Conformation pattern changes in R1-pS262 tau peptide induced endogenous tau aggregation,synaptic damage,and cognitive impairments

Abstract

Background

To date, the effect of tau phosphorylation at different amino acid sites on the conformation and function of tau is still unclear in Alzheimer's disease (AD). Protein fingerprinting, also known as the protein folding shape code (PFSC) method, is a protein structure prediction technique based on protein sequence, which can reveal proteins’ most likely spatial conformation.

Objective

To investigate the effect of phosphorylation on tau protein conformation using PFSC technology and further analyze the differences in the effect of phosphorylation on tau aggregation at specific sites.

Methods

We performed a conformational analysis of wild-type and simulated mutant hTau441 using the PFSC method and synthesized the phosphorylated and non-phosphorylated tau fragments by the chemical solid phase method.

Results

We found that the number of Ser262 protein fingerprints increased from six in tau S262A to nine in tau S262E, together with increased conformational changes and enhanced flexibility. The in vitro Thioflavin S assay showed that phosphorylated tau fragments R1-pS262 possessed a stronger activity of inducing tau aggregation. In contrast to the non-phosphorylated tau fragment R1-nS262, R1-pS262 promoted endogenous tau aggregation and decreased synaptic proteins. In rats, R1-pS262 caused cognitive impairments and neuronal loss in addition to endogenous tau aggregation and synaptic damage.

Conclusions

Our study firstly reports that tau phosphorylation at Ser262 induces tau aggregation, and phosphorylated tau fragments R1-pS262 directly result in neuropathological changes. These provide new clues to the pathogenesis of tauopathy, such as AD, and a new molecular target for possible intervention.

Keywords

Alzheimer's disease phosphorylation protein folding shape code R1-pS262 tau aggregation

Introduction

Alzheimer's disease (AD) is one of the most common progressive neurodegenerative diseases and is currently one of the major health threats with a rapidly aging population worldwide. Its clinical symptoms are memory loss, abnormal mental behavior, cognitive dysfunction, decline in daily life ability, and so on.¹ Lesions caused by abnormally hyperphosphorylated tau are a necessary condition for the clinical manifestations of AD. Even in the absence of amyloid-β (Aβ), tau pathology can independently lead to dementia, and the level of neurofibrillary tangles (NFTs) positively correlated with the degree of clinical dementia in AD.^2,3 Therefore, tau pathology is central to the disease process of AD. Aggregation of abnormally hyperphosphorylated tau forms NFTs, which leads to neuronal degeneration and, ultimately, memory decline, cognitive defect, and other symptoms.⁴ However, the underlying mechanisms have not yet been fully clarified.

The protein folding shape code (PFSC) method is a promising technique for protein structure prediction based on amino acid sequence, which can predict the most likely spatial conformation of a protein based on the difference of amino acids at a specific site.⁵ The method first defines five amino acid residues as folding units, detects the characteristics of the local folding of proteins, and then extends the local folding to the entire protein system to discover all possible folding conformations. The PFSC actually represents the folded shape of five amino acid residues.⁶ This technique can achieve high throughput screening of a large number of protein data and might play an important role in accelerating biomedical research, especially in the study of drug targets for antibody vaccines.⁶ The PFSC method is a powerful tool to analyze protein conformation, which could exhibit the local structural folding features in detail. Researchers have reported that they utilized the PFSC to analyze Aβ peptide and found that mouse Aβ₄₂ is less likely to polymerize compared to that of humans.⁷

The full-length sequence of hTau40 contains 441 amino acids. Previous studies have shown that tau presents a paperclip-like structure under physiological conditions.⁸ However, under pathological conditions, tau is abnormally modified by phosphorylation, hematoxylation, and ubiquitination, which will change the conformation of tau and promote its aggregation.^9–11 However, in different tau pathologies, the conformation of the fibrillar tau proteins is different,¹² suggesting that tau possesses multiple conformations to form aggregates. In the process of AD, tau protein is phosphorylated at multiple sites, resulting in changes in the spatial conformation of the protein, which may be one of the mechanisms leading to AD. Therefore, we aimed to investigate the effect of phosphorylation on tau protein conformation using PFSC technology and further analyze the differences in the effect of phosphorylation on tau aggregation at specific sites.

In the current study, conformational analysis of hTau441 and simulated mutant proteins was performed using protein fingerprint coding technology. Thioflavin S assay combined with protein fingerprint code data prompted that the greater the number of fingerprint codes, the greater the conformational variability, the stronger the flexibility, and the easier it is to promote tau aggregation. We synthesized and chose phosphorylated tau fragments R1-pS262 and non-phosphorylated tau fragment R1-nS262 to explore tau aggregation, synaptic morphology and function, and cognition of rats, respectively, in vitro and in vivo. Our data provide new clues and molecular targets for further study of AD and other tauopathy.

Methods

Reagents

The polypeptides used here are listed in Table 1 and were generously supplied by Wuhan Baiyixin Biotechnology Co., Ltd All antibodies employed in this study are listed in Table 2.

Table 1.

Polypeptides used in this study.

Name	Site	Sequence	MW	Purity
R3-normal	306–336	VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ	3247.72	99.490%
R1-nS262	254–274	KNVKSKIGSTENLKHQPGGGK	2207.49	98.610%
R1-pS262	254–274	KNVKSKIG-pST ENLKHQPGGGK	2287.49	97.596%
CTF-nS396/nS404	390–410	AEIVYKSPVVSGDTSPRHLSN	2256.47	96.764%
CTF-pS396/pS404	390–410	AEIVYK-pSPVVSGDT-pSPRHLSN	2416.47	98.419%
AT8-nS202/nT205	190–211	KSGDRSGYSSPGSPGTPGSRSR	2180.25	95.493%
AT8-pS202/pT205	190–211	KSGDRSGYSSPG-pSPG-pTPGSRSR	2099.00	94.584%
AT100-nT212/nS214	207–217	GSRSRTPSLPT	1158.26	97.752%
AT100-pT212/pS214	207–217	GSRSR-pTP-pSLPT	1318.26	98.805%

Table 2.

Antibodies employed in this study.

