies [
69,
70]. The use of small D-amino acid peptides to prevent the pathological fibrillization of Tau may provide an alternative to small molecule non-peptide compounds [
71,
72]. D-peptides are proven to be protease stable and less immunogenic than L-peptides which make them suitable for in vivo applications. The novel D-amino acid peptides we described here inhibited the aggregation not only of Tau
FL, but also of disease-relevant Tau mutants.
ISAD1 and its synthesized reversed form show binding to both non-fibrillized Tau
FL and filaments of Tau
FL in ELISA studies. Both D-peptides bind to Tau
FL filaments with an estimated EC
50 in the low micromolar range (Fig.
1). We found that ISAD1 binds to the PHF6 motif of Tau (Sup. Fig.
3A) and inhibits the fibrillization of PHF6, but not of PHF6* despite the close vicinity and high sequence similarity of these two hexapeptides. PHF6 (
306VQIVYK
311) is known to strongly promote Tau aggregation into β-structured filaments [
6,
7], while most other parts Tau protein are unstructured. This could be the reason why phages in the selection process bind more preferentially to this structured sequence motif.
We illustrated the binding mode of ISAD1 to PHF6 fibrils by in silico modeling according to previously described Tau aggregation inhibitor peptides [
9,
50] selected against PHF6. The modeling data indicated a similar sequence motif and binding mode in blocking PHF6 fibrillization as previously described PHF6-addressing D-peptides TLKIVW [
9] and TD28 [
50]. The modeling demonstrated the similarity between the three D-peptide complexes with PHF6. The formation of stabilizing backbone hydrogen bonds to PHF6 is allowed by binding in parallel β-sheet conformation. Since the interface is formed between a D- and L-peptide, a rippled β-sheet is created [
73] which was shown by quantum chemistry calculations to exhibit favorable interaction energies in the PHF6 system [
74]. A second effect, which blocks lateral fibril growth, results from the steric repulsion between the D-peptides and the second stack of β-strands (Fig.
3). It has been suggested that such steric repulsion between L2 in TLK or M6 in TD28, respectively, and V306’/I308’ of the second stack of β-strands represents key feature blocking further fibril growth [
9,
50]. For ISAD1, the corresponding residue F3 shows a similar spatial orientation that may lead to a similar effect preventing the fibril or oligomers from further growth. Notably, this sequence motif was absent in the PHF6*-binding peptides MMD3 and MMD3rev reported in Malhis et al. [
52].
Our NMR data show that ISAD1 does bind Tau monomers in solution only moderately. Since the ELISA was carried out with high local concentrations of Tau that could lead to oligomerization, it is likely that Tau was immobilized as a mixture of monomeric and oligomeric forms, and the affinity measured by ELISA reflects the binding of ISAD1 to this mixture. The NMR spectra also support the claim that ISAD1 promotes the formation of high molecular weight oligomers of Tau. The reference Tau
FL spectrum contained a mixture of monomeric and oligomeric Tau, oligomer formation being favored in the absence of reducing agents. No marked increase in
I/
Io values was evident and there are generally many
I/
Io values below 1 especially for the mole ratio 1:30 Tau:ISAD1 (Sup. Fig.
2B). This suggests that at high concentrations of ISAD1 the affinity of ISAD1 for monomeric Tau is moderate, and Tau oligomer formation is favored in the presence of ISAD1.
Aggregation of Tau is the primary hallmark for the disease pathology in AD and other tauopathies. A significant inhibition of the aggregation by therapeutic molecules is considered beneficial. Our novel peptide ISAD1 inhibited the fibrillization of Tau
FL and pro-aggregant Tau
RDΔK (IC
50 of ~3 μM) in concentrations which are typical for peptide inhibitors. Interestingly, ISAD1 inhibited fibrillization of Tau more efficiently than other PHF6 addressing peptides: TLKIVW 54.1 μM [
9] and APT 5.9 μM [
50].
Familial mutations of Tau cause the rapid aggregation and progression of the diseases. This makes it necessary to find a D-peptide that can inhibit the aggregation of such pro-aggregant mutants of Tau. To date, only our D-peptides have been proven to simultaneously inhibit the aggregation of three pro-aggregant Tau forms (Tau
FLΔK, Tau
FL-A152T, Tau
FL-P301L) found in FTD, AD, PSP, and FTDP-17 diseases (Fig.
