Lipidized Prolactin-Releasing Peptide Agonist Attenuates Hypothermia-Induced Tau Hyperphosphorylation in Neurons

Abstract

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases, characterized by the accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles. These tangles mainly consist of hyperphosphorylated tau protein. As it induces tau hyperphosphorylation in vitro and in vivo, hypothermia is a useful tool for screening potential neuroprotective compounds that ameliorate tau pathology. In this study, we examined the effect of prolactin-releasing peptide (PrRP), its lipidized analog palm¹¹-PrRP31 and glucagon-like-peptide-1 agonist liraglutide, substances with anorexigenic and antidiabetic properties, on tau phosphorylation and on the main kinases and phosphatases involved in AD development. Our study was conducted in a neuroblastoma cell line SH-SY5Y and rat primary neuronal cultures under normothermic and hypothermic conditions. Hypothermia induced a significant increase in tau phosphorylation at the pThr212 and pSer396/pSer404 epitopes. The palmitoylated analogs liraglutide and palm¹¹-PrRP31 attenuated tau hyperphosphorylation, suggesting their potential use in the treatment of neurodegenerative diseases.

Keywords

Hypothermia lipidization primary neuronal culture prolactin-releasing peptide SH-SY5Y tau phosphorylation

INTRODUCTION

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases associated with memory loss and cognitive decline [1]. AD is characterized by the extracellular accumulation of amyloid-β peptides as amyloid plaques [2] and intraneuronal neurofibrillary tangles (NFT) of hyperphosphorylated tau protein [3]. The main factors implicated in the development of AD are aging [4], type 2 diabetes mellitus (T2DM), and obesity [5, 6]. Epidemiological studies suggest that T2DM is an important risk factor for the development of tau hyperphosphorylation and affects AD pathology, but the precise mechanism remains unclear [7, 8]. Hence, novel studies investigated the effect of T2DM on tau phosphorylation in db/db and ob/ob mice [9, 10]. Hyperphagic obese db/db mice with a mutation in the leptin receptor develop severe insulin resistance and represent a model of spontaneous T2DM [11]. Similarly, leptin-deficient ob/ob mice are hyperphagic and obese and mimic features of mild T2DM [12]. Both models displayed tau hyperphosphorylation at multiple epitopes that was linked to hypothermia. Several other studies supported the hypothesis that hypothermia increased tau phosphorylation and contributed to pathology in the brain [4 , 13– 16].

Hypothermia, defined as subnormal body temperature, is classified into different ranges from mild (32– 34°C) to profound (4– 14°C) [5]. Planel et al. reported that hypothermia can be induced by alterations in glucose metabolism [14] or anesthesia [17]. Metabolically active mouse brain slices, SH-SY5Y or SH-SY5Y overexpressing 3R-Tau cells displayed tau hyperphosphorylation at the epitope pS199/pS202/pT205, pThr181, pSer202, and pSer396/pSer404 after incubation at 30°C [13]. In AD, tau is hyperphosphorylated by the main kinases glycogen synthase kinase-3β (GSK-3β) [18] and cyclin-dependent kinase-5 (Cdk5) [19]. However, hypothermia induces tau hyperphosphorylation by a significant inhibition of protein phosphatase 2A (PP2A) [17, 20] rather than by activation of tau kinases. Several studies have demonstrated that anti-T2DM drugs, such as glucagon-like-peptide-1 (GLP-1) analogs, have shown potential neuroprotective benefits and increased the activation of the insulin signaling cascade [21 –23]. The antidiabetic drug liraglutide, GLP-1 analog, was reported to reduce tau phosphorylation and improve memory and learning in several models of AD-like pathology [24 –26]. Liraglutide acts centrally as anorexigenic compound and has weight-lowering properties [23, 27]. We have therefore suggested if prolactin-releasing peptide (PrRP), another anorexigenic neuropeptide [28] with antidiabetic properties [29], could reduce tau phosphorylation and we searched for the molecular mechanism of the action. PrRP is linked to anorexigenic hormone leptin in the regulation of food intake and metabolism and leptin regulates its expression [30]. Leptin also decreases tau phosphorylation and amyloid-β peptides formation in vitro and in vivo [31 –33].

Previously, we reported that a lipidized analog of PrRP, palm¹-PrRP31, decreased hippocampal tau phosphorylation in mice with monosodium glutamate (MSG)-induced obesity [21]. Moreover, in the THY-Tau22 transgenic mouse model of AD-like tauopathy [34], the novel lipidized analog palm¹¹-PrRP31 attenuated tau hyperphosphorylation at different epitopes in the hippocampus [35]. Those findings suggest that, besides GLP-1 analogs, other anorexigenic compounds could be used as a possible treatment for neurodegenerative diseases.

