A Novel PSEN1 K311R Mutation Discovered in Chinese Families with Late-Onset Alzheimer’s Disease Affects Amyloid-β Production and Tau Phosphorylation

Abstract

Background: Presenilin-1 (PSEN1) is the most frequently mutated gene in familial Alzheimer’s disease (AD), whereas only several novel mutations have been reported in China and functional studies were seldom conducted.

Objective: We describe a novel PSEN1 K311R mutation in two Chinese families with late-onset AD and its functional impact on amyloid-β protein precursor (AβPP) processing and tau phosphorylation.

Methods: The mutation was detected by direct sequencing of PSEN1 exon 9. HEK293 cells stably expressing wild-type APP695 (HEK293-APP695_wt) were transfected with plasmids containing human wild-type PSEN1, PSEN1 K311R mutation, and PSEN1 E280A mutation to compare the K311R mutation’s effects on AβPP processing with other groups. In addition, each group of cells were co-transfected with plasmids harboring PSEN1 and human wild-type MAPT complementary DNA to study the mutation’s impacts on tau phosphorylation.

Results: The K311R mutation was detected in probands of two late-onset AD families. Expression of the K311R or E280A mutation increased amyloid-β (Aβ)₄₂ levels but decreased Aβ₄₀ levels, resulting in an overall increase in the Aβ₄₂/Aβ₄₀ ratio compared to those in wild-type PSEN1 transfected cells (p < 0.05). The K311R or E280A mutation also increased the levels of phosphorylated tau compared to wild-type PSEN1 (p < 0.05).

Conclusion: The K311R mutation might contribute to AD pathogenesis by overproducing toxic Aβ species and enhancing tau phosphorylation. Further in-depth studies are needed to decipher the pathogenic mechanisms of the K311R mutation in terms of AβPP cleavage, tau phosphorylation, and other presenilin-1 mediated functional pathways.

Keywords

Amyloid-β familial Alzheimer’s disease mutation presenilin tau

INTRODUCTION

The study of familial Alzheimer’s disease (FAD), in particular the discovery of disease-causing gene mutations, can help elucidate the etiology of AD. Presenilin-1 (PSEN1), presenilin-2 (PSEN2), and amyloid precursor protein (APP) are three causative genes that have been identified to date [1]. PSEN1 ranks as the most frequently mutated gene among the three, with more than 200 pathogenic mutations reported in the Alzheimer Disease & Frontotemporal Dementia Mutation Database (http://www.molgen.ua.ac.be/ADMutations/) [2].

Intriguingly, carriers of the PSEN1 mutation have a broad spectrum of clinical manifestations. Age at symptom onset ranges from <30 to >60 years [3]. Although worsening memory is usually shared among patients with these mutations, atypical clinical features such as seizures, extrapyramidal symptoms, behavioral and psychiatric symptoms, spastic paraparesis, myoclonus, cerebellar ataxia, and aphasia are unevenly presented across patients with mutations in different exons. Moreover, some clinical features have frequently been described for a particular mutation, such as headache in 73% of E280A carriers [4]. These findings indicate that the pathological impact of a PSEN1 mutation is intrinsically associated with its structural location. The effects of a PSEN1 mutation on amyloid-β production vary with the mutation [5]. Moreover, some mutations may impair signaling pathways other than those for amyloid-β protein precursor (AβPP) processing, such as Notch and β-catenin [6], suggesting that these mutations may affect γ-secretase activity in various ways. Hence, it is valuable to document the clinical features and explore the functional impacts of novel PSEN1 mutations for a deeper insight into AD pathogenesis.

Novel PSEN1 mutations are far less frequently reported in Chinese people than in Caucasians. Of all mutations reported in Chinese families with FAD, function tests have only been conducted on the V97L mutation [7]. Thus, the pathogenic mechanisms of most mutations detected in Chinese people remain unknown.

