Cystatin C promotes cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsin Ia/Ib pathway

  • Authors:
    • Guangna Yu
    • Xingyuan Sun
    • Li Li
    • Lijuan Huang
    • Hongbin Liu
    • Shuying Wang
    • Zhanjun Ren
    • Yanjiao Zhang
  • View Affiliations

  • Published online on: December 31, 2019     https://doi.org/10.3892/etm.2019.8403
  • Pages: 2282-2290
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Although higher serum level of cystatin C (CysC) was observed in patients with cerebral microbleeds, its associated role in the disease has not been elucidated. In this work, a rat model of cerebral microbleeds was created with the aim of investigating effects of CysC on cognitive function in rats with cerebral microbleeds and the underlying mechanism. Serum samples of patients with cerebral microbleeds and healthy people of the same age were collected. Levels of cystatin C expression in these samples were measured using CysC kits. Moreover, 48 spontaneously hypertensive rats (SHRs) bred under specific pathogen‑free (SPF) conditions were randomly divided into 4 groups: sham surgery control group (sham), model group (CMB), model + empty vector control group (CMB + vehicle), and model + cystatin C overexpression group (CMB + CysC). Expression levels of CysC in hippocampus of rats in each group were measured by western blot analysis. The Y‑maze was used to evaluate cognitive function of rats. Hippocampal long‑term potentiation (LTP) in rats was assessed by the electrophysiological assay. Alterations in levels of p‑ERK1/2 and p‑synapsin Ia/b proteins associated with cognitive function were identified by western blot analysis. The serum levels of CysC in patients with cerebral microbleeds were significantly upregulated (P<0.001). After injection of CysC, its expression levels in rat hippocampus were significantly increased (P<0.001), which enhanced the decline in learning and memory function, as well as the decrease of LTP in the rat model of cerebral microbleeds (P<0.001). Western blot results showed that injection of CysC further reduced the levels of p‑ERK1/2 and p‑synapsin Ia/b in the rat model of microbleeds (P<0.001). CysC was up regulated in serum of patients with cerebral microbleeds. It promoted cognitive dysfunction in rats with microbleeds by inhibiting ERK/synapsin Ia/Ib pathway.

Introduction

Cerebral microbleeds (CMB) are subclinical lesions caused by deposition of hemosiderin and other substances in the brain parenchyma as a result of leakage of intravascular components into the surrounding space following damage of the cerebral small vessel wall (1). The prevalence rate of CMB in healthy adults is ~5% (2). CMB is rarely detected in young people (aged <40 years) (3), but the prevalence rate and case number increase with age. In a Rotterdam Scan study, the prevalence rate of CMB increased from 18% in people aged 60–69 years to 38% in people aged ≥80 years (4). CMB occurred to 34% of patients with ischemic stroke and 60% of patients with intracerebral hemorrhage (2), indicating that CMB is associated with the severity of underlying vascular diseases. Association of CMB with stroke was reported in many studies (5,6). Among patients with dementia, the prevalence rate of CMB is ~14% in patients with mild cognitive impairment and 23% in patients with Alzheimer's disease (7,8). The increased prevalence rate of CMB in the elderly indicates its association with conditions of deep perforating vessels and cerebral amyloid angiopathy. It was reported that CMB is spatially associated with amyloid deposition areas in the brain (9,10), and thus it was speculated that CMB may pose a risk of developing dementia (411). Two hypotheses were proposed for possible mechanism. Firstly, CMB may have an impact on the complex cortical connections, leading to disruption of the neural network (12,13). Secondly, it may induce conditions of deep perforating vessels and cerebral amyloid angiopathy (14). As far as we know, there are few reports on specific molecular mechanisms.

Cystatin C (CysC) is a member of the type 2 cystatin superfamily of cysteine protease inhibitors. It is a small protein (13 kDa) produced and secreted by almost all karyocytes in the body (15,16). Cystatin C is a potent competitive cysteine protease inhibitor that primarily modulates extracellular protease activity. Secreted extracellular cystatin C (CysC) can also regulate target tissue homeostasis after in vivo cellular uptake (17,18). CysC is a negative regulator of angiogenesis and endothelial cell homeostasis both in vitro and in vivo (16). Clinically, the levels of CysC in serum of patients with CMB and patients with stroke were found significantly higher than the normal level in healthy people (1922). Elevated expression of CysC is a common response of the body to injury. However, researchers have not yet reached a consensus about its implication and mechanism. It was reported that CysC played a neuroprotective role in preclinical disease models (23,24). It was also reported that CysC was negatively correlated with cognitive function (25,26), and expression of CysC in the elderly was higher than that in young people (27,28). As far as we know, there has been no report on the role of CysC in cognitive development in CMB patients.

In this study, the difference in expression of CysC in serum of patients with CMB and healthy subjects was confirmed. CMB model mice were treated with CysC drug, followed by investigation of the effect of CysC on the cognitive function of CMB mice and the molecular mechanism.

