Activation of Neuropeptide Y2 Receptor Can Inhibit Global Cerebral Ischemia-Induced Brain Injury

Abstract

Cardiopulmonary arrest (CA) can greatly impact a patient’s life, causing long-term disability and death. Although multi-faceted treatment strategies against CA have improved survival rates, the prognosis of CA remains poor. We previously reported asphyxial cardiac arrest (ACA) can cause excessive activation of the sympathetic nervous system (SNS) in the brain, which contributes to cerebral blood flow (CBF) derangements such as hypoperfusion and, consequently, neurological deficits. Here, we report excessive activation of the SNS can cause enhanced neuropeptide Y levels. In fact, mRNA and protein levels of neuropeptide Y (NPY, a 36-amino acid neuropeptide) in the hippocampus were elevated after ACA-induced SNS activation, resulting in a reduced blood supply to the brain. Post-treatment with peptide YY_3–36 (PYY_3–36), a pre-synaptic NPY2 receptor agonist, after ACA inhibited NPY release and restored brain circulation. Moreover, PYY_3–36 decreased neuroinflammatory cytokines, alleviated mitochondrial dysfunction, and improved neuronal survival and neurological outcomes. Overall, NPY is detrimental during/after ACA, but attenuation of NPY release via PYY_3–36 affords neuroprotection. The consequences of PYY_3–36 inhibit ACA-induced 1) hypoperfusion, 2) neuroinflammation, 3) mitochondrial dysfunction, 4) neuronal cell death, and 5) neurological deficits. The present study provides novel insights to further our understanding of NPY’s role in ischemic brain injury.

Introduction

According to the World Health Organization (WHO) statistics, cardiovascular disease (CVD) is the number one killer globally with 17.9 million deaths each year. Cardiopulmonary arrest (CA) is one of the leading causes of death in the United States, affecting over 393,872 people per year (Virani et al. 2021). The economic impact of cardiovascular disease will cause a projected loss of $1.1 trillion by 2035 (Dunbar et al. 2018), demonstrating the urgent need to provide effective multi-faceted therapies. Although the aftermath of whole-body ischemia following CA has a devastating impact on most organs, the brain is the most susceptible due to its dependence on major ionic fluctuations in the form of electrical activity that utilize large amounts of energy. 60% of CA patients die from extensive brain injury and only 3–10% of survivors resume their former lifestyles (Krause et al. 1986; Nichol et al. 2008); these major disabilities reflect wide-spread neuronal cell death in the brain.

The major challenge of post-resuscitative care is the highly complicated and multi-factorial pathological processes of neuronal cell death due to CA, which can lead to decreased cognition such as learning/memory deficits. While the exact mechanisms of CA-induced brain injury remain unclear, it is believed that hypoperfusion, a decrease in cerebral blood flow (CBF) lasting hours to days after CA, plays a vital role in ischemia-mediated neuronal cell death and its consequent neurological deficits (Harukuni and Bhardwaj 2006; Manole et al. 2009; R. H. Lee et al. 2017; R. H. Lee et al. 2019; R. H. Lee et al. 2020). Thus, pharmacological interventions to alleviate hypoperfusion or enhance CBF following CA may promote neuronal survival in the brain and functional recovery (R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2014a; Lin et al. 2014b; R. H. Lee et al. 2020).

CBF control is mediated by perivascular innervations from both the sympathetic and parasympathetic nervous system (T. J. Lee et al. 1980; T. J. Lee et al. 1976; T. J.-F. Lee 1994; Faraci et al. 1987b). These nerves control vascular tone via presynaptic neurotransmitter(s) release either on the same or neighboring nerves or on postsynaptic vascular smooth muscle of cerebral arteries (R. H. Lee et al. 2011; R. H. Lee et al. 2012; C. Y. Wu et al. 2014). Changes in PCO₂, PO_2, and pH levels in the blood during/after CA has been shown to affect tonic activity of chemoreceptors, leading to enhanced sympathetic activity (Patel et al. 2020; Pelletier 1972). We previously discovered that this enhanced perivascular sympathetic nerve activity after CA is detrimental to brain circulation and functional outcomes (R. H. Lee et al. 2017). Therefore, we surgically reduced perivascular sympathetic nerve activity through bilateral decentralization of the superior cervical ganglion (the major ganglia in the sympathetic nervous system innervating cerebral arteries (T. J. Lee et al. 1976; Faraci et al. 1987a), which enhanced post-resuscitative CBF, inhibited neuronal cell death, and improved learning/memory (R. H. Lee et al. 2017).