Here, we present a novel PSEN1 (K311R) missense mutation in two Chinese families with late-onset Alzheimer’s disease (LOAD). The mutation was located within the hydrophilic loop (HL) domain of the PSEN1 C-terminal cytoplasmic loop (CL) where mutations are seldom found. The mutation promoted amyloidogenic processing of AβPP and tau phosphorylation in HEK293-APP695_wt cells in vitro. This is the first report of a mutation at this site, and our results will help unravel the potential pathogenic mechanisms of mutations in the CL hydrophilic domain.

MATERIALS AND METHODS

Families were enrolled at the Memory Clinic of Xuan Wu Hospital, Beijing, China. Probable AD was diagnosed according to the National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria [8]. Subjects who met the following criteria were enrolled in the cognitively normal group: age >65 years, no cognitive decline and no history of neurodegenerative disease or cerebral amyloid angiopathy. Individuals who met the following criteria were enrolled in the late-onset sporadic Alzheimer’s disease (LOSAD) group: age >65 years, diagnosis of probable AD according to the NINCDS-ADRDA criteria and no history of familial hereditary disease.

This study was approved by the Ethics Committee of Xuan Wu Hospital. Written informed consent was obtained from each participant.

Genetic testing

DNA was extracted from blood samples of available family members. Polymerase chain reaction (PCR) was performed to amplify exons 3–12 of PSEN1 and PSEN2 and exons 16–17 of APP, followed by direct sequencing. After the mutation was identified, it was screened for in all patients. The APOE genotype was determined by HhaI restriction enzyme isoform genotyping [9].

Construction of the expression plasmids

The PSEN1 K311R or E280A mutation was introduced into the pCDNA 3.1 (zeo+)-EGFP expression vector encoding human wild-type (WT) PSEN1 using the site-directed mutation method (Stratagene, La Jolla, CA, USA), as a result, all the PSEN1 encoding expression vectors were EGFP-tagged. The human WT MAPT complementary DNA (cDNA) was amplified from the total RNA of human neuroblastoma SH-SY5Y cells with the reverse transcription PCR method by using the following primers: 5^′-CGGAATTCATGGCTGAGCCCCGCCAGGAG-3^′ (forward) and 5^′-CCGCTCGAGTCACAAACCCTGCTTGGCCAGGGA-3^′ (reverse) for human MAPT. The amplification products were cloned into the pCDNA 3.1 (zeo+) expression vectors. Both WT and mutant cDNA sequences were verified.

Cell culture and transfection

HEK293 cells stably transfected with human WT APP695 were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 300μg/mL G418 (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified incubator with 5% CO₂. Transfections were performed in 6-well plates. Each well of cells were transiently transfected with 3μg plasmids containing WT PSEN1 and the K311R and E280A mutations using 6μl Lipofectamine 2000 (Invitrogen), respectively. For the analysis of tau phosphorylation, cells were co-transfected with 3μg of each PSEN1 and MAPT plasmids per well using 8μl Lipofectamine 2000. Cells transfected with mock plasmid and non-transfected cells (blank) were used as controls. The cells and medium were harvested 48 h post-transfection for further analysis. Transfection efficiency was tested by real-time quantitative PCR (rt-qPCR) method.

Total RNA isolation and rt-qPCR

Total RNA was isolated from freshly harvested cells using TRIzol reagent (Invitrogen). cDNA was synthesized using the SuperScript First-Strand Synthesis Kit (Promega, Madison, WI, USA). Each cDNA sample was amplified using SYBR Green (TransGen Biotech Co., Ltd, Beijing, China) on the ABI 7500 Real-time PCR System (Applied Biosystem, CA, USA). Briefly, the reaction conditions consisted of 2μl of cDNA and 0.2μM primers in a final volume of 20μl of supermix. Each cycle consisted of denaturation at 95°C for 15 s, annealing at 60°C for 20 s, and extension at 72°C for 10 s. cDNA amplification was performed by using the following primers: 5^′-GCATGGCTCATCTTGGCT-3^′ (forward) and 5^′-CGGAAGGAAGCTGCAGAA-3^′ (reverse) for human PSEN1. β-actin was used as an endogenous control to normalize each sample. The experiment was performed by three independent experiments. The results were analyzed using the comparative CT method (2^-ΔΔCt).