Materials and methods

Materials
Subjects

Serum samples of 60 patients with cerebral microbleeds, including 32 males and 28 females, were collected. Furthermore, serum samples of 60 healthy subjects of similar age were collected, including 29 males and 31 females. Fasting blood samples were drawn in the morning from all subjects after an 8-h overnight fast. All subjects signed informed consent. Patients who met the following criteria (29) were eligible for the study: i) patients whose age was ≥18 years but <65 years; ii) patients who were diagnosed with brain microbleeds (CMB) by MRI in accordance with the diagnostic criteria of CMB; and iii) patients' family agreed to participate in the study and signed an informed consent form. Patients who met the following criteria were excluded from this study: i) patients who did not take an MRI exam; ii) patients who had intracerebral hemorrhage due to abnormal structures in the brain; iii) patients who were experiencing parenchymal hemorrhage due to intracranial aneurysm rupture; iv) patients who had cerebral bleeding due to traumatic brain injury; v) patients who had circulatory system diseases; vi) patients who had moyamoya disease; vii) patients who were pre-treated with anticoagulant therapy; and viii) patients who had severe respiratory diseases, advanced cancers, severe liver and kidney dysfunction, severe heart dysfunction, hyperthyroidism or severe endocrine system diseases. The study was approved by the Ethics Committee of The Third Affiliated Hospital of Qiqihar Medical University (Qiqihar, China).

Animals

Spontaneously hypertensive rats (SHR) were purchased from Shanghai Experimental Animal Center affiliated with Chinese Academy of Sciences. The animals were given a batch number of SYXK Black 2008004 and were raised and reproduced in our institution's laboratory animal center.

Reagents

Materials and reagents used in this study were purchased from commercial sources: Human cystatin C kit (item no. ab179883) from Abcam; CysC drug from Enzo Life Sciences; RIPA lysis buffer from Beyotime Biotechnology Co., Ltd.; sucrose, glucose, KCl, NaHCO3, NaH2PO4, CaCl2 and MgCl2 from Sigma-Aldrich; Merck KGaA; rat brain stereotaxic device from Anhui Zhenghua Biological Instrument Co., Ltd.; primary antibodies to cystatin C (item no. ab109508), p-extracellular signal-regulated kinase 1/2 (ERK1/2) (item no. ab223500), synapsin I (item no. ab8), and p-synapsinI-S549 (item no. ab119370) from Abcam; and β-actin primary antibody (item no. 66009-1-Ig) from Proteintech.

Methods
Blood sample collection and processing

Venous blood was drawn from subjects into a 15 ml tube after more than 8 h fast. Immediately after collection, the blood was centrifuged at 1,006.2 × g at room temperature (25–28°C) for 20 min. The supernatant was carefully collected, aliquoted into 200 µl, and stored at −80°C. Multiple freeze-thaw cycles were avoided. In accordance with the CysC kit manual, absorbance of the sample was measured at 450 nm using a microplate reader, and the expression level of CysC in the serum was calculated.

Animal housing and handling

Following conditions were provided in SHR housing. All the SHRs were housed in a specific pathogen-free room maintained at 18–26°C with a daily temperature difference of ≤3°C. The housing was kept at a relative humidity of 40–70%, a noise level of ≤60 dB, and a 12 h light (150–300 Lux)/12 h dark cycle. The animals were maintained with free access to food and water. All experimental procedures were consistent with experimental animal welfare and ethical principles.

The animals were randomly divided into 4 groups: sham surgery control group (sham), model group (CMB), model + empty vector control group (CMB + vehicle), and model + cystatin C overexpression group (CMB + CysC). Of each group, 12 animals were used for behavioral experiment, 6 for electrophysiological experiment, and the remaining 6 for molecular biology experiments.

Following protocol was for establishing the animal model. Forty-eight specific pathogen-free SHRs at 10 weeks of age were selected, of which 36 were randomly chosen for the rat model with brain microbleeds. A published protocol was followed (30). Rats were first restricted from food and water for 8 h prior to the experiment. The rats were anesthetized with 1% sodium pentobarbital via intraperitoneal injection at 0.6 ml/10 g. After induction of deep anesthesia, the rats were fixed on a stereotactic instrument. The skin area in the head was prepared and disinfected for surgery. Two holes were drilled 5 mm behind the anterior fontanelle, one at 2 mm left and the other at 2 mm right of the midline. To elicit microbleed formation, the rat brain was pierced perpendicularly to a depth of 4 mm from the dura mater using stainless steel needles of 474 and 159 µm in diameter, respectively, at both sides of the midline. Bone wax was used to patch the holes, and the skin over it was closed with sutures. The other 12 rats were used for sham surgery which was the same as the procedure described above except that there was no piercing of stainless-steel needles into the brain.

To elicit cystatin C overexpression, following steps were performed. The drug CysC was dissolved in physiological saline to a final concentration of 50 µg/ml (31). The lateral ventricle was injected with 10 µl of the drug solution. The same amount of saline was injected into the brain of rats in the negative control group. The lateral ventricle injection was at 0.8 mm behind the anterior fontanelle, 1.5 mm distal to the midline, and 4.5 mm deep.