The consequences of the enhanced activity of perivascular sympathetic nerves following CA include a rise in catecholamine levels [norepinephrine (NE)] in the synaptic cleft (Sadoshima and Heistad 1983; Kern et al. 1989). NPY can stimulate postsynaptic NPY receptors on vascular smooth muscle cells to induce long-lasting and potent vasoconstriction 100X more potent than other sympathetic neurotransmitters including NE (Hakanson et al. 1986; Tanaka et al. 1997). We sought to inhibit NPY release after asphyxia cardiac arrest (ACA), an animal model of CA, via post-treatment with PYY_3–36 [specific presynaptic NPY-2 receptor (NPY2R) agonist] to investigate NPY as a modulator mediating CA-induced hypoperfusion and subsequent brain injury. NPY2Rs are G_i/G_o protein-coupled receptors located mainly on the presynaptic nerve terminals (Parker and Balasubramaniam 2008). Activation of NPY2 receptors by PYY_3–36 or other NPY2R agonists (i.e. truncated C-terminal fragments of NPY and PYY) (Abounader et al. 1995) inhibits presynaptic release of NPY to negatively modulate NPY signaling (Brothers and Wahlestedt 2010). The consequences of PYY_3–36 inhibit ACA-induced hypoperfusion, neuroinflammation, mitochondrial dysfunction, neuronal cell death, and neurological deficits. Altogether, using PYY_3–36 to inhibit NPY release following CA holds therapeutic value against ischemic brain injury.

Materials and methods

Chemicals

PYY_3–36 (catalog number: 1618) was obtained from Tocris Bioscience (Bristol, UK) and dissolved in sterile water. PYY_3–36 was further diluted from a PYY_3–36 stock solution (1 μg/μl) with sterile water to the appropriate concentration before injection. Since others have reported high blood-brain-barrier permeability of PYY_3–36 (Chelikani et al. 2005; Nonaka et al. 2003), rats received an intravenous (IV) single bolus injection of PYY_3–36 at 40 μg/kg via femoral vein immediately after resuscitation to study the effects of PYY_3–36 on ACA-induced brain injury. The dosage of PYY_3–36 (40 μg/kg) used in the present study was derived from our preliminary study. We tested 3 different dosages (10, 40, and 120 μg/kg) (Henry et al. 2015; Halatchev and Cone 2005) and found that PYY_3–36 at 40 μg/kg reached a plateau to resolve learning/memory deficits after ACA.

Animal preparation

All animal experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (2011). All experimental procedures were approved by the Institutional Animal Care and Use Committee (Louisiana State University Health Sciences Center in Shreveport and West Virginia School of Osteopathic Medicine). Male Sprague-Dawley rats (270–350 g and 9–10 weeks old) were obtained from Charles River Laboratories (Wilmington, MA). Rats were housed in the animal facilities for 1 week after arrival with complete access to standard rat chow and water (Lin et al. 2010).