Protein isolation and analysis

Cells were lysed in RIPA buffer with 1 mM PMSF and 1x phosphatase inhibitors cocktail PhoSTOP (Roche, Basel, Switzerland) on ice for 30 min. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was transferred to a fresh tube and stored at – 80°C. The proteins were quantified using the BCA assay (Thermo Fisher Scientific Inc., Rockford, IL, USA). Levels of Aβ₄₀ and Aβ₄₂ in conditioned medium were quantified using sandwich enzyme-linked immunosorbent assay (ELISA) kits (Aβ₄₀/Aβ₄₂ ELISA kits, IBL, Hamburg, Germany) following the manufacturer’s instructions. The Aβ₄₂/Aβ₄₀ ratio was calculated for each group of cells. Western blotting was performed to detect the expression levels of soluble (sAβPP)β, β-secretase, presenilin-1, total tau, and phosphorylated tau (p-tau) according to the following protocol: cell lysates or medium (50μg of protein) were heated to 60°C for 5 min with loading buffer, loaded on a 12% SDS-polyacrylamide gel, separated by electrophoresis and transferred to nitrocellulose membranes (Millipore Co., Billerica, MA, USA). After blocking nonspecific sites, the membranes were incubated consecutively with primary and secondary antibodies. After washing, the immune complexes were detected using HRP chemiluminescent substrates (Millipore). Rabbit polyclonal antibodies against sAβPPβ (1 : 250, SIG-39138, Covance, Princeton, NJ, USA), rabbit monoclonal antibodies against PSEN1 (1 : 250, ab134195, Abcam, Cambridge, MA, USA), GSK-3β (1 : 2000, ab32391, Abcam), p-Ser9-GSK-3β (1 : 1000, ab75814, Abcam), CDK5 (1 : 500, ab40773, Abcam), mouse monoclonal antibodies against β-secretase (1 : 250, ab201946, Abcam), total tau (1 : 250. ab80579, Abcam), p-tau at Ser-202/Thr-205 (1 : 1000, MN1020, Pierce, Rockford, IL, USA), p-tau at Thr-181 (1 : 500, 5H9L11, Invitrogen), and β-actin (1 : 1000, sc-81178, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for western blotting.

Tau phosphorylation kinases GSK-3β and CDK5 activity assays

Activity of GSK-3β was measured using GENMED GSK-3β Activity Assay Kit (GENMED Scientifics Inc., Arlington, MA, USA), based on its ability to phosphorylate its targeting sequence GPHRSTPESRAAV in the presence of ATP and GSK-3α inhibitor Aloisine A. Activity of CDK5 was measured using GENMED CDK5/P25 Activity Assay Kit (GENMED), based on its ability to phosphorylate its targeting sequence PKTPKKAKKL in the presence of ATP and CDK5/P35 inhibitor Purvalanol. Then through a series of reactions involving pyruvate kinase and lactate dehydrogenase, the phosphorylated products in the system oxidize NADH (nicotinamide adenine dinucleotide, reduced form) to NAD (nicotinamide adenine dinucleotide). The disappearance of NADH is detected by measuring a decrease in extinction at 340 nm.

Statistical analysis

Comparisons among groups were performed by one-way analysis of variance, followed by Tukey’s post-hoc test using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). A p-value <0.05 was considered significant. Quantity one software (Bio-Rad, Hercules, CA, USA) was utilized to analyze western blotting results.