Although an in vivo study offers an integrated approach, the presence of both neuronal and glial cells in the brain makes it difficult to decipher the direct effect of palm¹¹-PrRP31 on tau phosphorylation in neurons. In this study, to focus selectively on the effect of palm¹¹-PrRP31 on tau phosphorylation in neuronal cells, we first examined the influence of moderate hypothermia at 30°C in two different in vitro models (human SH-SY5Y neuroblastoma cells and differentiated primary neuronal cultures from embryonic rat cortex), and then analyzed the effect of PrRP, palm¹¹-PrRP31 and liraglutide as a comparator. We further examined the effect of PrRP and palm¹¹-PrRP31 on the main kinases and phosphatase involved in the development of AD in normothermic or hypothermic conditions.

MATERIAL AND METHODS

Peptide synthesis

Human PrRP31 and palm¹¹-PrRP31, a palmitoylated analog of hPrRP31, were synthesized at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic (IOCB AS CR), as described by [36]. The GLP-1 analogue liraglutide (Victoza^®, Novo Nordisk A/S, Bagsværd, Denmark) (6 mg/mL) was obtained from a pharmacy. All peptides were dissolved in water.

Neuroblastoma cells

The SH-SY5Y neuroblastoma cell line (ATCC^® CRL-2266™) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 2 mM L-glutamine, and 1% nonessential amino acids in a 5% CO₂ humidified incubator at 37°C. Cells were seeded at 200,000 cells/well in 24-well plates coated with poly-L-lysine (0.1 mg/mL) and maintained at 37°C until the beginning of the experiment.

Rat primary neuronal culture

Primary neuronal cultures from rat embryonic brain cortex (primary neurons) were prepared from 16– 17-day-old embryos from Wistar rats (Janvier Labs, Le Genest-Saint-Isle, France). The brain cortex was dissected out and carefully dissociated in culture medium (neurobasal medium supplemented with 2% B27, 200 mM L-glutamine and 1% antibiotic-antimycotic agent; Thermo Fisher Scientific Inc., Waltham, MA, USA) with a Pasteur pipette as described previously [37]. Subsequently, cells were seeded at 200,000 cells/well in 24-well plates coated with poly-D-lysine (0.1 mg/mL) and laminin (20 μg/mL) for immunoblotting or at 80,000 cells/well in coated 24-well plates with slides for immunocytochemistry. Plates were maintained in a 5% CO₂ humidified incubator at 37°C until 13 days in vitro (DIV13).

Treatment and hypothermia

On the day of the experiment, the SH-SY5Y cells or primary cortical neurons seeded in 24-well plates were treated with either 0.1 μM liraglutide, hPrRP31 or palm¹¹-PrRP31 and incubated in growth media for 2 h. Plates were kept in a 5% CO₂ humidified incubator either at 37°C for normothermic or at 30°C for moderate hypothermic conditions. The experiment was performed at least twice, and the controls and compounds were tested in duplicate or triplicate.

Sample preparation and immunoblotting

At the end of the experiment, the SH-SY5Y or primary cortical neurons were rapidly washed 3 times with phosphate buffered saline (PBS) (pH 7.4) at 37°C or 30°C and lysed in a lysis buffer as described previously [38]. Electrophoresis and western blots were carried out according to [35] using a Criterion system with a 26-well 4– 15% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA) at a constant voltage of 200 V or 100 V. Nitrocellulose membranes were blocked in 5% nonfat milk or BSA at room temperature for 1 h. Subsequently, membranes were incubated overnight with primary antibody at 4°C, according to the manufacturer’s instructions (Table 1). A list of used antibodies and their appropriate dilutions is shown in (Table 1). The next day, the membranes were incubated with an HRP-linked secondary antibody for 1 h at room temperature. Chemiluminescence was visualized with a ChemiDoc™ System (Bio-Rad) and quantified using Image Lab Software (Bio-Rad). Band intensities were normalized using β-actin as an internal loading control.