Y-maze test

Behavioral experiment was conducted 5 days after rat model creation. Y-maze test was performed using a Y-maze apparatus. The Y-maze as a labyrinth was composed of three arms of equal length, i.e. arm I, arm II and arm III, as well as their junction area. The bottom of the box was covered with an electric grid (0.2 cm in wire diameter, 14 cm in length and 1 cm in grid spacing). There were multiple buttons on the Y-maze control panel such as I, II, III and 0, as well as a voltage adjusting knob and a time control knob. When button I, II or III was pressed, a signal light corresponding to that arm became on, indicating the arm was not energized and therefore was a safe zone, while the other two arms and the junction area were dark and energized and therefore were non-safe zones. When the button 0 was pressed, the junction area was energized while the three arms were not. Five seconds after the signal light was on, electricity (60 v) was on in the grid. Each rat was tested 20 times a day for 2 days. Day 1 test was for study, and day 2 test was for memory retention. Safe zones were changed in a random and alternating way. In the beginning of the test, two arms including the one where the rat hid were energized. The animal would escape to the light area after receiving an electric shock. A test was completed when the rat stayed in the safe zone for 30 sec. Then next test started from where the rat was. It would be a correct reaction if the rat fled directly to the safe zone after an electric shock or ran to the safe zone within 10 sec after the shock. It would be an incorrect reaction if the rat escaped to any other arm without lights.

Testing indicators are listed below: i) Error number (EN) was defined as the number of icorrect reactions in all reactions. ii) Total reaction time (TRT) was defined as sum of the time required to complete correct reactions and incorrect reactions. Reaction time referred to the time taken from when the signal light was on to when the rat escaped to the safe zone. iii) Active avoidance rate (AAR) was defined as the percentage of times a rat completed its escape reaction within 5 sec after the light was on but the arm was not energized.

Electrophysiology
Preparation of acute hippocampal slices

Artificial cerebrospinal fluid (ACSF) for anatomical use was made containing 210 mM sucrose, 12 mM glucose, 2 mM KCl, 24 mM NaHCO3, 1 mM NaH2PO4, 0.5 mM CaCl2 and 7 mM MgCl2. A mixed gas of 5% CO2/95% O2 was bubbled through the ACSF for at least half an hour at an osmotic pressure of 310–320 Osm in order to saturate it with oxygen. The pH was adjusted to 7.4. A rat was rapidly decapitated, and the head was placed in ice-cold artificial cerebrospinal fluid where the brain tissue was dissected out. The well-extracted brain tissue was transferred to fresh ice-cold and oxygen-saturated artificial cerebrospinal fluid. The brain tissue was trimmed with a scalpel while it was completely submerged in ACSF. The trimmed brain tissue was fixed on a sample tray, and a 2% agar block was used to hold the ventral side of the brain to prevent the slice from tilting. Ice-cold artificial cerebrospinal fluid for anatomical use was poured into the sample tray to completely submerge the brain tissue. The sample tray together with the brain tissue was mounted on a microtome. A mixed gas of 5% CO2/95% O2 was bubbled through the ACSF. After a blade was installed and parameters such as slicing speed and oscillation frequency were set, the brain tissue was cut into coronal slices of 400 µm thickness.

MED64 planar microelectrode array recording system was purchased from Alpha Med Science, Japan and used for electrophysiological recordings (32). ACSF for recording use was prepared containing 120 mM NaCl, 27 mM NaHCO3, 20 mM glucose, 1 mM NaH2PO4, 3 mM KCl, 2.6 mM CaCl2 and 1 mM MgCl2. A mixed gas of 5% CO2/95% O2 was bubbled through the ACSF for half an hour at an osmotic pressure of 310–320 Osm, followed by pH adjustment to 7.4. The well-cut brain slices were incubated in the ACSF for recording use at room temperature for at least one hour to restore brain slice activity. A slice was selected, and after it was placed on the MED64 probe, the slice was infused continuously. At this stage it was ready for recording. A stimulation site in the hippocampal CA3 region was selected. All the sites in the CA1 region were for recording. An input-output (I-O) curve was constructed. The current corresponding to 30–50% of the maximum excitatory post-synaptic potential amplitude was chosen as the subsequent stimulation intensity. High-frequency stimulation was delivered to the brain slice after baseline recording. The change in amplitude was recorded during the 60 min period after stimulation.

Western blot analysis

Cells were lysed with ultrasound in RIPA lysis buffer. Protein concentration was measured using the BCA kit. Samples were subjected to electrophoresis, membrane transfer, blocking, incubation with antibodies and image development. Incubation with primary antibodies, i.e. anti-cystatin C (dilution factor, 1:20,000), p-ERK1/2 (dilution factor, 1:400), synapsin I (dilution factor, 1:1,000), p-synapsinI-S549 (dilution factor, 1:1,000), and β-actin (dilution factor, 1:10,000), was maintained at 4°C overnight. After washing, the membrane was incubated with corresponding secondary antibody (dilution factor, 1:4,000), followed by exposure and image development. Quantity One software was used to analyze the image in Grayscale format.

Statistical analysis

Experimental data were expressed as mean ± standard deviation (mean ± SEM). Statistical analysis was performed using SPSS 16.6 statistical software. The results were analyzed using one-way ANOVA. A Bonferroni test was used for comparison between groups. A difference was statistically significant at P<0.05, P<0.01 and P<0.001. GraphPad Prism 5 software was used for graph drawing.

Results

Measurement of CysC level in serum of patients with cerebral microbleeds (CMB)

CysC expression levels in serum of clinically healthy subjects and patients with cerebral microbleeds were measured. The results in Fig. 1 show that compared with the control group, expression level of CysC in serum of patients with cerebral microbleeds increased significantly. The difference was statistically significant.