RESULTS

Clinical features

Two patients from two different generations were presented in one of the families (Fig. 1A). The proband (patient II 1) was a 73-year-old male who had visited our hospital complaining of progressive memory decline over the past 4 years. He scored 21/30 on the Montreal Cognitive Assessment (MoCA), which was below the recommended cut-off value of 22 adjusted for education. He only remembered 7 words from the WHO-UCLA Delayed Recall Memory Test, which was near the recommended cut-off value of 6. No other cognitive domains were impaired. His mother had developed memory loss around the age of 70 years, became progressively disoriented, incontinent, and bed-ridden around the age of 80 years, and died at the age of 83 years. The proband’s two younger siblings were cognitively normal without complaints.

Another family showed a similar pattern of inheritance. Two patients from two different generations were presented (Fig. 1B). The proband (patient II 2) was a 79-year-old male who complained of memory decline over the past 2 years. He scored 25/30 and 22/30 on the Mini-Mental State Examination and MoCA, respectively, both of which were below the cut-off values. He recalled 13 words on the Verbal Learning Test (cut-off, 18), but no words were recalled on the Delayed Recall Memory Test. Other cognitive domains were intact. His mother experienced memory loss at the age of 75 years, followed by progressive impairment of executive functions and visual spatial skills. Disease duration was 13 years. The proband’s older sister was cognitively normal. The clinical characteristics of the K311R mutation carriers are listed in Table 1.

Genetic analysis

A PSEN1 K311R (c.932A>G, Lys311Arg) heterozygous missense mutation was identified in the probands of the two families with LOAD, but was absent in one unaffected member (family 2, patient II 1), indicating that the mutation may segregate with disease status. The mutation was also not found in 100 cognitively normal individuals or 100 patients with LOSAD, suggesting that it may not be a common polymorphism. No PSEN2 or APP mutations were detected. The APOE genotypes of the two probands were ɛ3/ɛ2 and ɛ4/ɛ3.

The K311R mutation affects Aβ production

The Aβ₄₀ level in conditioned medium from cells transfected with the PSEN1 K311R mutation plasmid (K311R group) was 0.47-fold lower 48 h post-transfection than that of cells transfected with the WT PSEN1 plasmid (p = 0.000). In contrast, the Aβ₄₂ level in medium from the K311R group increased 2.48-fold, and the Aβ₄₂/Aβ₄₀ ratio increased 5.30-fold compared to those in the WT group (p = 0.000, p = 0.000 respectively) (Fig. 2). PSEN1 E280A is a well-known mutation that was first detected in a large Colombian early-onset AD cohort consisting of more than 100 affected family members. Functional studies have confirmed that the E280A mutation affects Aβ production. In our experiment, cells transfected with the PSEN1 E280A mutation plasmid (E280A group) showed a 0.36-fold decrease in Aβ₄₀ level (p = 0.000), a 3.40-fold increase in Aβ₄₂ level (p = 0.000) and a 9.48-fold increase in the Aβ₄₂/Aβ₄₀ ratio (p = 0.000) in medium compared to the WT group (Fig. 2), which is consistent with previous studies. These results suggest that the effects of the K311R mutation on Aβ production are weaker than those of the E280A mutation.

The K311R mutation does not affect β-secretase expression

β-secretase level in the cell lysates and sAβPPβ level in the medium were not different between the K311R and WT groups, whereas these levels were significantly higher in the E280A group (Fig. 3A-C) than in the WT group (p = 0.000 and p = 0.021, respectively), indicating that the K311R mutation may have less of an effect on β-secretase expression than the E280A mutation.

The K311R mutation does not affect presenilin-1 expression or endoproteolysis

No differences were observed in message RNA or protein (Fig. 3A, D, E) levels among the WT, K311R, or E280A groups (p > 0.05), suggesting that the K311R mutation does not affect presenilin-1 expression or endoproteolysis.