Table 1

List of primary antibodies used for immunoblotting and their dilutions

Antibody	Epitope	Dilution for WB	Provider
AD2 rabbit Ab	pS396/pS404	1:1 000 5% milk TBS/tween-20	Produced in Lille (INSERM Laboratory UMR-S 1172)
AT180 mouse Ab	pT231/pS235	1:1000 5% BSA TBS/tween-20	Thermo Fisher Scientific Inc., Waltham, MA, USA
AT270 mouse Ab	pT181	1:1 000 5% milk TBS/tween-20	Thermo Fisher Scientific Inc., Waltham, MA, USA
AT8 mouse Ab	pS199/pS202/pT205	1:1 000 5% milk TBS/tween-20	Thermo Fisher Scientific Inc., Waltham, MA, USA
CP13 mouse Ab	pS202	1:1 000 5% milk TBS/tween-20	Dr. Peter Davies
Phospho-GSK-3β rabbit Ab	pS9	1:1 000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
GSK-3β rabbit Ab		1:1 000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
Phospho-p44/42 MAPK (ERK1/2) mouse Ab	pT202/pT204	1:2000 5% milk TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
p44/42 MAPK (ERK1/2) mouse mAb		1:2000 5% milk TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
PHF-1 mouse Ab	pS396/pS404	1:1000 5% milk TBS/tween-20	Dr. Peter Davies
Phospho-p38 MAPK rabbit Ab	pT180/pY182	1:1000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
p38 MAPK rabbit Ab		1:1000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
PP2A subunit C rabbit Ab		1:1000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
Phospho-SAPK/JNK rabbit mAb	pT183/pY185	1:1000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
SAPK/JNK rabbit Ab		1:1000 5% BSA TBS/tween-20	Cell Signaling Technology, Beverly, MA, USA
Phospho-Tau (Thr212) rabbit Ab	pT212	1:1 000 5% BSA TBS/tween-20	Thermo Fisher Scientific Inc., Waltham, MA, USA
Phospho-Tau (Ser396) rabbit Ab	pS396	1:1 000 5% BSA TBS/tween-20	Thermo Fisher Scientific Inc., Waltham, MA, USA
Tau 5 mouse Ab		1:10 000 5% milk TBS/tween-20	Thermo Fisher Scientific Inc., Waltham, MA, USA

Immunocytochemistry

Primary cortical neurons used for immunocytochemistry were carefully washed 3 times with PBS at 37°C or 30°C and then fixed for 20 min in 4% paraformaldehyde at room temperature. Next, the cells were washed 3 times with 50 mM NH₄Cl in PBS/1% BSA and then permeabilized with 0.1% Triton X-100 in PBS/1% BSA for 10 min. After 1 h of blocking in 1% BSA in PBS buffer, the cells were incubated overnight at 4°C with the appropriate antibody (AT8, pThr212, pSer396, AT180; all from Thermo Fisher Scientific Inc.) diluted 1:400 in PBS/1% BSA. Then, the cells were incubated for 1 h with the secondary antibody, mouse Alexa 488 (#A11001) or rabbit Alexa 568 (#A11011) (both from Thermo Fisher Scientific Inc.). The nuclei were stained with Vectashield/DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, Burlingame, CA, USA). Images were acquired on a Zeiss confocal laser-scanning microscope LSM 710 using a 488 nm Argon laser and a 405 nm ultraviolet laser with the same laser intensities to compare the images at the Lille 2 University (Plate-forme d’Imagerie Moléculaire et Cellulaire) and on a Zeiss confocal laser-scanning microscope LSM 780 using same conditions at the IOCB AS CR.

Analysis of data and statistics

Data from the immunoblots of tau epitopes were normalized to β-actin as a loading control and subsequently divided by total tau (Tau 5 antibody) normalized to β-actin. Data from the immunoblots of kinases and phosphatases were normalized to β-actin. All data are presented as the means of the percentage of the normothermic nontreated controls±SEMs (n = 2– 3, in each of two independent experiments). Statistical analysis was performed by unpaired t-test using Graph-Pad Prism Software (San Diego, CA, USA); p < 0.05 was considered statistically significant.

RESULTS

A lipidized PrRP analog, palm¹¹-PrRP31, attenuates hypothermia-induced tau hyperphosphorylation at the epitope pThr212 in the SH-SY5Y cells

To examine the effect of treatment with liraglutide, hPrRP31 or palm¹¹-PrRP31 on tau phosphorylation, the SH-SY5Y cells were incubated with the compounds in normothermic (37°C) or hypothermic (30°C) conditions for 2 h and cell lysates were analyzed by immunoblots (n = 2– 3, two independent experiments). Representative immunoblots of analyzed tau epitopes and their quantification are shown in (Fig. 1A and B).