Asphyxial Cardiac Arrest

Rats were anesthetized with 4% isoflurane and a mixture of O₂ and N₂O (30:70), then subjected to endotracheal intubation. After intubation, isoflurane was reduced to 1.5% and the respiration rate was maintained at 60 breaths/min by mechanical ventilation (VentElite Small Animal Ventilator Harvard Apparatus, Holliston, MA). To monitor physiological parameters (i.e. mean arterial pressure, blood gas, and glucose levels) and to administer drugs/solutions, the right femoral vein and artery were cannulated using a polyethylene tubing (PE-50) catheter (Becton Dickinson, Franklin Lakes, NJ). Cisatracurium besylate (Nimbex, Abbott Laboratories, Chicago, IL) at 0.27 mg/kg was administered IV every 10 min throughout the surgical procedure to immobilize the rat. Heating pads and lamps were utilized to maintain head and body temperatures at 36.5–37.3°C. ACA was induced by disconnecting the ventilator from endotracheal intubation for 6 mins. Epinephrine at 0.005 mg/kg was administered via femoral vein 6 mins after asphyxia, followed by manual chest compressions coupled with mechanical ventilation with 100% O₂ at 80 breaths/min. Continuous chest compressions were performed until mean arterial pressure reached 60 mmHg. IV infusion of sodium bicarbonate (1 meq/kg iv) was then utilized to alleviate respiratory acidosis in the rats. Approximately 180 rats had ACA in the present study. The asphyxial cardiac arrest (ACA) model is a mild to moderate form of cardiac arrest. The survival rate after ACA is approximately 80%. In this study, we did not observe any differences in survival rate between groups.

Enzyme-linked Immunosorbent Assay (ELISA) for the measurement of NE

EDTA (1.5 mg/ml)-treated blood samples (1.5 ml) were collected from the femoral artery 10 mins before/after and 24 hrs after ACA. Blood samples were then centrifuged at 2000 g for 15 mins for the measurement of plasma NE levels. To measure NE levels, the hippocampus was homogenized in the ddH₂O containing 1 mM EDTA and 4 mM Na₂O₅S₂ and centrifuged at 19,000 g for 15 minutes. The supernatant was collected and stored at −80°C. NE levels were determined via Noradrenaline Research ELISA™ (LDN® immunoassays and services, Nordhorn, Grafschaft Bentheim, Germany) measured at 450 nm.

Reverse transcription real-time polymerase chain reaction

To measure relative NPY mRNA levels, the RNA from hippocampus was extracted at either 1, 3, or 7 days after ACA/ACA+PYY_3–36 using a RNeasy Mini Kit and a RNase-Free DNase Set (Qiagen, Valencia, California, USA). Total RNA (0.5 μg) was utilized to generate cDNA via SuperScript™ III First-Strand Synthesis System (Invitrogen, Carlsbad, California, USA). Real-time polymerase chain reaction analysis was performed using a CFX96™ Touch Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA) coupled with iQ SYBR Green Supermix (Bio-rad, Hercules, California, USA) with a total reaction volume of 20 μL. Thermal cycle conditions were as follows: initial denaturation at 95 °C for 3 mins followed by 40 cycles of 95°C for 10 s and 60 °C for 30 s. Each experiment was repeated 3 times (triplicate). Relative mRNA levels of NPY were calculated according to the 2^−ΔΔCt method (Livak and Schmittgen 2001). β-actin was elected as an endogenous control. Changes in the NPY mRNA levels after ACA in the presence/absence of PYY_3–36 were further normalized with control rats. Primer sequences of NPY and are describe as follows: NPY: forward 5’-CCTGTCCCACCCAATGCA-3’, reverse 5’-CATATATACAACGACAACAAGGGAAATG-3’ (Vermijlen et al. 2004); β-actin: forward 5’-CCCTAAGGCCAACCGTGAA-3’, reverse 5’-AGAGGCATACAGGGACAACACA-3’.

ELISA for the measurement of NPY levels

To measure NPY levels, rats were sacrificed at either 1, 3, or 7 days after ACA. The entire hippocampus was homogenized using a glass homogenizer in the ice-cold (4°C) T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific™, Waltham, MA). The homogenate was then centrifuged at 10,000 x g at 4°C for 5 min, and the supernatant was collected for the measurement of NPY concentration using Rat/Mouse Neuropeptide Y (NPY) ELISA (MilliporeSigma, Burlington, MA). The supernatant was diluted with the assay buffer to desired concentration for ELISA analysis. The absorbance was measured at 450 and 590 nm and the NPY levels were calculated based on the absorbance units.