The K311R mutation affects tau phosphorylation

The tau level in the cell lysate did not differ among any of the groups. However, both the p-tau (AT8) level and p-tau (Thr181) level were significantly higher in the K311R and E280A groups than in the WT group (p = 0.035, p = 0.000 and p = 0.000, p = 0.002, respectively) (Fig. 4A-D). Further, we analyzed the protein levels and activities of GSK-3β and CDK5, which are two major kinases involved in tau phosphorylation. The level of GSK-3β was not significantly altered in any of the groups (Fig. 4A, E), whereas, the activity of GSK-3β was enhanced by 2.92-fold in E280A group (p = 0.004) compared with WT group (Fig. 5A). Correspondingly, the level of the inactivated form of GSK-3β (p-Ser9-GSK3β) was significantly lower in the K311R and E280A groups than in the WT group (p = 0.000 and p = 0.000, respectively) (Fig. 4A, F). The protein level and activity of CDK5 showed no significant changes among all groups (Fig. 4A, G, Fig. 5B).

DISCUSSION

We report a novel PSEN1 missense mutation (K311R) in two Chinese families with LOAD, which has not previously been reported. The mutation was heterozygous in the probands of both families but was not present in one cognitively normal family member, 100 normal individuals or 100 patients with LOSAD, suggesting a possible link between the mutation and AD. Due to a lack of data from deceased patients and additional family members, it is difficult to confirm the segregation pattern of this mutation.

The K311R mutation is located in the HL domain of the CL. The CL is located between the sixth and seventh transmembrane domains, which are multifunctional domains containing sites for phosphorylation, caspase cleavage, and partner binding [10]. A number of mutations have been described within the CL, particularly in the N-terminal one-third of the hydrophobic loop domain [11]. However, mutations are less frequently reported in the remaining two-thirds of the HL domain, particularly in exon 9 where the K311R mutation is located. Most carriers of mutations in the HL domain present with a relatively late age at onset (range, 47–57 years) compared to those carrying mutations in the hydrophobic loop domain (range, 26–69 years) [11]. In addition, most reports have included no more than two patients or only sporadic cases have been described. The clinical spectrum and biological effects of mutations in the HL domain are summarized in Table 2. In our study, the age at onset of each proband was >65 years, and only two patients had the mutation, which is consistent with the clinical features of previously described mutations in the HL.

Conditioned medium of cells transfected with the K311R mutation plasmids showed increased Aβ₄₂ levels and decreased Aβ₄₀ levels, indicating that the K311R mutation affects γ-secretase-mediated production of Aβ₄₂ and Aβ₄₀. It is still unknown how the K311R mutation affects Aβ production. The functional impact of HL domain mutations on Aβ production is controversial. One study concluded that HL-deleted PSEN does not significantly alter Aβ production [6]. However, other studies have demonstrated that mutations leading to exon 9 skipping enhance Aβ₄₂ and reduce Aβ₄₀ production [12]. Deleting exon 10-encoded segments drastically reduces γ-secretase cleavage at the Aβ₄₀ site [21] and impairs Aβ₃₉ and Aβ₃₈ production [22]. In addition, our study also found that E280A mutation, which located within the HL domain, increased Aβ₄₂ levels and the Aβ₄₂/Aβ₄₀ ratio but decreased Aβ₄₀ levels. These findings suggest that structural variation within the HL may help explain the inconsistent effects of these mutations on Aβ production.

Structural predictions of the presenilin-1 CL have revealed that the HL contains multiple alpha helical regions, including helix α (M292–N297), helix β (E356–L369) and helix 7 (G384–A398) [23]. Intriguingly, as shown in Table 2, most of the mutations reported in exons 9 and 10 are located in and around helix α or β, indicating that alpha helix structures may play an important role in γ-secretase cleavage of AβPP. In support of this notion, L392V, a mutation located within helix 7, reduces the helix content of the HL and causes early-onset FAD [24]. In addition,most residues of these alpha helices are embedded within hydrophobic SDS micelles; residues near helix α (R308–N312), where the K311R mutation is located, are also likely to be embedded [23], suggesting that residue K311 might be located within or in close proximity to the membrane near helix α. Thus, we hypothesized that the K311R mutation could alter the structural conformation near helix α and affect Aβ production in a pattern similar to mutations within helix α, such as S290C [12]. However, the effect may be weak, as substituting arginine for lysine does not change the polarity of the residue. Further studies are needed to clarify the mechanism by which the K311R mutation affects Aβ production.