Fig.1

Palm¹¹-PrRP31 attenuates hypothermia-induced tau hyperphosphorylation in SH-SY5Y cells. Immunoblots of phosphorylated tau epitopes (A) and their quantifications (B). SH-SY5Y were subjected to normothermic conditions (37°C) or hypothermia (30°C) for 2 h and incubated either with 0.1 μM liraglutide (lira), hPrRP31 (hP31), palm¹¹-PrRP31 (palm¹¹-P31), or medium alone. Phosphorylation of tau protein was determined by specific antibodies AT270, PHF-1, pThr212, and pSer396. Data were normalized to total tau (Tau 5) and to loading control, β-actin. Data are presented as mean of percentage of normothermic non-treated control±SEM (n = 2– 3, in each of two independent experiments). Statistical analysis was calculated by unpaired t-test. ^**p < 0.01 treatment 37°C or control 30°C versus control 37°C, ^#p < 0.05, ^# # #p < 0.001 treatment 30°C versus control 30°C.

Levels of phosphorylated tau at all analyzed epitopes were significantly increased under hypothermic conditions at 30°C (pThr181 (AT270) +91%, pSer396/pSer404 (PHF-1) +43%, pThr212 +90%, pSer396 +77%) (Fig. 1A and B, Supplementary Table 1A). Incubation with liraglutide, hPrRP31, or palm¹¹-PrRP31 did not change the levels of phosphorylated tau epitopes in normothermic conditions. On the other hand, natural PrRP31 and lipidized palm¹¹-PrRP31 decreased phosphorylation at pThr212 in hypothermia (Fig. 1A and B, Supplementary Table 1A).

Liraglutide, hPrRP31, and palm¹¹-PrRP31 differentially affect the phosphorylation of the kinases MAPK/ERK1/2 and p38 and the expression of the phosphatase PP2A in the SH-SY5Y cells in control or hypothermic conditions

As we wanted to determine the molecular mechanism of tau hyperphosphorylation and the impact of our compounds, we next examined the expression of major tau kinases, which have been implicated in AD pathogenesis in control or hypothermic conditions. These kinases include phospho-GSK-3β (Ser9), whose kinase activity is inhibited upon phosphorylation of its Ser9, GSK-3β, and Cdk5. We also investigated mitogen-activated protein kinases (MAPKs), which are mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2) and its active phosphorylated form pMAPK/ERK1/2, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and its activated phosphorylated form pSAPK/JNK, p38 and its activated phosphorylated form phospho-p38 (p-p38). We next examined the expression of phosphatase PP2A subunit C in control or hypothermic conditions. These kinases and phosphatase are responsible for the phosphorylation or dephosphorylation of tau in vitro.

Neither hypothermia nor treatment with liraglutide, hPrRP31 or palm¹¹-PrRP31 caused changes in the level of the main tau kinases, Cdk5 and GSK-3β (pSer9) (Fig. 2A and B, Supplementary Table 1B). After incubation in hypothermic conditions, there were no significant changes in pMAPK/ERK1/2 (Fig. 2A and B, Supplementary Table 1B), but treatment with liraglutide, hPrRP31 and palm¹¹-PrRP31 significantly increased phosphorylation of MAPK/ERK1/2 at 37°C. Moreover, palm¹¹-PrRP31 increased the levels of phosphorylated MAPK/ERK 1/2 at 30°C. The expression of pSAPK/JNK and p-p38 was significantly reduced (– 44% and – 43%, respectively) in hypothermic conditions. Treatment with liraglutide and palm¹¹-PrRP31 increased the level of p38 at 30°C (Fig. 2A and B, Supplementary Table 1B).

Fig.2

Palm¹¹-PrRP31 does not affect the activity of main tau kinases in SH-SY5Y cells. Immunoblots of phosphorylated kinases or PP2A (C) and their quantifications (B). SH-SY5Y were subjected to normothermic conditions (37°C) or hypothermia (30°C) for 2 h and incubated either with 0.1 μM liraglutide (lira), hPrRP31 (hP31), palm¹¹-PrRP31 (palm¹¹-P31), or medium alone. Phosphorylation of tau protein kinases or PP2A was determined by immunoblotting using specific antibodies and was normalized to β-actin. Data are presented as mean of percentage of normothermic non-treated control±SEM (n = 2– 3, in each of two independent experiments). Statistical analysis was calculated by unpaired t-test. ^*p < 0.05, ^**p < 0.01 treatment 37 C or control 30°C versus control 37°C, ^#p < 0.05, ^##p < 0.01 treatment 30°C versus control 30°C.