Antibodies	Specificity	Type	Dilution	Sources
Tau5	Total tau	mAb	1:1500	Zen-bio
Anti-pT231	p-tau (T231)	mAb	1:1500	Zen-bio
Anti-pS262	p-tau (S262)	mAb	1:1500	Zen-bio
Anti-pS396	p-tau (S396)	mAb	1:1500	Zen-bio
GRIN1	NMDAR1	mAb	1:800	Thermo
GRIN2B	NMDAR2B	mAb	1:800	Thermo
Synaptophysin	Synaptophysin	mAb	1:800	Abcam
Synapsin-1	Synapsin-1	pAb	1:800	Abcam
PSD95	Post Synaptic Density95	mAb	1:800	Cell Signaling Technology
GAPDH	GAPDH	mAb	1:1000	Beyotime
β-actin	β-actin	mAb	1:1000	Santa-Cruz

mAb: monoclonal antibody; pAb: polyclonal antibody

Animals

Eight-week-old male Sprague Dawley rats (SD) were provided by the Animal Colony of Tongji Medical College. All animals are kept in a suitable environment with a normal circadian rhythm (ambient temperature: 18 ∼ 22°C, light cycle: 12 h /12 h) to ensure free movement, food, and water. Animals were randomly group into three groups: the R1-pS262 treatment group, the R1-nS262 treatment group, and the normal saline control group.

All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology, and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Thioflavin staining

Th-S is a fluorescent dye, when combined with a protein fiber β folded sheet, it can emit fluorescence at a wavelength of 490 nm under excitation light of 440 nm. Therefore, the Th-S method is often used to quantitatively detect amyloid fiber production. After mixing the reagent according to the experimental design, the fluorescence kinetics of Th-S can be measured with the fluorescence spectrometer. The test conditions are set as follows: constant temperature at 37°C, excitation wavelength at 440 nm, emission wavelength at 490 nm, and slit width of excitation wavelength and emission wavelength at 10 nm. The concentration of R3 peptide was 2.4 mM, the concentration of phosphorylated or non-phosphorylated peptide was 2.4 mM, the concentration of Th-S was 320 μM, the concentration of Tris-HCL (pH7.4) was 50 mM, and the concentration of heparin sodium was 10 mg/ml. The running time is 12 h, and the data is recorded every 5 min.

Primary neuron culture of hippocampus

Pregnant rats at about 17–18 days gestation were anaesthetized and euthanized. Subsequently, the hippocampal tissues of the fetal rats were carefully collected. After treatment with 0.25% trypsin solution (vol/vol), neurons were collected and then cultured in a specialized neurobasal medium supplemented with 2% (vol/vol) B-27 and 1×GlutaMAX for polypeptide treatment.

Cell culture and polypeptide treatment

The cultured HEK293/tau cells was exposed to R1-pS262, R1-nS262, or normal saline solutions for 48 h, respectively. Then the cells were lysed with RIPA lysis buffer, and the protein concentration in the resulting lysis solution was quantified by BCA method.

Intracerebral stereotaxic injection

Rats were sedated with an intraperitoneal injection (ip) of 6% chloral hydrate. The scalp was detached from the brain and fascia was excised between the midpoints of the binocular junction, revealing the fontanel of the skull. The scalp was sterilized using a swab soaked in 1.5% hydrogen peroxide. Stereoscopic positioning of the injection site was confirmed, followed by gentle drilling into each side using a dental drill, producing wells suitable for injection. The fontanel is the origin of coordinates, and the suture is the reference line. According to the coordinates of CA1 in the hippocampus in the injection map of rats, + 1.3 mm (A/P), ± 2 mm (M/L), −4 mm (D/V), Hamilton needle was connected to a microinjection pump, and the experimental rats were automatically injected with a volume of 2 μ L R1-pS262 at a concentration of 2400 μM or a volume of 2 μL. The concentration of R1-nS262 was 2.4 mM, and the control group was injected with 2 μL normal saline. The injection rate was 100 nL/min, and the needle was left for 10 min after injection to prevent the polypeptide from spreading or backflow into the extracranial space. After injection in the right CA1 region, the left CA1 region was injected.

Behavioral test

Open field test. The effect of R1-pS262 or R1-nS262 administration on the exploratory behavior of experimental rats was evaluated using established methods. Experimental rats were transferred to a test chamber and allowed to acclimate. Then, OFT was performed. The activity trajectories of the animals in the chamber were observed and captured by a camera system for at least 5 min to assess the impact on their exploration behavior.

Novel objective recognition test. To assess the impact of R1-pS262 or R1-nS262 treatment on the recognition and memory capabilities of experimental animals for specific environmental conditions, we employed NOR experiments. Five minutes after detection in the 100 cm × 100 cm × 70 cm box, a new object in the NOR detection box was replaced. The activity trajectories of the experimental rats in the box for at least 5 min were captured by a camera to assess their recognition and memory capabilities.

Fear conditioning test. To assess the impact of R1-pS262 or R1-nS262 administration on episodic memory in rats, a literature-based FCT was implemented. Experimental rats first underwent a 10-s auditory stimulation followed by a 3-s electrical current of 0.8 mA. After 24 h, rats’ body stiffness duration during post-sound electrical stimulation was measured to assess the impact on episodic memory.

Morris Water Maze test. To determine the effect of R1-pS262 or R1-nS262 treatment on the memory function of experimental animals in terms of spatial recognition and location, we performed the literature-based MWM method. After a continuous experimental training of 5 d × 4 times/day, the time for rats to reach the platform position for the first time and the number of crossings were recorded to assess the impact on memory function.

Sarkosyl-insoluble tau fractionation

HEK293/tau cells were cultured in the presence of R1-pS262 or R1-nS262 for 48 h. The cells were then washed with PBS and lysed with RIPA lysis buffer. The protein content of lysate was quantified using BCA method. Subsequently, 1% volume of sarkosyl was added to the cell lysate, and the sarkosyl-insoluble and sarkosyl-soluble fractions of tau were isolated by centrifugation at 100,000×g for 1 h at 4 °C.