We found no differences in message RNA or protein levels among the WT, K311R or E280A groups, suggesting that the K311R mutation does not affect PSEN1 expression or endoproteolysis. Endoproteolytic cleavage of presenilin-1 occurs at amino acids 291–299 in the HL [25], where the K311R mutation locates to but may not be affected.

In addition, we found that tau phosphorylation increased in the K311R and E280A mutation groups. Tau phosphorylation may be the consequence of Aβ₄₂ overproduction or an increase in the Aβ₄₂/Aβ₄₀ ratio [26]. In vitro treatment of primary hippocampal neurons with Aβ markedly induces the phosphorylation of tau at Ser202 and Ser396/404, resulting in the loss of microtubule binding capacity [27]. In addition, Aβ₄₂ can elicit rapid and reversible tau phosphorylation at Ser202, Thr181, and Thr231 in serum-deprived human SK-N-MC neuroblastoma cells and hippocampal synaptosomes [28]. Studies have demonstrated that Aβ might induce tau phosphorylation through the inhibition of PI3-kinase signaling and subsequent activation of GSK-3β, and blockade of GSK-3β activity prevents Aβ-induced neurodegeneration [29]. In our study, the activity of GSK-3β was enhanced in E280A group and the level of inactivated p-Ser9-GSK-3β decreased in the K311R and E280A mutation groups, which further support the notion that tau hyperphosphorylation might be the consequence of Aβ overproduction. Whereas, other studies have shown that presenilin-1 can directly bind tau and GSK-3β in the same region within residues 250–298, and mutations in this region can increase the ability of presenilin-1 to bind GSK-3β [30]. Presenilin-1 also forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association, and mutations like E280A can significantly inhibit the presenilin-1-dependent PI3K/Akt activation [31]. These studies suggested that PSEN1 mutations might affect tau phosphorylation by regulating the interactions with PI3K/Akt/GSK-3β signaling independent of Aβ. In support of this, PSEN1 I213T and c.548G>T knock-in mice exhibited neurofibrillary tangle-like tau pathology in the absence of Aβ deposition [32, 33]. In our study, the K311R mutation locates outside of GSK-3β binding region and it is still unknown whether this mutation affects the interaction between presenilin-1 and PI3K. The activity and level of CDK5 remained unchanged in mutation groups, which is consistent with previous studies that CDK5 pathway is unaffected by PSEN1 mutations [32].

The study has some limitations. Due to the unavailability of DNA samples from some of the family members, it was difficult for us to confirm the segregation status of K311R mutation with disease. In addition, our study used only one type of cell model, it would be better if our results had been confirmed in other cell models or animal models.

In summary, we report a novel PSEN1 K311R mutation in two Chinese families with LOAD. The mutation was located within the HL domain of PSEN1 CL and enhanced Aβ₄₂ production and tau phosphorylation in HEK293-APP695_wt cells. Future studies are needed to clarify the underlying mechanisms associated with impaired AβPP cleavage and enhanced tau phosphorylation to explore the functional impact of the mutation on other presenilin-1-related signaling pathways.

Footnotes

ACKNOWLEDGMENTS

This study was supported by CHINA-CANADA Joint Initiative on Alzheimer’s Disease and Related Disorders (81261120571), the Key Project of the National Natural Science Foundation of China (81530036), the National Key Scientific Instrument and Equipment Development Project (31627803), Key medical professional development plan of Beijing Municipal Administration of Hospitals (ZY201301), Mission Program of Beijing Municipal Administration of Hospitals (SML20150801), Beijing Scholars Program and Beijing Municipal Science & Technology Commission (Z161100000216137).

Authors’ disclosures available online ().

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