CysC expression in rat hippocampus

Expression levels of CysC in hippocampus of rats in each group were measured. As shown in Fig. 2, expression level of CysC in the CMB model group increased slightly after model creation. When rats in the CMB model group were administered with CysC, the expression level of CysC in hippocampus increased significantly.

Cognitive function evaluation of rats in each group

Cognitive functions, including learning and memory, were evaluated in each group using the Y-maze test. Results showed that compared with the sham group, the number of incorrect reactions (Fig. 3A and B) and the total reaction time (Fig. 3C and D) increased, while the active avoidance rate (Fig. 3E and F) decreased in learning and memory test in the CMB model group, indicating impairment of rat learning and memory function. After administration of drug CysC, the number of incorrect reactions and the total reaction time (Fig. 3A3D) further increased, while the active avoidance rate (Fig. 3E and F) further decreased, indicating that CysC aggravated impairment of learning and memory function in the model rats.

Recording rat hippocampal LTP

LTP is widely considered one of the major cellular mechanisms that underlies learning and memory. In this study, changes in LTP were recorded in each group using slice electrophysiological techniques. As shown in Fig. 4, compared with the sham group, LTP of the CMB model group was significantly reduced. Compared with the CMB model group, LTP of the cystatin C overexpression group showed further reduction as well.

ERK/synapsinIa/b pathway expression

Western blot analysis was performed to observe changes in expression of p-ERK and p-synapsinIa/b (Ser549) which are associated with cognitive functions. As shown in Fig. 5, compared with the sham group, expression levels of p-ERK and p-synapsinIa/b (Ser549) were significantly reduced in the CMB model group. Compared with the CMB model group, CysC overexpression led to a further reduction in expression levels of p-ERK and p-synapsinIa/b (Ser549).

Discussion

In clinic, expression level of cystatin C (CysC) was found significantly higher in serum of patients with cerebral microbleeds (CMB) than in healthy subjects (Fig. 1). In this study, a rat CMB model was created, and was injected with CysC in the hippocampus aimed at studying the impact of CysC on rat cognitive functions (Fig. 2). In behavioral study, cognitive dysfunction was observed in the CMB model rats. After administration of CysC, the learning and memory functions were further deteriorated in the rats (Fig. 3). In electrophysiological study, a decrease in hippocampal LTP was found in the CMB model rats. The hippocampal LTP was further decreased after administration of CysC in the model rats (Fig. 4). Among biomarkers associated with learning and memory functions, it was found that the expression levels of phosphorylated ERK1/2 and phosphorylated synapsin Ia/b (Ser549) were reduced in hippocampus of the CMB model rats. After administration of CysC, these levels were further reduced (Fig. 5). These findings suggested that overexpression of CysC can promote cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsin Ia/b pathway.

CMB is the main cause of mild vascular cognitive impairment and vascular dementia (33). The underlying mechanism may be that CMB affects specific cognitive domain functions such as executive functions and attention, and CMB may also damage the cerebral white matter association fiber bundles, cholinergic fiber bundles, and frontal subcortical circuitry (34). In addition, CMB is often associated with neurodegenerative diseases such as Alzheimer's disease, aggravating cortical atrophy and increasing the risk of cognitive impairment (35). The present study demonstrated learning and memory dysfunction in CMB model rats through Y-maze learning and memory tests, indicating association of CMB with rat cognitive functions. Our findings were consistent with related literature reports. For example, CMB was reported to cause reduced cognitive ability and increased risk of dementia (36,37). CMB may play a role in development of cerebrovascular diseases and neurodegenerative diseases. Its detection rate in Alzheimer's disease is twice that in the same age control group (38). In electrophysiology, LTP is a synaptic plasticity mechanism underlying learning and memory (3940). In this study, LTP in the CA1 region of rat hippocampus was recorded using slice electrophysiological techniques. It was found that CMB caused a decrease in LTP, which was consistent with the behavioral test results.

CysC is an important endogenous inhibitor of cysteine protease activity (41). Its function is still unclear in the brain, but it is associated with neuronal degeneration and nervous system repair. CysC is highly expressed in patients with epilepsy and neurodegenerative diseases. According to literature, high expression was also found in facial nerve transection, perforation pathway transection, pituitary resection, and animal models of transient cerebral ischemia and epilepsy (42). There is an opinion that high CysC expression in injury or disease may imply an intrinsic neuroprotection that counteracts disease progression. It was also reported that CysC exerts a protective effect when neurons are under attack by inducing autophagy (31,43). Several studies have demonstrated that upregulated expression of CysC is positively correlated with cerebrovascular diseases (44,45). In this study, overexpression of CysC in rat serum and hippocampus was achieved by injection of external CysC aimed at studying its impact on cognitive functions in CMB model rats. It was found that overexpression of CysC aggravated learning and memory dysfunction and LTP reduction in CMB model rats. Effects of CysC on neurons have been reported inconsistently in different studies. The inconsistency may be due to several reasons. The first may be related to disease progression. CysC may play different roles in different disease states. The second may be related to the way an animal model was created, and different CysC concentrations were used. The third is that more likely CysC has a different effect on neurons in different diseases.