Western blotting

The brain tissue of experimental rats were carefully collected. Protein was extracted with RIPA lysis buffer. The protein was extracted by SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. Then, the membrane was subjected to primary antibody binding and secondary antibody binding according to a conventional western blotting protocol to quantify the expression level of the target protein in tissues.

Nissl staining

The experimental rats were anaesthetized and sacrificed. The brain tissues were fixed with paraformaldehyde and dehydrated with sucrose solution following the protocol of the Beyotime Nissl Staining Kit. The staining of Nissl bodies within the cytoplasm of neurons was strictly completed accordance with the manufacturer's instructions (Wuhan Good-bio Technology CO, LTD, Wuhan, Hubei, China).

Statistical analysis

Data were expressed as Mean ± SEM and analyzed using GraphPad prism9 statistical software (GraphPad Software, San Diego, CA, USA). One-way ANOVA were performed for differences among three groups. A Student's t-test was used for Western blot analyses and assessment of behavioral tests. Significance was accepted at the 95% confidence level (p < 0.05)

Results

Effect of tau protein phosphorylation on its conformational and structural characteristics

In order to identify all the conformational and structural characteristics of tau after folding and aggregation induced by phosphorylation, the full-length human tau (2N4R tau, wild type) was analyzed using the protein fingerprinting technique. In this study, we first obtained the full-length hTau441 sequence (serial number: P10636-8) from the Uniprot website and then submitted the sequence to the protein fingerprinting technology website http://micropht.com/ to obtain the fingerprint codes of all potential structures of tau (Supplemental Figure 1A). This indicates that the more letters under each amino acid, the more conformational changes that can exist at that location and the more flexible it is. Conversely, the fewer the letters, the more rigid the conformational position of the amino acid, which is not easily changed. Fingerprint codes are represented by 26 letters and a character $ shown in the Supplemental Table 1, in which Letter A represents the typical α helical structure, Letter B represents the β slice, Letters V/J/Y/P/D/H represent similar α helical structures, Letters E/G/S/M/V/J represent the similar β slice, Letters X/U/R/I/F/L/O/C/Z/W/T/K/$/N/Q represents the irregular four-fold folding shape. The first line of letters below the amino acid represents the most likely conformation.

Phosphorylation causes tau to lose its physiological function and promote its aggregation.^13,14 Previous studies have shown that 85 amino acids of hTau441 were potential phosphorylation sites.^11,15,16 To evaluate the conformational changes of tau after phosphorylation, the 85 potential phosphorylation sites were modified to alanine (A), simulating non-phosphorylated status, or glutamate (E), simulating phosphorylated status, and then the simulated mutant protein sequences were analyzed by PFSC method (Supplemental Figure 2 and 3 and Table 3). Phosphorylated tau at T181 can be used as a blood biomarker to predict the pathology of AD, and its phosphorylation is crucial to phosphorylation at other sites and plays a leading role.^17,18 The fingerprint code of T181 is JPW, and after mimicking mutation to A, its fingerprint code is PWJ, and the number of fingerprint codes is unchanged, indicating that the non-phosphorylation of this site has little effect on the structure of tau protein. After mimicking mutation to E, the secondary structure of the site transforms into a β sheet, and the number of fingerprint codes increases from 3 to 7, becoming more flexible. These data partially indicate that phosphorylation at Thr181 promoted the flexibility of tau protein structure, enhanced tau variability, and then affected the phosphorylation at other sites and tau aggregation. The recognition sites of AT8 antibodies commonly used in the pathological detection of AD are Ser202 and Thr205.¹⁹ Although there was no significant change in the number of fingerprint codes after Ser202/Thr205 mutation to E, the irregular four-fold shape of Ser202 and Thr205 was found to be reduced, as suggested by PFSC analysis. Phosphorylation of Ser262 strongly reduces binding of tau to microtubules, supporting that Ser262 site of hTau441 is another important site closely related to AD pathology.²⁰ After mutation, the number of Ser262 protein fingerprints increased from 6 in tau S262A to 9 in tau S262E, implying that the number of fingerprint codes increased after simulated phosphorylation; that is, the site conformation increased, and the flexibility was enhanced. Moreover, phosphorylation of tau at S396/S404 is associated with the prion-like properties of tau, promoting the seeding capacity of tau aggregates.^21,22 After mimicking phosphorylation mutation, the conformation of S396 changes from β sheet to α helical structure. However, the Ser404E mutation exhibited a reduced number of fingerprint codes, leading to more fixed structural changes and enhanced rigidity. GSK3β and PKA, two major tau kinases, are responsible for tau phosphorylation at Thr212/Ser214, which is recognized by the AT100 antibody, and the formation of the AT100 antigenic determinant requires a change in tau conformation.^23,24 Compared to T212A/ S214A, the number of fingerprint codes in T212E/S214E was increased, while the PFSC structure of T212E/S214E shifted from the irregular four-fold shape and α-helical structure to β-sheet structure.

Table 3.

Comparison of the PFSC results of hTau441 protein with two variants with serine phosphorylation sites mutated to alanine (A) and glutamate (E).