As expected, the expression of PP2A was inhibited (– 26%) at low temperature (Fig. 2A and B, Supplementary Table 1B). Only natural PrRP31 significantly reduced PP2A at 37°C compared to the normothermic control (Fig. 2A and B, Supplementary Table 1B).

Human PrRP31 and palm¹¹-PrRP31 attenuate hypothermia-induced tau hyperphosphorylation in rat primary neuronal cultures

As we observed a significant decrease in hyperphosphorylated tau after treatment with palm¹¹-PrRP in the neuroblastoma SH-SY5Y cells, we further examined its effect on tau phosphorylation in differentiated neurons from rat primary neuronal cultures. Immunoblots were performed from lysates of rat primary neurons (n = 2, two independent experiments). The cells were incubated with liraglutide, hPrRP31, or palm¹¹-PrRP31 in normothermic (37°C) or hypothermic (30°C) conditions for 2 h. Representative immunoblots and their quantification are shown in (Fig. 3A and B). Levels of phosphorylated tau were significantly increased at all analyzed epitopes (pSer396/pSer404 (AD2) +103%, pThr181 (AT270) +37%, pSer202 (CP13) +20%, pSer396/pSer404 (PHF-1) +17%, pThr212 +90%) in hypothermic conditions at 30°C (Supplementary Table 1A). AD2 and PHF-1 antibodies recognize the same epitopes at pSer396/pSer404 [40], and they revealed similar results. Lipidized palm¹¹-PrRP31 caused a decrease in tau hyperphosphorylation at the epitopes pThr181 (– 49%), pSer202 (– 41%), pSer396/pSer404 (– 35%) and pThr212 (– 94%) at 30°C when compared with the hypothermic control (Fig. 3B, Supplementary Table 1A). However, natural hPrRP31 did not cause any changes in hyperphosphorylated tau levels in hypothermic conditions. Interestingly, treatment with hPrRP31 and palm¹¹-PrRP31 decreased the phosphorylation of pSer202 (CP13), pSer396/pSer404 and pThr212 in normothermic (37°C) conditions (Supplementary Table 1A). Liraglutide was used as a comparator and decreased phosphorylation of pSer202 and pSer396/pSer404 when compared with the control at 37°C.

Fig.3

Palm¹¹-PrRP31 attenuates hypothermia-induced tau hyperphosphorylation in rat primary neurons. Immunoblots of phosphorylated tau epitopes (A) and their quantifications (B). Primary cortical neurons were incubated either with 0.1 μM liraglutide (lira), hPrRP31 (hP31), palm¹¹-PrRP31 (palm¹¹-P31), or medium alone (control) in normothermic conditions (37°C) or hypothermia (30°C) for 2 h. Phosphorylation of tau protein was determined using specific antibodies AD2, AT270, CP13, PHF-1, and pThr212. Data are normalized to total Tau (Tau 5) and to loading control, β-actin and are presented as mean of percentage of normothermic non-treated control±SEM (n = 2, in each of two independent experiments). Statistical analysis was calculated by unpaired t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 treatment 37°C or control 30°C versus control 37°C, ^#p < 0.05 treatment 30°C versus control 30°C.

To confirm the decreasing effects of hPrRP31 and palm¹¹-PrRP31 on hypothermia-induced tau hyperphosphorylation, we performed immunocytochemistry on rat primary cortical neurons in similar normothermic and hypothermic conditions. In agreement with the immunoblots, immunocytochemistry data showed a similar tendency of hypothermia to increase the phosphorylation of tau at epitope pThr212 (Fig. 4C). Incubation at 30°C for 2 h also increased the phosphorylation at pThr231/pSer235 (AT180) (Fig. 4A), pSer396 (Fig. 4B), and pSer199/pSer202/pThr205 (AT8) (Fig. 4C) in cortical neurons. Treatment with hPrRP31 and palm¹¹-PrRP31 decreased the signal compared to the hypothermic control at 30°C for all the tested epitopes.

Fig.4

The phosphorylation of tau protein at AT180 (pThr231/pSer235) (A), as well as at pSer396 (B) and AT8 (pS199/pS202/pT205), pThr212 (C) was decreased after palm¹¹-PrRP31 treatment in hypothermic conditions compared with hypothermic non-treated control (30°C). The immunohistochemical staining of protein tau phosphorylated at pThr231 (A), pSer396 (B) and AT8, pThr212 (C) in rat primary cortical neurons (n = 2, in each of two independent experiments) in normothermic (37°C) and hypothermic (30°C) conditions. The nuclei were stained with Vectashield/DAPI (4,6-diamidino-2-phenylindole).