Extracellular signal-regulated kinase 1/2 (ERK1/2) is a signal transduction protein belonging to the mitogen-activated protein kinase (MAPK) family. It is ubiquitous in various tissues and participates in cell proliferation and differentiation. Activation of ERK can mediate physiological functions of nutrient-related factor receptors and multiple growth factor receptors. There are two ERK isoforms, i.e. ERK1 and ERK2 (46,47). MAPKs are serine/threonine protein kinases in cells, which also include two signal transduction pathways, i.e. the c-Jun N-terminal kinase (JNK) and p38 MAPK, in addition to ERK. The MAPK/ERK pathway in the brain can communicate a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell, thereby participating in the regulation of neuronal apoptosis and proliferation. This pathway is closely associated with learning and memory functions (48). The ERK signaling pathway is activated by synaptic activity in learning and induction of LTP, but its activity is reduced in defected LTP and learning (4951). Delayed audiogenic seizure development in a genetic rat model is associated with overactivation of ERK1/2 and disturbances in glutamatergic signaling (52). In the present study, it was found that the level of p-ERK1/2 was decreased after CMB model creation, and overexpression of CysC further decreased the level of p-ERK1/2, indicating that ERK signaling was involved in the promoting process of cognitive dysfunction in CMB rats due to CysC overexpression.

Synapsin I is a key regulator of synaptic vesicle dynamics in the presynaptic terminals, regulating synaptic transmission by modulating the storage and mobilization of synaptic vesicles (53). It can selectively control synaptic maturation of long-range projections in the lateral amygdala (54). Synapsin I binds to a variety of upstream molecules, through which it participates in LTP and learning and memory functions. Among these upstream molecules, leucine-rich repeat kinase 2 (LRRK2) is a large multi-domain scaffold protein. LRRK2 exhibits GTPase and kinase activity, involving in synaptic kinetics. LRRK2 can modulate glutamate release from presynaptic sites by interacting with synapsin I via its WD40 domain (55). It was reported in literature that the modulatory function of neurofibrin on learning is via its regulation of the ERK-synapsin I pathway and GABA release (56). ERK phosphorylates mammalian synapsin I at positions 4 and 5 of domain B and at position 6 of domain D (57). In addition, PKA and CaMKII can also act by phosphorylating synapsin I (58). In this study, a decrease was observed in the level of p-synapsin Ia/b (Ser549) after CMB model creation. According to literature, the Ser549 site is targeted by ERK and Cdk1 (59). Overexpression of CysC further decreased the level of p-synapsin Ia/b (Ser549), indicating that synapsin I was involved in the promoting process of cognitive dysfunction in CMB rats due to CysC overexpression.

Although this work demonstrated the promoting effect of CysC on cognitive impairment and the involvement of the ERK-synapsin I signal pathway, it does not rule out the possibility of CysC having other mechanisms of promoting cognitive dysfunction in the CMB model rats. Thus, further work is still necessary for clarification.

In conclusion, this study preliminarily demonstrated that CysC overexpression can promote cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsinIa/b pathway. Based on these findings, it is postulated that reducing the expression level of CysC in serum of CMB patients may help alleviate the symptoms and slow down disease progression of cognitive impairment.

Acknowledgements

Not applicable.

Funding

This study was supported by Qiqihar Science and Technology Project (SFGG-201952).

Availability of data and materials

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

Authors' contributions

GY wrote the manuscript. XS and LL conceived and designed the study. LH and HL were responsible for the collection and analysis of the experimental data. SW interpreted the data and drafted the manuscript. ZR and YZ revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of The Third Affiliated Hospital of Qiqihar Medical University (Qiqihar, China). All subjects signed informed consent.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Moulin S and Cordonnier C: Role of cerebral microbleeds for intracerebral haemorrhage and dementia. Curr Neurol Neurosci Rep. 19:512019. View Article : Google Scholar : PubMed/NCBI

2 

Cordonnier C, Al-Shahi Salman R and Wardlaw J: Spontaneous brain microbleeds: Systematic review, subgroup analyses and standards for study design and reporting. Brain. 130:1988–2003. 2007. View Article : Google Scholar : PubMed/NCBI

3 

Daugherty AM and Raz N: Incident risk and progression of cerebral microbleeds in healthy adults: A multi-occasion longitudinal study. Neurobiol Aging. 59:22–29. 2017. View Article : Google Scholar : PubMed/NCBI

4 

Vernooij MW, van der Lugt A, Ikram MA, Wielopolski PA, Niessen WJ, Hofman A, Krestin GP and Breteler MM: Prevalence and risk factors of cerebral microbleeds: The Rotterdam Scan Study. Neurology. 70:1208–1214. 2008. View Article : Google Scholar : PubMed/NCBI

5 

Charidimou A, Imaizumi T, Moulin S, Biffi A, Samarasekera N, Yakushiji Y, Peeters A, Vandermeeren Y, Laloux P, Baron JC, et al: Brain hemorrhage recurrence, small vessel disease type, and cerebral microbleeds: A meta-analysis. Neurology. 89:820–829. 2017. View Article : Google Scholar : PubMed/NCBI

6 

Charidimou A, Kakar P, Fox Z and Werring DJ: Cerebral microbleeds and recurrent stroke risk: Systematic review and meta-analysis of prospective ischemic stroke and transient ischemic attack cohorts. Stroke. 44:995–1001. 2013. View Article : Google Scholar : PubMed/NCBI

7 

Cordonnier C and van der Flier WM: Brain microbleeds and Alzheimer's disease: Innocent observation or key player? Brain. 134:335–344. 2011. View Article : Google Scholar : PubMed/NCBI