No	Phosphorylation Site	Sequence	Fingerprint code			Fingerprint code number
No	Phosphorylation Site	Sequence	Wild Type	Mutated to A	Mutated to E	Wild Type	Mutated to A	Mutated to E
1	T17	AGTYG	CZRDS	BZAPDWYVCSJF	JZCPVDWYA	5	12	9
2	Y18	GTYGL	DRAWJS	AQWDCPRVBJSY$EFZ	APQJCYZVSDBEW	6	16	13
3	Y29	GGYTM	WBEVPAJR	YZABD	AR	8	5	2
4	T30	GYTMH	A	D	D	1	1	1
5	T39	GDTDA	PVAJYBQW	VJYADSZ	VPJAY	8	7	5
6	S46	KESPL	WPLJYACEBF	CWP	PJBCW	10	3	5
7	T50	LQTPT	WPC	YJWACEPL	PZCWJSA	3	8	7
8	T52	TPTED	WVCB	AYZBWVJS	PCSJW	4	8	5
9	S56	DGSEE	WAJYBPVS	B$DYPAZ	PSJWCADVRY	8	7	10
10	S61	PGSET	PYBJAERCFDSWL	WCYZAVBPJ	AYBPZJSVWEU	13	9	11
11	T63	SETSD	WAEPBJQVY	AJPZCYDVBS	ADCJVQSWBEPYFRL	9	10	15
12	S64	ETSDA	YVBWAP	ADJWVYC	AYDVEQJPC	6	7	9
13	S68	AKSTP	JALSQWP	WJASPBRVDFLYE	PW	7	13	2
14	T69	KSTPT	CWEBP	CAWJLBPSYV	JP	5	10	2
15	T71	TPTAE	WCYLA	JAPYSVCEZWDBRF	JBLSAEPV	5	14	8
16	T76	DVTAP	APJWV	ACDEBRY	VWBJAPE	5	7	7
17	T95	PHTEI	A	ADBR	A	1	4	1
18	T101	EGTTA	WYJCESB	AYCDJZVSREW	ACEZSWBJVFILRDQ	7	11	15
19	T102	GTTAE	ABZSERWY	ADEPVCBR	ABDSJRZEPVW	8	8	11
20	T111	GDTPS	CWPFJS	PJCAWYSV	BJ	6	8	2
21	S113	TPSLE	APVBW	ADCYBJVSW	ACSV	5	9	4
22	T123	HVTQA	W	VADW	A	1	4	1
23	S129	MVSKS	ADW	AD	AEJDB	3	2	5
24	S131	SKSKD	PCJWZB	ABSWDYJCE	JBSAWDCE	6	9	8
25	T135	DGTGS	FADPSBVCJYR	REVPCWBADS	CVAWY	11	10	5
26	S137	TGSDD	WPJCQBSY	BCEWYVJAIPRLSU	ACJYBS	8	14	6
27	T149	GKTKI	JZERSABDY	YESCVRJPWLAZ	ERABPSJ	9	12	7
28	T153	IATPR	ABW	CSW	WJCYV	3	3	5
29	T169	NATRI	EASVB	ADVY	ADSE	5	4	4
30	T175	AKTPP	PCJSWRBE	PC	P	8	2	1
31	T181	PKTPP	JPW	PWJ	BCEWJPS	3	3	7
32	S184	PPSSG	JAVBCSEY	CYASVWBJZE	ACPWYV	8	10	6
33	S185	PSSGE	PBSJCYWQUERVAZ	AYEZPC$SWD	YWAUZPS	14	10	7
34	S191	PKSGD	P	AYSDZR	YZQPAV	1	6	6
35	S195	DRSGY	VU	JAPQY$UDWS	AP	2	10	2
36	Y197	SGYSS	BCJUWEYV	AEZDYJPBSCVWRILFHUQ	ASJCYDB	8	19	7
37	S198	GYSSP	WCVAEJ	ACDERVBWYLS	JPVBWYCAES	6	11	10
38	S199	YSSPG	JVPSYB	JCPAVWYSQ	JCPWAS	6	9	6
39	S202	PGSPG	PCJVWB	SBJPWAC	PWJCF	6	7	5
40	T205	PGTPG	PWCJSFBR	SBJPWAC	PWJCF	8	7	5
41	S208	PGSRS	YPVAJBSWCFQZ	DABPYJZ$C	YAJ	12	9	3
42	S210	SRSRT	ASDCY	ADVEJSBPWYZ	ADS	5	11	3
43	T212	SRTPS	CWBPSEFJ	YPCJSA	AJVBBESR	8	6	8
44	S214	TPSLP	JVP	SVAWCER	EBCSLWBYV	3	7	9
45	T217	LPTPP	CPJSR	CWSPBEJL	PCAJBW	5	8	6
46	T220	PPTRE	WBCA	CYSV	ASYZ	4	4	4
47	T231	VRTPP	CWP	J	WPAL	3	1	4
48	S235	PKSPS	CWERBFLSY	CPWSBJE	PJWCVABY	9	7	8
49	S237	SPSSA	ACWJVSBE	ACVDJWBYSERPLQ	ADBSVY	8	14	6
50	S238	PSSAK	AJVW	ADLR	AJQD$ZSYV	4	4	9
51	S241	AKSRL	PDAV	ADBEJPS	AJDP	4	7	4
52	T245	LQTAP	SBE	AVCSDE	CAWD	3	6	4
53	S258	VKSKI	APWBJSYDVCZ	AJDBWCPE	AYDVSBJP	11	8	8
54	S262	IGSTE	ASJWPYZFBCRV	AYSZBD	WYSJPZBVC	12	6	9
55	T263	GSTEN	AVLYD	D	Q$PA	5	1	4
56	S285	DLSNV	APCVW	ADCPQS$J	A	5	3	1
57	S289	VQSKC	VAW	W	A	3	1	1
58	S293	CGSKD	ACSWDPLQY	VAYBCDJPZW	J	9	10	1
59	S305	GGSVQ	CSBFARJYW	AJCSFQV$ELPR	JRCADESBV	10	12	9
60	Y310	IVYKP	JBWPC	AV	WCVLE	5	2	5
61	S316	DLSKV	ABJVWDPS	CWABSDP	ADVW	8	7	4
62	T319	KVTSK	EBVCAPW	AESVBJWYP	AWDBCSP	7	9	5
63	S320	VTSKC	E	AD	YSVA	1	2	4
64	S324	CGSLG	SAJD	Z	B	4	1	1
65	S341	VKSEK	ERJBPWSCAD	ASDUYEJPW	APJSV$EY	10	9	8
66	S352	VQSKI	B	AEB	DAV	1	3	3
67	S356	IGSLD	SBAJC	ADV	ABCS	5	3	4
68	T361	NITHV	EBAJL	AD	D	5	2	1
69	T373	IETHK	VEBASJY	DA	AD	7	2	2
70	T377	KLTFR	BE	ABEDSQW	WACD	2	7	4
71	T386	AKTDH	B	ACD	SYBV	1	3	4
72	Y394	IVYKS	EAB	ASD	WESDVBYJQ	3	3	9
73	S396	YKSPV	BCS	PCJWASV	PJCWV	3	7	5
74	S400	VVSGD	WYVJBCZAS	EYUQWBPCZA	YVWPBJAUZ	9	10	9
75	T403	GDTSP	BZDPVJ$WY	PAVBCJSWLY	PVJYW	9	10	5
76	S404	DTSPR	CPJ	CAPWJ	W	3	5	1
77	S409	HLSNV	PVAWC	AYV	VWCA	5	3	4
78	S412	NVSST	ACWBESY	ADCEBPJ	ACVSD	7	7	5
79	S413	VSSTG	ZAYEVSPBLWCJR	ASDCW$FBEJRV	ADJSPBVWY$CEQR	13	12	14
80	T414	SSTGS	YVJWPCUAQ	AQJYVDWPECUSBRFIHL$Z	PWSA$VJYZ	9	20	9
81	S416	TGSID	BPJSVACEL	AJPBSEUVWDR	ARBCYSDP	9	11	8
82	S422	VDSPQ	WSJ	CJPW	AYWJ	3	4	4
83	T427	LATLA	DAECPW	ADBEQL	ADEVBRSCLQ	6	6	10
84	S433	EVSAS	CASDBEWF	ADRSCEWF	AEDSVBUCWPZJR	8	8	13
85	S435	SASLA	EADBLSCJW	ADJES$QBCP	ADEWVRS	9	10	7