Palm¹¹-PrRP31 affects the expression of the main tau kinases and phosphatase in rat primary cortical neurons in normothermic conditions

We next examined the levels of the main tau kinases Cdk5, GSK-3β, pGSK-3β, MAPK/ERK1/2, pMAPK/ERK1/2, SAPK/JNK, pSAPK/JNK, p38, and p-p38 or phosphatase PP2A on immunoblots from lysates of rat primary cortical neurons (n = 2, two independent experiments). There were no significant changes in Cdk5 induced by hypothermia or treatment with liraglutide, hPrRP31 or palm¹¹-PrRP31 in hypothermic conditions at 30°C. Interestingly, incubation with hPrRP31 and palm¹¹-PrRP31 showed decreased levels of Cdk5 (– 47%, – 52% respectively) in normothermic conditions (Supplementary Table 1B). The low temperature increased the phosphorylation of GSK-3β at Ser9 (+29%), which led to partial inhibition of its kinase activity, compared to the normothermic control (37°C) (Fig. 5A and B, Supplementary Table 1B). Incubation with liraglutide, hPrRP31 or palm¹¹-PrRP31 did not have any effect. Neither hypothermic conditions nor liraglutide, hPrRP31 or palm¹¹-PrRP31 treatment caused any significant changes in phosphorylated MAPK/ERK1/2 or p38 (Fig. 5A and B, Supplementary Table 1B). The level of pSAPK/JNK was not affected by hypothermic conditions. Treatment with palm¹¹-PrRP31 lowered the level of pSAPK/JNK (– 35%) at 37°C but not at 30°C (Fig. 3A, B). As expected, the expression of PP2A was strongly inhibited (– 57%) at the lower temperature; treatment with all of the compounds tended to increase PP2A levels even though it was nonsignificant (Fig. 5A, B). Similarly to what was observed in the SH-SY5Y cells, palm¹¹-PrRP31 reduced levels of PP2A in normothermic condition (Fig. 5A and B, Supplementary Table 1B).

Fig.5

Activation of kinases implicated in tau hyperphosphorylation after treatment with palm¹¹-PrRP31 in normothermic (37°C) and hypothermic (30°C) conditions in primary cortical neurons. Immunoblots of kinases (A) and quantification of western blots (B). Phosphorylation of tau protein kinases was determined by immunoblotting using specific antibodies. Data are presented as mean of percentage of normothermic non-treated control±SEM (n = 2, in each of two independent experiments). Statistical analysis was calculated by unpaired t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 treatment 37°C or control 30°C versus control 37°C, ^#p < 0.05 treatment 30°C versus control 30°C.

DISCUSSION

AD and other dementias represent an increasing and still unresolved health problem. A variety of cellular models of AD-like pathology has helped to study key aspects of tau hyperphosphorylation or amyloid-β formation. Many in vitro and in vivo studies have identified hypothermia as a useful tool to study the molecular mechanisms involved in tau hyperphosphorylation, which is implicated in the development of AD [5, 14]. Besides, studies have found that elderly individuals are susceptible to hypothermia, which may be another risk factor contributing to AD pathogenesis [4, 41]. A decreased body temperature has higher impact on tau hyperphosphorylation in old animals compared to the young ones [4]. This indicates that lower body temperature enhances the risk of AD development in the old age, where the individuals are more susceptible to the changes in temperature. Moreover, tau hyperphosphorylation at multiple epitopes was observed in db/db and ob/ob mice where it was linked to hypothermia because of T2DM [9, 10].

In this study, we investigated the impact of natural PrRP31, our potent lipidized PrRP analog, palm¹¹-PrRP31, and liraglutide as a comparator, on hypothermia-induced tau hyperphosphorylation. Moderate hypothermia (from 26– 33°C) represents a suitable model for inducing strong tau hyperphosphorylation at multiple epitopes [14]. We also studied the molecular mechanisms of kinases and phosphatase in two neuronal models— neuroblastoma SH-SY5Y cells and differentiated cortical neurons from rat primary neuronal cultures. In accordance with a previous study [13], we confirmed hypothermia-induced tau hyperphosphorylation at multiple epitopes (pThr212, pThr231/pSer235, pThr181, pSer202, and pSer396/pSer404) both in the SH-SY5Y cells and rat primary cortical neurons. AD-related tau phosphorylation was assigned previously to the epitopes pSer202/pThr205, pThr231, pThr181, and pSer396/pSer404 [42]. Among those, pThr231 and pSer262 were labeled as early pathological tau modifications [43], while other tau phosphorylation sites pThr181, pSer202/pThr205, and pSer396/pSer404 were associated with a later stage of the disease [43].