8 

Staekenborg SS, Koedam EL, Henneman WJ, Stokman P, Barkhof F, Scheltens P and van der Flier WM: Progression of mild cognitive impairment to dementia: Contribution of cerebrovascular disease compared with medial temporal lobe atrophy. Stroke. 40:1269–1274. 2009. View Article : Google Scholar : PubMed/NCBI

9 

Dierksen GA, Skehan ME, Khan MA, Jeng J, Nandigam RN, Becker JA, Kumar A, Neal KL, Betensky RA, Frosch MP, et al: Spatial relation between microbleeds and amyloid deposits in amyloid angiopathy. Ann Neurol. 68:545–548. 2010. View Article : Google Scholar : PubMed/NCBI

10 

Gurol ME, Dierksen G, Betensky R, Gidicsin C, Halpin A, Becker A, Carmasin J, Ayres A, Schwab K, Viswanathan A, et al: Predicting sites of new hemorrhage with amyloid imaging in cerebral amyloid angiopathy. Neurology. 79:320–326. 2012. View Article : Google Scholar : PubMed/NCBI

11 

Greenberg SM, Vernooij MW, Cordonnier C, Viswanathan A, Al-Shahi Salman R, Warach S, Launer LJ, Van Buchem MA and Breteler MM; Microbleed Study Group, : Cerebral microbleeds: A guide to detection and interpretation. Lancet Neurol. 8:165–174. 2009. View Article : Google Scholar : PubMed/NCBI

12 

Lawrence AJ, Patel B, Morris RG, MacKinnon AD, Rich PM, Barrick TR and Markus HS: Mechanisms of cognitive impairment in cerebral small vessel disease: Multimodal MRI results from the St. George's cognition and neuroimaging in stroke (SCANS) study. PLoS One. 8:e610142013. View Article : Google Scholar : PubMed/NCBI

13 

Lawrence AJ, Chung AW, Morris RG, Markus HS and Barrick TR: Structural network efficiency is associated with cognitive impairment in small-vessel disease. Neurology. 83:304–311. 2014. View Article : Google Scholar : PubMed/NCBI

14 

Poels MM, Ikram MA, van der Lugt A, Hofman A, Niessen WJ, Krestin GP, Breteler MM and Vernooij MW: Cerebral microbleeds are associated with worse cognitive function: The Rotterdam Scan Study. Neurology. 78:326–333. 2012. View Article : Google Scholar : PubMed/NCBI

15 

Leem AY, Park MS, Park BH, Jung WJ, Chung KS, Kim SY, Kim EY, Jung JY, Kang YA, Kim YS, et al: Value of serum Cystatin C measurement in the diagnosis of sepsis-induced kidney injury and prediction of renal function recovery. Yonsei Med J. 58:604–612. 2017. View Article : Google Scholar : PubMed/NCBI

16 

Benndorf RA: Renal Biomarker and Angiostatic Mediator? Cystatin C as a negative regulator of vascular endothelial cell homeostasis and angiogenesis. J Am Heart Assoc. 7:e0109972018. View Article : Google Scholar : PubMed/NCBI

17 

Ekström U, Wallin H, Lorenzo J, Holmqvist B, Abrahamson M and Avilés FX: Internalization of cystatin C in human cell lines. FEBS J. 275:4571–4582. 2008. View Article : Google Scholar : PubMed/NCBI

18 

Mathews PM and Levy E: Cystatin C in aging and in Alzheimer's disease. Ageing Res Rev. 32:38–50. 2016. View Article : Google Scholar : PubMed/NCBI

19 

Oh MY, Lee H, Kim JS, Ryu WS, Lee SH, Ko SB, Kim C, Kim CH and Yoon BW: Cystatin C, a novel indicator of renal function, reflects severity of cerebral microbleeds. BMC Neurol. 14:1272014. View Article : Google Scholar : PubMed/NCBI

20 

Yang S, Cai J, Lu R, Wu J, Zhang M and Zhou X: Association between serum cystatin C level and total magnetic resonance imaging burden of cerebral small vessel disease in patients with acute lacunar stroke. J Stroke Cerebrovasc Dis. 26:186–191. 2017. View Article : Google Scholar : PubMed/NCBI

21 

Zhang JB, Jü XH, Wang J, Sun HR and Li F: Serum cystatin C and cerebral microbleeds in patients with acute cerebral stroke. J Clin Neurosci. 21:268–273. 2014. View Article : Google Scholar : PubMed/NCBI

22 

Zhang JB, Liu LF, Li ZG, Sun HR and Jü XH: Associations between biomarkers of renal function with cerebral microbleeds in hypertensive patients. Am J Hypertens. 28:739–745. 2015. View Article : Google Scholar : PubMed/NCBI

23 

Fang Z, Deng J, Wu Z, Dong B, Wang S, Chen X, Nie H, Dong H, Xiong L and Cystatin C: Cystatin C is a crucial endogenous protective determinant against stroke. Stroke. 48:436–444. 2017. View Article : Google Scholar : PubMed/NCBI

24 

Zou J, Chen Z, Wei X, Chen Z, Fu Y, Yang X, Chen D, Wang R, Jenner P, Lu JH, et al: Cystatin C as a potential therapeutic mediator against Parkinson's disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units. Cell Death Dis. 8:e28542017. View Article : Google Scholar : PubMed/NCBI