Differences in the aggregation of R3 fragments caused by phosphorylated and non-phosphorylated tau peptides

To further investigate whether the flexibility enhancement is more likely to lead to tau aggregation and the effect of phosphorylation at those sites mentioned above on tau aggregation, we first synthesized the corresponding polypeptides by the chemical solid phase method. These include the phosphorylated polypeptide R1-pS262 and non-phosphorylated polypeptide R1-nS262, the C-terminal phosphorylated polypeptide CTF-pS396/pS404 and the non-phosphorylated polypeptide CTF-nS396/nS404, the phosphorylated polypeptide AT8-pS202/pT205 and non-phosphorylated polypeptide AT8-nS202/nT205, the phosphorylated polypeptide AT100-pT212/pS214 and non-phosphorylated polypeptide AT100-nT212/nS214 (Table 2). The third microtubule-binding repeat (R3) domain of tau is confirmed as the most aggregation-favorable sequence of the full-length tau. Therefore, we also synthesized R3 peptides to evaluate their effects on R3 fragment aggregation.

The application of heparin is a commonly used method to induce tau aggregation in vitro.²⁵ Thioflavin S (Th-S) assay was performed to detect the degree of protein aggregation. As the easiest sequence to undergo tau aggregation, R3 polypeptide demonstrated the ability to self-aggregate over time (Figure 1A-H). However, the phosphorylated and non-phosphorylated peptides did not possess the ability to self-aggregate (Figure 1A-H). Interestingly, R1-pS262 and R1-nS262 promoted the aggregation of R3 polypeptide, and R1-pS262 had a stronger effect on R3 polypeptide aggregation than R1-nS262 (Figure 1A). According to the fingerprint code of S262, the number of fingerprint codes increased when the site is mutated from A to E (Figure 1B), indicating more conformational changes and stronger flexibility. CTF-pS396/pS404 and CTF-nS396/nS404 also promoted the aggregation of R3 polypeptide, but still, the aggregation degree of the phosphorylated fragment was significantly weaker than that of the non-phosphorylated fragment (Figure 1C). According to the fingerprint code of S396, the number of S396E fingerprint codes became less than that of S396A (Figure 1D), indicating non-phosphorylation state of S396 meant more conformational changes and stronger flexibility. The results of AT8-pS202/pT205 and AT8-nS202/nT205 were consistent with those of CTF-pS396/pS404 and CTF-nS396/nS404 (Figure 1E, F). AT100-pT212/pS214 and AT100-nT212/nS214 accelerated the aggregation of R3 polypeptide, and the phosphorylated peptide had a significantly higher aggregation effect on R3 polypeptide than the non-phosphorylated peptide, while the number of fingerprint codes increased when T212A/S214A was mutated to T212E/S214E (Figure 1G, H). Taken together, these findings suggested that the more fingerprint codes, the greater conformational change, and stronger flexibility, which means easier aggregation.

Figure 1.

Investigating the impact of phosphorylated peptides on R3 fragment aggregation. (A) ThS staining results of R3 peptide fragment aggregation during concubation with R1-pS262 + R3 or R1-nS262 + R3. (B) The number of protein fingerprints of R1-S262A and R1-S262E. (C) ThS staining results of R3 peptide fragment aggregation during concubation with CTF-pS396/pS404 + R3 or CTF-nS396/nS404 + R3. (D) The number of protein fingerprints of CTF-S396A/pS404A and CTF-S396E/S404E. (E) ThS staining results of R3 peptide fragment aggregation during concubation with AT8-pS202/pT205 + R3 or AT8-nS202/nT205 + R3. (F) The number of Protein folding shape code of AT8-S202A/T205A and AT8-S202E/T205E. (G) ThS staining results of R3 peptide fragment aggregation during concubation with AT100-pT212/pS214 + R3 or AT100-nT212/nS214 + R3. (H) The number of Protein folding shape code of AT100-T212A/S214A and AT100-T212E/S214E.

R1-pS262 promoted tau aggregation in vitro

The tau fibers isolated from the brains of AD and Pick disease patients exhibit different conformations, with tau being highly phosphorylated at Ser262 in AD, while there is no significant increase in phosphorylation in Pick disease patients, suggesting that Ser262 is a distinctive phosphorylation site of tau which may differentiate AD from other neurodegenerative disease. Meanwhile, phosphorylation of tau at Ser262 promotes microtubule depolymerization.²⁰ To further explore the effect of tau phosphorylation at a single site on aggregation and toxicity in biological systems, R1-pS262 and R1-nS262 were selected for cell and animal experiments in this study. Firstly, HEK293/tau cells were treated with R1-pS262 and R1-nS262. Western blotting showed that R1-pS262 and R1-nS262 did not affect levels of tau phosphorylation at Thr231, Ser262, and Ser396, and total tau also did not change (Figure 2A-C). However, R1-pS262 treatment significantly increased the level of insoluble tau, while R1-nS262 treatment did not affect insoluble tau compared to the control group (Figure 2A, D). In addition, we also detected tau phosphorylation and solubility in primary hippocampal neurons. Consistent with the result from HEK293/tau cells, R1-pS262 and R1-nS262 did not affect levels of tau phosphorylation, only R1-pS262 promoted tau aggregation (Figure 2E-H). These findings indicated that phosphorylation of tau at Ser62 promoted tau aggregation in vitro.