Here, in primary cortical neurons, palm¹¹-PrRP31 attenuated hypothermia-induced tau hyperphosphorylation at the epitopes pThr212, pThr231, pThr181, pSer202, and pSer396/pSer404. Palm¹¹-PrRP31 and natural PrRP had a similar effect in normothermic conditions at 37°C in rat cortical neurons. These results were in agreement with our previous in vivo study, which showed that palm¹¹-PrRP31 inhibited the phosphorylation of pThr231 and pSer396/pSer404 in THY-Tau22 mice with overexpressed human tau [35] and suggest that palm¹¹-PrRP31-induced inhibition of tau phosphorylation observed in vivo is mediated through a direct effect on neurons. Taken together, these findings support the presumption that palm¹¹-PrRP31 is a potential tool to attenuate the tau hyperphosphorylation implicated in neurological diseases. Due to decreased stability in rat plasma, natural PrRP31 had a lower effect on the attenuation of tau phosphorylation compared to lipidized PrRP analogs [44]. In our study, GLP-1 agonist liraglutide, used as a comparator to palm¹¹-PrRP31, decreased tau hyperphosphorylation at the epitopes pThr181, pSer202 and pSer396/pSer404 in primary cortical neurons in normothermic conditions. Our results are in agreement with an in vivo study [45], where liraglutide ameliorated hippocampal tau hyperphosphorylation in rats with streptozotocin-induced sporadic AD and confirm that liraglutide, similarly as it has been mentioned for palm¹¹-PrRP31, affects directly neurons.

In the SH-SY5Y cell line, palm¹¹-PrRP31 reduced tau hyperphosphorylation only at the pThr212 epitope. Globally, liraglutide, hPrRP31, and palm¹¹-PrRP31 showed less potential to decrease phosphorylation at different tau epitopes in the SH-SY5Y cells compared to primary cortical neurons. Contrarily to differentiated neurons, the SH-SY5Y cells are dividing cells that do not express mature neuronal markers. In this study, results obtained in differentiated primary cortical neurons better reflect those previously obtained in vivo [35], confirming the accuracy of this cellular model to study cellular mechanisms involved in brain [46].

Hypothermia-induced tau hyperphosphorylation is caused by a dysfunction of the kinase/phosphatase system [13 , 17] in which inhibition of tau phosphatase PP2A outweighs activation of tau kinases. The major kinases phosphorylating tau in AD are GSK-3β [47] and Cdk5 [48]. GSK-3β kinase activity is upregulated by phosphorylation at Tyr216 and downregulated by Ser9 phosphorylation, which leads to its partial inhibition [49]. Here, we investigated the impact of liraglutide, hPrRP31 and palm¹¹-PrRP31 on the main tau kinases in normothermic and hypothermic conditions. Similar to our previous in vivo studies [21, 35], we did not observe any changes in GSK-3β Ser9 phosphorylation after treatment with the tested substances either in the primary cortical neurons or in the SH-SY5Y cells under both normo- and hypothermic conditions. Regarding Cdk5 in primary cortical neurons, its kinase level was reduced significantly after treatment with hPrRP31 and palm¹¹-PrRP31 in normothermic conditions, but not in hypothermic conditions where it only tended to an attenuating effect. No significant changes in Cdk5 were induced by hypothermia or by treatment with the tested compounds in the SH-SY5Y cells. This result might point to PrRP as a substance that can lower Cdk5 in vitro in neurons and physiological conditions. Palm¹¹-PrRP31 attenuated phosphorylation of pThr212, pThr231, pSer396, and pSer404, all targets of the Cdk5 kinase activity [39]. We could then speculate that palm¹¹-PrRP31 affects Cdk5, but further studies are needed.

Other kinases involved in tau phosphorylation in AD are MAPK kinases MAPK/ERK1/2, SAPK/JNK, and p38 [14, 48]. All MAPKs mentioned were previously reported to be inactive under hypothermic conditions in brain extracts [14]. We observed a similar trend in primary cortical neurons in this study. On the other hand, in neuroblastoma SH-SY5Y cells, activation of SAPK/JNK and p38 was paradoxically decreased under hypothermic conditions. The treatment with liraglutide increased the kinase activity of p38 and so did the treatment with palm¹¹-PrRP31 only in the SH-SY5Y cells. At normothermic conditions, the activation of MAPK/ERK1/2 occurred in the SH-SY5Y cells after treatment with liraglutide, hPrRP31, and palm¹¹-PrRP31 in accordance with previous studies. PrRP was shown previously to activate preferably MAPK/ERK1/2 and its target CREB (cAMP response element-binding protein) in rat pituitary RC-4B/C cells [50]. Liraglutide caused activation of pMAPK/ERK1/2 signaling within the nucleus tractus solitarii [51].