25 

Kono S, Adachi H, Enomoto M, Fukami A, Kumagai E, Nakamura S, Nohara Y, Morikawa N, Nakao E, Sakaue A, et al: Impact of cystatin C and microalbuminuria on cognitive impairment in the population of community-dwelling Japanese. Atherosclerosis. 265:71–77. 2017. View Article : Google Scholar : PubMed/NCBI

26 

Yin Z, Yan Z, Liang Y, Jiang H, Cai C, Song A, Feng L and Qiu C: Interactive effects of diabetes and impaired kidney function on cognitive performance in old age: A population-based study. BMC Geriatr. 16:72016. View Article : Google Scholar : PubMed/NCBI

27 

Stephan Y, Sutin AR and Terracciano A: Subjective age and cystatin C among older adults. J Gerontol B Psychol Sci Soc Sci. 74:382–388. 2019. View Article : Google Scholar : PubMed/NCBI

28 

Wei Y, Wei YK and Zhu J: Early markers of kidney dysfunction and cognitive impairment among older adults. J Neurol Sci. 375:209–214. 2017. View Article : Google Scholar : PubMed/NCBI

29 

Park YS, Chung MS and Choi BS: MRI assessment of cerebral small vessel disease in patients with spontaneous intracerebral hemorrhage. Yonsei Med J. 60:774–781. 2019. View Article : Google Scholar : PubMed/NCBI

30 

Tóth A, Berente Z, Bogner P, Környei B, Balogh B, Czeiter E, Amrein K, Dóczi T, Büki A and Schwarcz A: Cerebral microbleeds temporarily become less visible or invisible in acute susceptibility weighted magnetic resonance imaging: A Rat Study. J Neurotrauma. 36:1670–1677. 2019. View Article : Google Scholar : PubMed/NCBI

31 

Liu Y, Li J, Wang Z, Yu Z and Chen G: Attenuation of early brain injury and learning deficits following experimental subarachnoid hemorrhage secondary to cystatin C: Possible involvement of the autophagy pathway. Mol Neurobiol. 49:1043–1054. 2014. View Article : Google Scholar : PubMed/NCBI

32 

Li XH, Matsuura T, Liu RH, Xue M and Zhuo M: Calcitonin gene-related peptide potentiated the excitatory transmission and network propagation in the anterior cingulate cortex of adult mice. Mol Pain. Feb 28–2019.(Epub ahead of print). doi: 10.1177/1744806919832718, 2019. View Article : Google Scholar

33 

Pantoni L: Cerebral small vessel disease: From pathogenesis and clinical characteristics to therapeutic challenges. Lancet Neurol. 9:689–701. 2010. View Article : Google Scholar : PubMed/NCBI

34 

Dey AK, Stamenova V, Turner G, Black SE and Levine B: Pathoconnectomics of cognitive impairment in small vessel disease: A systematic review. Alzheimers Dement. 12:831–845. 2016. View Article : Google Scholar : PubMed/NCBI

35 

Roseborough A, Ramirez J, Black SE and Edwards JD: Associations between amyloid β and white matter hyperintensities: A systematic review. Alzheimers Dement. 13:1154–1167. 2017. View Article : Google Scholar : PubMed/NCBI

36 

Akoudad S, Wolters FJ, Viswanathan A, de Bruijn RF, van der Lugt A, Hofman A, Koudstaal PJ, Ikram MA and Vernooij MW: Association of cerebral microbleeds with cognitive decline and dementia. JAMA Neurol. 73:934–943. 2016. View Article : Google Scholar : PubMed/NCBI

37 

Cai Z, Wang C, He W, Tu H, Tang Z, Xiao M and Yan LJ: Cerebral small vessel disease and Alzheimer's disease. Clin Interv Aging. 10:1695–1704. 2015. View Article : Google Scholar : PubMed/NCBI

38 

Love S and Miners JS: Small vessel disease, neurovascular regulation and cognitive impairment: Post-mortem studies reveal a complex relationship, still poorly understood. Clin Sci (Lond). 131:1579–1589. 2017. View Article : Google Scholar : PubMed/NCBI

39 

Cobar LF, Yuan L and Tashiro A: Place cells and long-term potentiation in the hippocampus. Neurobiol Learn Mem. 138:206–214. 2017. View Article : Google Scholar : PubMed/NCBI

40 

Lømo T: Discovering long-term potentiation (LTP) - recollections and reflections on what came after. Acta Physiol (Oxf). 222:2222018. View Article : Google Scholar

41 

Abrahamson M, Alvarez-Fernandez M and Nathanson CM: Cystatins. Biochem Soc Symp. 70:179–199. 2003. View Article : Google Scholar

42 

Levy E, Jaskolski M and Grubb A: The role of cystatin C in cerebral amyloid angiopathy and stroke: Cell biology and animal models. Brain Pathol. 16:60–70. 2006. View Article : Google Scholar : PubMed/NCBI

43 

Tizon B, Sahoo S, Yu H, Gauthier S, Kumar AR, Mohan P, Figliola M, Pawlik M, Grubb A, Uchiyama Y, et al: Induction of autophagy by cystatin C: A mechanism that protects murine primary cortical neurons and neuronal cell lines. PLoS One. 5:e98192010. View Article : Google Scholar : PubMed/NCBI