Figure 2.

In Vitro promotion tau aggregation by phosphorylated polypeptide R1-pS262. (A) Western blots were carried out with antibodies against phosphorylated tau protein at different sites, soluble tau protein and insoluble tau protein in HEK-293/tau cell lysate. (B-D) Quantification of blots in (A) are shown. n = 3 per group. *p < 0.05, **p < 0.01. All data represent mean ± SEM. (E) Western blots were carried out with antibodies against phosphorylated tau protein at different sites in primary neuronal lysate. (F-H) Quantification of blots in (A) are shown. n = 4 per group, **p < 0.01. All data represent mean ± SEM.

R1-pS262 impaired synapse-associated proteins in primary neurons

Previous studies have shown that tau aggregation can lead to synaptic damage.^26,27 To investigate the effects of these peptides on synapses, primary neurons were treated with R1-pS262 and R1-nS262, respectively. The levels of synapse-associated proteins were detected by western blot assay (Figure 3A), and the quantitative analysis showed that R1-pS262 significantly reduced the levels of GRIN1, GRIN2B, Synapsin1, and PSD-95 compared to the control group (Figure 3B-F), but the level of synaptophysin remained unchanged (Figure 3D). However, R1-nS262 had no significant effect on the above synapse-associated protein levels (Figure 3B-F). Together with its ability to promote tau aggregation, our data suggest that R1-pS262 leads to synaptic damage probably by promoting tau aggregation in treated primary neurons.

Figure 3.

Reduced synapse associated proteins levels in primary hippocampal neurons induced by phosphorylated polypeptide R1-pS262. (A) Western blot results of GRIN1, GRIN2B, Synaptophysin, Synapsin-1 and PSD-95. (B-F) Quantification of blots in (A) are shown. n = 4 per group, *p < 0.05. All data represent mean ± SEM.

R1-pS262 impaired cognitive function in rats

A large number of studies have reported that tau aggregation and synaptic insult impair cognition.^28,29 To determine the effects of Ser262 phosphorylated and non-phosphorylated peptides R1-pS262 and R1-nS262, respectively, on animal behavior and cognitive function, normal saline, R1-pS262, and R1-nS262 were injected into the CA1 hippocampal region of SD rats respectively. Behavioral tests were performed 30 days after injection (Figure 4A). In the Open-field test, which evaluates anxiety and exploratory activity levels, both R1-pS262 and R1-nS262 rats displayed similar patterns of anxiety and exploratory activity as normal saline control animals (Figure 4B, C). Fear conditioning test results showed a lower freezing time in the R1-pS262 rats than in the R1-nS262 and control rats (Figure 4D), indicating that R1-pS262 impaired scene-dependent fear memory. The results of the NOR showed a decreased curiosity for exploring new things within 24 h, as indicated by the decreased time spent discovering a new object in the R1-pS262 but not in the R1-nS262 group compared to the control group (Figure 4E). The data from the MWM support that R1-pS262 rats exhibit an increased latency to find the hidden platform, while no significant difference in the R1-nS262 rats was found compared with the control group (Figure 4F, G). On the 6th day, the test data showed a significant decrease in both the time spent in the target quadrant and the number of target platform crossings in the R1-pS262 group compared to the control group (Figure 4H, I). The results together showed that R1-pS262 treatment impaired spatial learning and memory in rats.

Figure 4.

Cognitive impairment induction in rats by phosphorylated polypeptide R1-pS262. (A) Sprague Dawley (SD) rats performed open field, new object recognition, Morris water maze, and fear conditioning test. (B-C) In the open field test, the average speed and total distance of the rats were recorded. (D) Fear conditioning was used to detect episodic memory in rats, and percentage freezing time in rats 24 h later was detected. The percentage freezing time of rats in the phosphorylated polypeptide group was lower than in the control group. (E) In the new object recognition test, the target resolution of phosphorylated polypeptide treated rats was reduced when compared to that of control rats. (F) The rats underwent learning and memory training for 1–5 days. (G) Motion trajectories of rats during the 6th day water maze test. (H) The movement time of the rats in the target area was measured on day 6 after the platform was removed. (I) The number of rats crossing the platform was measured on day 6 after the platform was removed. n = 8–12 per group. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001. All data represent mean ± SEM.

R1-pS262 induced tau aggregation, synaptic damage, and neuronal loss in vivo

To assess whether learning and memory impairment of R1-pS262 rats is associated with tau phosphorylation and aggregation, we carried out immunoblotting. Consistent with western blotting results from cell experiments (Figure 2), neither R1-pS262 nor R1-nS262 altered tau phosphorylation at Thr231, Ser262, Ser396, and total tau levels compared to control animals (Figure 5A-C). However, R1-pS262 but not R1-nS262 increased the insoluble tau level in the hippocampus (Figure 5A, D). These findings suggest that Ser262 phosphorylation promoted tau aggregation without affecting tau phosphorylation at other sites. To further clarify the effect of phosphorylated peptides on synapses, we also detected the synaptic proteins in the hippocampus of peptides-treated rats. Similar to results from primary neurons, we found that R1-pS262 treatment significantly downregulated the levels of synaptophysin, synapsin1, and PSD-95 in the hippocampus of rats compared with the control group, while no change in R1-nS262 rats was observed (Figure 5E, F). The data further supported that R1-pS262 impaired synapses. Previous studies have shown that tau accumulation is associated with neuronal loss.³⁰ Therefore, we detected the effects of peptides on the number of hippocampal neurons. Nissl staining showed that R1-pS262 treatment resulted in a significant decrease in the number of neurons in DG, CA1, and CA3 brain regions of the hippocampus compared with the control group, but no changes in R1-nS262 rats (Figure 5G, H). These results suggest that R1-pS262 treatment leads to neuronal loss in treated rats.