5A–C) in a concentration-dependent manner which makes our D-peptides unique and proves their therapeutic potential. Mutations ΔK280 and P301L are each located on R2 of the repeat domain, close to a possible binding site of ISAD1 while mutation A152T is located in the proline-rich domain within Tau. The inhibitory potencies are almost the same for Tau
FL and Tau
RDΔK (IC
50 of 2.9 μM), despite the fact that the two Tau constructs have different aggregation efficiencies.
Based on DLS and pelleting assays, we observed that the ISAD1 and its reversed form prevent fiber formation of Tau
FL and Tau
RDΔK and instead induce the formation of higher molecular weight off-pathway Tau oligomers (Fig.
6B) which are non-fibrillar in nature (ThS negative), similar to other previously described peptides [
43,
52]. We had demonstrated by atomic force microscopy (AFM) that in case of the MMD3 peptide, the aggregates formed are amorphous clumps of off-pathway high-n oligomers [
52] which cause large signals in DLS experiments, also observed for the ISAD1and its reversed form.
In order to be used as a therapeutic agent for neurodegenerative diseases, the D-peptides need to cross the blood-brain barrier (BBB), should be actively taken up by neurons, and should be non-toxic to brain cells. Peptides do in general not cross membranes very well, but the naturally occurring transcription factor domain penetratin, HIV-Tat, or synthetic cationic peptides have been described as cell penetrating peptides [
75‐
77].
Interestingly, D-peptides investigated previously have also been shown to cross the BBB in combination with high bioavailability and drug exposure to the brain [
45]. The novel D-peptides developed in this study were demonstrated to cross the membranes of N2a-Tau
RDΔK cells efficiently (Fig.
7), although the mechanism of penetration is still unclear. Since the N2a-Tau
RDΔK cells take up the peptides uniformly, we assume that the uptake of the peptides occurs through bulk endocytosis. Our D-peptides neither caused a change in cell viability (Fig.
8A) nor cell membrane integrity (Fig.
8B) even at high concentrations, suggesting high tolerability by neuronal cells. Additionally, ISAD1 and its reversed form prevented the cytotoxic potential of Tau aggregates by promoting off-pathway high-n oligomers, evident from the enhanced cell viability (Fig.
9A) in N2a-Tau
RDΔK cells, improved cell membrane integrity (Fig.
9B), and prevent the ROS elevation (Fig.
9C). Compared to other Tau-derived peptides previously published [
78‐
80], ISAD1 demonstrates similar prevention of toxicity by maintaining cell viability and metabolic activity also in the presence of cellular Tau
RDΔK expression. The N2a-Tau
RDΔK cell model of Tau pathology is well established and useful in the screening and study of therapeutic compounds, such as Tau aggregation inhibitors [
81,
82]. Earlier studies with N2a-Tau
RDΔK cells had shown a time-dependent increase in cell death [
81]. In N2a-Tau
RDΔK cells, the expression of Tau
RDΔK starts at 24 h after protein induction and its overexpression is the trigger for dimerization and aggregation. The treatment of N2a-Tau
RDΔK cells with ISAD1 and its reversed form decreased the toxicity of cellular Tau
RDΔK in a dose-dependent manner, as seen by the parameters of cell viability and LDH release (Fig.
10). This demonstrates that toxic effects of Tau
RDΔK aggregates can be suppressed by the D-peptides.
In conclusion, especially our novel D-amino acid peptide ISAD1 inhibits fibril formation of pro-aggregant toxic Tau, is non-toxic to cells, and prevents the toxic effects of Tau by promoting off-pathway aggregate formation which makes our D-peptide a potential therapeutic molecule to prevent Tau pathology in AD and other Tau-associated diseases. However, ISAD1rev might have a limited therapeutic potential due to significant lower inhibition of Tau aggregation. More details like BBB transfer and the efficacy of ISAD1 have to be investigated in future in vivo treatment studies to further elucidate the peptide mechanism of action and its full therapeutic potential.
Limitations
This report has some limitations. The study resulted in very promising findings in vitro and in cell culture; however, BBB permeability and efficiency in vivo still need to be investigated.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.