Tau can be dephosphorylated mainly by PP2A, and conversely, inhibition of PP2A in cell cultures, neuronal cultures and in vivo results in hyperphosphorylation of tau at multiple epitopes, as reviewed by [52]. Previously, two in vivo studies reported that under hypothermia, tau hyperphosphorylation was attributed to decreased PP2A activity [14, 17]. Hypothermia exponentially decreases the activities of phosphatases, while activities of tau kinases decrease only linearly [5, 14]. In both SH-SY5Y cells and primary cortical neurons, we confirmed significant decrease of PP2A during hypothermia in our study. However, in normothermic conditions, natural PrRP31 decreased the expression of the PP2A subunit C in the SH-SY5Y cells, and palm¹¹-PrRP31 treatment decreased its expression in primary cortical neurons. Therefore, the decrease of the phosphorylation of the tau epitopes pThr212, pThr231, pSer396, and pSer404 after the treatment in normothermic conditions is not due to changes in PP2A level but rather by inhibition of kinases.

Model of hypothermia is a useful tool for screening potential neuroprotective compounds that might ameliorate tau pathology. In conclusion, both tested palmitoylated analogs, liraglutide and palm¹¹-PrRP were potentially neuroprotective because they attenuated tau phosphorylation at several epitopes suggesting their potential in AD treatment.

Footnotes

ACKNOWLEDGMENTS

This study was supported by LabEx (Excellence Laboratory), DISTALZ (Development of Innovative Strategies for a Transdisciplinary Approach to Alzheimer’s Disease) and INSERM (Institut National de la Santé et de la Recherche Médicale) and by the Grant Agency of the Czech Republic (grant number 16-00918S) and the Academy of Sciences of the Czech Republic (RVO: 61388963 and RVO: 67985823). We thank Séverine Bégard and Sarah Lieger (Université Lille, INSERM, CHU Lille, UMR – S 1172 – Jean Pierre Aubert Research Centre) for their help in preparation of the rat neuronal cultures and Dr. Peter Davies (Litwin-Zucker Center for the study of Alzheimer’s Disease, The Feinstein Institute for Medical Research, New York, NY, USA) for kind providing of antibodies. We are grateful to the IMPRT (Institut de Médecine Prédictive et de Recherche Thérapeutique, Lille) for access to the confocal microscopy platform and animal facility.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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Lipidized Prolactin-Releasing Peptide Agonist Attenuates Hypothermia-Induced Tau Hyperphosphorylation in Neurons

Abstract

Keywords

INTRODUCTION

MATERIAL AND METHODS

Peptide synthesis

Neuroblastoma cells

Rat primary neuronal culture

Treatment and hypothermia

Sample preparation and immunoblotting

Immunocytochemistry

Analysis of data and statistics

RESULTS

A lipidized PrRP analog, palm11-PrRP31, attenuates hypothermia-induced tau hyperphosphorylation at the epitope pThr212 in the SH-SY5Y cells

Liraglutide, hPrRP31, and palm11-PrRP31 differentially affect the phosphorylation of the kinases MAPK/ERK1/2 and p38 and the expression of the phosphatase PP2A in the SH-SY5Y cells in control or hypothermic conditions

Human PrRP31 and palm11-PrRP31 attenuate hypothermia-induced tau hyperphosphorylation in rat primary neuronal cultures

Palm11-PrRP31 affects the expression of the main tau kinases and phosphatase in rat primary cortical neurons in normothermic conditions

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

A lipidized PrRP analog, palm¹¹-PrRP31, attenuates hypothermia-induced tau hyperphosphorylation at the epitope pThr212 in the SH-SY5Y cells

Liraglutide, hPrRP31, and palm¹¹-PrRP31 differentially affect the phosphorylation of the kinases MAPK/ERK1/2 and p38 and the expression of the phosphatase PP2A in the SH-SY5Y cells in control or hypothermic conditions

Human PrRP31 and palm¹¹-PrRP31 attenuate hypothermia-induced tau hyperphosphorylation in rat primary neuronal cultures

Palm¹¹-PrRP31 affects the expression of the main tau kinases and phosphatase in rat primary cortical neurons in normothermic conditions