Figure 5.

Phosphorylated polypeptide R1-pS262 induces tau aggregation, synaptic damage and neuron loss in vivo. (A) Western blotting was performed to assess tau protein phosphorylation levels at different sites in hippocampal homogenate and soluble/insoluble tau. (B-D) Quantification of blots in (A) are shown. n = 3 per group. (E) Western blotting of Synaptophysin, Synapsin-1, PSD-95, and β-actin. (F) Quantification of blots in (A) are shown. n = 4 per group. (G) Representative images of Nissl staining of rat brain sections with scale bars of 500 μm and magnification scale of 200 μm. (H) The quantitative analysis of the number of neurons in DG, CA1, and CA3 regions of hippocampus. The cell numbers in the DG, CA1, and CA3 area were counted by Image software in 2–3 brain sections from each rat hippocampus. Quantification of blots in (A) are shown. n = 3 per group. ns indicates no significant difference. **p < 0.01,***p < 0.001. All data represent mean ± SEM.

Discussion

The incidence of AD is rapidly increasing with the aging population, and there is still a lack of effective drugs for the treatment of AD in clinics. The abnormal hyperphosphorylation of tau protein is the key pathological process of AD, and there is no ideal report on drug research on it. The reason is that the effect of abnormal phosphorylation of tau protein on its conformation is unknown. In the current study, we employed the PFSC method to reveal the most likely spatial conformation of wild-type and simulated mutant hTau441. The chemical solid phase method was used to synthesize the phosphorylated and non-phosphorylated tau fragments. PFSC analysis from tau Ser202/Thr205/Thr212/Ser214/Ser262/Ser396/Ser404 and Thioflavin S assay on the peptides showed that the more fingerprint codes, the greater conformational change and higher flexibility, which may promote the aggregation of tau fragments. Since phosphorylation of tau at Ser262 promotes more phosphorylation sites of tau to kinases,³¹ phosphorylation of Ser262 strongly reduces binding of tau to microtubules,²⁰ and tau phosphorylation at Ser262 acts as a key role in Aβ₄₂-induced tau pathology,³² we used tau Ser262 peptides to investigate the effect of phosphorylation of Ser262 in tau toxicity. We found that phosphorylated tau fragments R1-pS262 rather than non-phosphorylated tau fragment R1-nS262 caused endogenous tau aggregation, neuronal loss, synaptic damage, and cognitive impairments.

Protein folding is one of the most challenging topics in the life sciences and has attracted a lot of attention since the recent progress of AlphaFold. Based on artificial intelligence, AlphaFold achieved a major breakthrough in accurately predicting the 3D structures of proteins based on protein sequences.³³ Given the flexibility of protein structures, many databases have accumulated information on protein or sequence regions of intrinsically disordered proteins. PFSC method, as a new technique, revealed changes in protein folding and, most likely, changes in conformation.⁵ In addition, this method can well reveal conformational differences caused by protein mutation, modification, and differentiation, which is very important for studying the mechanism of abnormal post-translational modifications of proteins in diseases like AD. In this study, we analyzed the fingerprints of wild-type hTau441 and eighty-five phosphorylated sites mutated hTau441 by using the PFSC method. Actually, phosphorylation does not imply a large number of fingerprint codes, and a small number of fingerprint codes is not synonymous with non-phosphorylation. For example, the number of fingerprints at phosphorylated Ser202/Thr205/Ser396/Ser404 sites was less than that at non-phosphorylated Ser202/Thr205/Ser396/Ser404 sites. In contrast, the number of fingerprints at phosphorylated Thr212/Ser214/Ser262 sites was more than that at non-phosphorylated Thr212/Ser214/Ser262 sites. However, phosphorylated peptides R1-pS262 and AT100-pT212/pS214 were more likely to promote R3 polypeptide aggregation than non-phosphorylated peptides R1-nS262 and AT100-nT212/nS214, while non-phosphorylated peptides AT8-nS202/nT205 and CTF-nS396/nS404 were more likely to promote R3 polypeptide aggregation than phosphorylated peptides AT8-pS202/pT205 and CTF-pS396/pS404, supporting that the more the fingerprint codes the more likely to promote tau aggregation.

To the best of our knowledge, the effect of real phosphorylated tau peptides on tau aggregation has not been reported. In this study, we synthesized phosphorylated tau peptides and examined their effects on tau aggregation and its downstream events at the cellular and animal levels. Our data indicate that phosphorylated tau polypeptide at S262 site R1-pS262 promoted endogenous tau aggregation but did not affect its phosphorylation level in vitro and in vivo. Moreover, it also caused synaptic damage and neuronal loss, culminating in the cognitive impairment observed in injected rats.

In summary, the present study suggests that predicting the fingerprint codes of phosphorylation sites of tau could indicate the probability of tau protein aggregation. This study is the first to report that the phosphorylated tau fragments R1-pS262 directly result in tau aggregation and downstream neuropathological alterations. Given that alterations of tau phosphorylation at Ser262 exert tau pathological events, blockage of its phosphorylation may provide a potential strategy for the treatment of tauopathies like AD.

Supplemental Material

sj-docx-1-alz-10.1177_13872877241307341 - Supplemental material for Conformation pattern changes in R1-pS262 tau peptide induced endogenous tau aggregation, synaptic damage, and cognitive impairments

Supplemental material, sj-docx-1-alz-10.1177_13872877241307341 for Conformation pattern changes in R1-pS262 tau peptide induced endogenous tau aggregation, synaptic damage, and cognitive impairments by Gang Wu, Yong Luo, Qian Guo, Mingming Yang, Yacoubou Abdoul Razak Mahaman, Yi Liu, Jian-Zhi Wang, Rong Liu, Xiang Gao and Xiaochuan Wang in Journal of Alzheimer's Disease

Footnotes

Acknowledgments

The authors are grateful to Mr Dan Ke and Ms. Qun Wang for helpful technical suggestions during the conduct of this study.