44 

Zhang Y and Sun L: Cystatin C in cerebrovascular disorders. Curr Neurovasc Res. 14:406–414. 2017. View Article : Google Scholar : PubMed/NCBI

45 

Osk Snorradottir A, Isaksson HJ, Kaeser SA, Skodras AA, Olafsson E, Palsdottir A and Thor Bragason B: Parenchymal cystatin C focal deposits and glial scar formation around brain arteries in hereditary cystatin C amyloid angiopathy. Brain Res. 1622:149–162. 2015. View Article : Google Scholar : PubMed/NCBI

46 

Li J, Zhang M and Ma J: Myricitrin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation and migration through suppressing PDGFRβ/Akt/Erk signaling. Int J Clin Exp Med. 8:21715–21723. 2015.PubMed/NCBI

47 

Frost EE, Zhou Z, Krasnesky K and Armstrong RC: Initiation of oligodendrocyte progenitor cell migration by a PDGF-A activated extracellular regulated kinase (ERK) signaling pathway. Neurochem Res. 34:169–181. 2009. View Article : Google Scholar : PubMed/NCBI

48 

Horikawa J and Ojima H: Cortical activation patterns evoked by temporally asymmetric sounds and their modulation by learning. eNeuro. 4:42017. View Article : Google Scholar

49 

Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM and Sweatt JD: The MAPK cascade is required for mammalian associative learning. Nat Neurosci. 1:602–609. 1998. View Article : Google Scholar : PubMed/NCBI

50 

English JD and Sweatt JD: A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem. 272:19103–19106. 1997. View Article : Google Scholar : PubMed/NCBI

51 

Selcher JC, Atkins CM, Trzaskos JM, Paylor R and Sweatt JD: A necessity for MAP kinase activation in mammalian spatial learning. Learn Mem. 6:478–490. 1999. View Article : Google Scholar : PubMed/NCBI

52 

Chernigovskaya EV, Korotkov AA, Dorofeeva NA, Gorbacheva EL, Kulikov AA and Glazova MV: Delayed audiogenic seizure development in a genetic rat model is associated with overactivation of ERK1/2 and disturbances in glutamatergic signaling. Epilepsy Behav. 99:1064942019. View Article : Google Scholar : PubMed/NCBI

53 

Song SH and Augustine GJ: Synapsin isoforms and synaptic vesicle trafficking. Mol Cells. 38:936–940. 2015. View Article : Google Scholar : PubMed/NCBI

54 

Lugarà E, De Fusco A, Lignani G, Benfenati F, Humeau Y and Synapsin I: Synapsin I controls synaptic maturation of long-range projections in the lateral amygdala in a targeted selective fashion. Front Cell Neurosci. 13:2202019. View Article : Google Scholar : PubMed/NCBI

55 

Marte A, Russo I, Rebosio C, Valente P, Belluzzi E, Pischedda F, Montani C, Lavarello C, Petretto A, Fedele E, et al: Leucine-rich repeat kinase 2 phosphorylation on synapsin I regulates glutamate release at pre-synaptic sites. J Neurochem. 150:264–281. 2019. View Article : Google Scholar : PubMed/NCBI

56 

Cui Y, Costa RM, Murphy GG, Elgersma Y, Zhu Y, Gutmann DH, Parada LF, Mody I and Silva AJ: Neurofibromin regulation of ERK signaling modulates GABA release and learning. Cell. 135:549–560. 2008. View Article : Google Scholar : PubMed/NCBI

57 

Giachello CN, Fiumara F, Giacomini C, Corradi A, Milanese C, Ghirardi M, Benfenati F and Montarolo PG: MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity. J Cell Sci. 123:881–893. 2010. View Article : Google Scholar : PubMed/NCBI

58 

Chen X, Wang X, Yang Y, Li Z, Zhang Y, Gao W, Xiao J and Li B: Schwann cells protect against CaMKII- and PKA-dependent acrylamide-induced synapsin I phosphorylation. Brain Res. 1701:18–27. 2018. View Article : Google Scholar : PubMed/NCBI

59 

Verstegen AM, Tagliatti E, Lignani G, Marte A, Stolero T, Atias M, Corradi A, Valtorta F, Gitler D, Onofri F, et al: Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses. J Neurosci. 34:7266–7280. 2014. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

March 2020
Volume 19 Issue 3

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
APA
Yu, G., Sun, X., Li, L., Huang, L., Liu, H., Wang, S. ... Zhang, Y. (2020). Cystatin C promotes cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsin Ia/Ib pathway. Experimental and Therapeutic Medicine, 19, 2282-2290. https://doi.org/10.3892/etm.2019.8403
MLA
Yu, G., Sun, X., Li, L., Huang, L., Liu, H., Wang, S., Ren, Z., Zhang, Y."Cystatin C promotes cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsin Ia/Ib pathway". Experimental and Therapeutic Medicine 19.3 (2020): 2282-2290.
Chicago
Yu, G., Sun, X., Li, L., Huang, L., Liu, H., Wang, S., Ren, Z., Zhang, Y."Cystatin C promotes cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsin Ia/Ib pathway". Experimental and Therapeutic Medicine 19, no. 3 (2020): 2282-2290. https://doi.org/10.3892/etm.2019.8403