ORCID iDs

Qian Guo

Mingming Yang

Yacoubou Abdoul Razak Mahaman

Jian-Zhi Wang

Rong Liu

Xiaochuan Wang

Author contributions

Gang Wu (Investigation; Methodology); Yong Luo (Investigation; Methodology); Qian Guo (Writing – review & editing); Mingming Yang (Methodology); Yacoubou Abdoul Razak Mahaman (Writing – review & editing); Yi Liu (Methodology); Jian-Zhi Wang (Formal analysis); Rong Liu (Formal analysis); Xiang Gao (Supervision); Xiaochuan Wang (Funding acquisition; Supervision; Writing – review & editing).

Data availability

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported in parts by grants from National Natural Science Foundation of China (82330041), grant from Science and Technology Innovation Team project to Xiaochuan Wang from Department of Science and Technology of Hubei Province (2022-72-18), grant from Laboratory Animal Research Project of Hubei (No.2023CFA007 to X.G).

Supplemental material

Supplemental material for this article is available online.

References

Long

Holtzman

. Alzheimer disease: an update on pathobiology and treatment strategies. Cell 2019; 179: 312–339.

Guo

Zhang

Zeng

, et al. Molecular and cellular mechanisms underlying the pathogenesis of Alzheimer's disease. Mol Neurodegener 2020; 15: 40.

Braak

. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 1991; 82: 239–259.

Goedert

. Tau protein and neurodegeneration. Semin Cell Dev Biol 2004; 15: 45–49.

Yang

. Comprehensive description of protein structures using protein folding shape code. Proteins 2008; 71: 1497–1518.

Yang

Cheng

Zhao

, et al. Comprehensive folding variations for protein folding. Proteins 2022; 90: 1851–1872.

Peng

Wang

, et al. Impact of alternating amino acid sequences on beta-amyloid-induced neurotoxicity and neuroinflammation in Alzheimer's disease. Aging (Albany NY) 2023; 15: 10580–10592.

Jeganathan

von Bergen

Brutlach

, et al. Global hairpin folding of tau in solution. Biochemistry 2006; 45: 2283–2293.

Mori

Kondo

Ihara

. Ubiquitin is a component of paired helical filaments in Alzheimer's disease. Science 1987; 235: 1641–1644.

10.

Min

Cho

Zhou

, et al. Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron 2010; 67: 953–966.

11.

Arakhamia

Lee

Carlomagno

, et al. Posttranslational modifications mediate the structural diversity of tauopathy strains. Cell 2020; 180: 633–644.e612.

12.

Shi

Zhang

Yang

, et al. Structure-based classification of tauopathies. Nature 2021; 598: 359–363.

13.

Lindwall

Cole

. Phosphorylation affects the ability of tau protein to promote microtubule assembly. J Biol Chem 1984; 259: 5301–5305.

14.

Liu

Sui

Dexheimer

, et al. Hyperphosphorylation renders tau prone to aggregate and to cause cell death. Mol Neurobiol 2020; 57: 4704–4719.

15.

Iqbal

Liu

Gong

. Tau and neurodegenerative disease: the story so far. Nat Rev Neurol 2016; 12: 15–27.

16.

Briner

Götz

Polanco

. Fyn kinase controls tau aggregation in vivo. Cell Rep 2020; 32: 108045.

17.

Janelidze

Mattsson

Palmqvist

, et al. Plasma P-tau181 in Alzheimer's disease: relationship to other biomarkers, differential diagnosis, neuropathology and longitudinal progression to Alzheimer's dementia. Nat Med 2020; 26: 379–386.

18.

Thijssen

La Joie

Wolf

, et al. Diagnostic value of plasma phosphorylated tau181 in Alzheimer's disease and frontotemporal lobar degeneration. Nat Med 2020; 26: 387–397.

19.

Braak

Alafuzoff

Arzberger

, et al. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol 2006; 112: 389–404.

20.

Biernat

Gustke

Drewes

, et al. Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 1993; 11: 153–163.

21.

Rosenqvist

Asuni

Andersson

, et al. Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau. Alzheimers Dement (N Y) 2018; 4: 521–534.

22.

Congdon

Sigurdsson

. Two novel Tau antibodies targeting the 396/404 region are primarily taken up by neurons and reduce Tau protein pathology. J Biol Chem 2013; 288: 33081–33095.

23.

Alonso

Di Clerico

, et al. Phosphorylation of tau at Thr212, Thr231, and Ser262 combined causes neurodegeneration. J Biol Chem 2010; 285: 30851–30860.

24.

Zheng-Fischhöfer

Biernat

Mandelkow

, et al. Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimer-specific epitope of antibody AT100 and requires a paired-helical-filament-like conformation. Eur J Biochem 1998; 252: 542–552.

25.

Fichou

Vigers

Goring

, et al. Heparin-induced tau filaments are structurally heterogeneous and differ from Alzheimer's disease filaments. Chem Commun (Camb) 2018; 54: 4573–4576.

26.

McInnes

Wierda

Snellinx

, et al. Synaptogyrin-3 mediates presynaptic dysfunction induced by tau. Neuron 2018; 97: 823–835.e828.

27.

Zhou

McInnes

Wierda

, et al. Tau association with synaptic vesicles causes presynaptic dysfunction. Nat Commun 2017; 8: 15295.

28.

Largo-Barrientos

Apóstolo

Creemers

, et al. Lowering synaptogyrin-3 expression rescues tau-induced memory defects and synaptic loss in the presence of microglial activation. Neuron 2021; 109: 767–777.e765.

29.

Ittner

Delerue

, et al. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models. Cell 2010; 142: 387–397.

30.

La Joie

Visani

Baker

, et al. Prospective longitudinal atrophy in Alzheimer's disease correlates with the intensity and topography of baseline tau-PET. Sci Transl Med 2020; 12: eaau5732.

31.

Lund

, et al. Role of individual MARK isoforms in phosphorylation of tau at ser²⁶² in Alzheimer's disease. Neuromolecular Med 2013; 15: 458–469.

32.

Iijima

Gatt

Iijima-Ando

. Tau Ser262 phosphorylation is critical for Abeta42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease. Hum Mol Genet 2010; 19: 2947–2957.

33.

Jumper

Evans

Pritzel

, et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021; 596: 583–589.

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