Laser speckle contrast imaging

Intra-vital laser speckle contrast imaging was performed to measure regional blood flow in the cortex. A stereotaxic device was utilized to stabilize the rat’s head. A longitudinal midline incision was made over the head to expose the entire scalp. The brain region of interest was exposed by drilling a circular cranial window (10 mm in diameter) at the frontoparietal cortex (1 mm lateral to the bregma), continuously irrigated with sterile saline to prevent overheating. The PeriCam PSI laser imager (Perimed, Jarfalla, Sweden) was then placed 10 cm above the brain region of interest. Cortical cerebral blood flow was measured by illuminating the brain tissue with a 785 nm laser. The brain perfusion was recorded for 5 mins and expressed as averaged pixel intensity).

Measurements of neuroinflammatory cytokines

Proteins extracted from the hippocampus were utilized to measure protein levels of ionized calcium binding adaptor molecule 1 (Iba1) and 10 inflammatory cytokines [i.e. interferon-γ (IFN- γ), Interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL10, IL-13, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α (TNF-α)] via Wes system (ProteinSimple, Biotechne®, Mineapolis, Minnesota, USA) (Beekman et al. 2018; Nevidalova et al. 2020) and Quantibody® Rat Inflammation Array 1 (RayBiotech, Peachtree Corners, Georgia, USA), respectively. The primary antibodies and their dilution utilized in the capillary-based immunoassay were as follows: rabbit polyclonal anti-IbaI (1:50) (GTX100042, Gentex, Zeeland, Michigan, USA) and rabbit monoclonal anti-β-actin (1:200) (4970, Cell Signaling Technology®, Danvers, Massachusetts, USA). Iba1 protein levels were normalized with β-actin as endogenous control. The array was visualized through a laser scanner utilizing Cy3 wavelength (532 nm, green channel). The data was analyzed using log-log regression curve.

Mitochondrial oxygen consumption rate analyses

Rats were sacrificed (anesthetized with isoflurane, then decapitated) 7 days after either ACA only or ACA+PYY_3–36 (40 μg/kg) surgery for hippocampal slices. Coronal slices of the hippocampus were sectioned into 200 μm thickness in the ice-cold (4°C) artificial cerebrospinal fluid (aCSF, 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl₂, 1 mM MgCl₂ hexahydrate, 0.4 mM KH₂PO₄, 5 mM HEPES, 10 mM glucose, 1 mg/ml of bovine serum albumin, pH 7.4) using a Leica VT1000E vibratome (Leica, Wetzlar, Germany). Tissue sections (1 mm in diameter) of the hippocampus were obtained using stainless steel biopsy punch (Sklar Instruments, West Chester, Pennsylvania, USA). The tissue sections were loaded individually into each well of the XF Islet Capture Microplate (101122–100; Agilent Technologies, Santa Clara, California, USA) containing 700 μl of aCSF buffer. The Islet Capture Microplate was acclimated at room temperature for 30 mins, followed by 37°C for 1 hr. All drugs/mitochondrial complex inhibitors were prepared and diluted in the aCSF, with their final working concentrations as follows: I) 1^st injection of aCSF to serve as a vehicle control, II) 2^nd injection of oligomycin (20 μg/ml) to inhibit mitochondrial complex V, III) 3^rd injection of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, a mitochondrial uncoupler) at 10 mM plus pyruvate (1 mM) to depolarize the mitochondria, IV) 4^th injection of antimycin A (20 μM) to inhibit complex III. Assay protocols contained a combination of 3-min mix, 3-min wait, and 2-min measure sequences. The oxygen consumption rate (OCR) was measured 3 times before 1^st and 2^nd injections, 8 times after 2^nd injection, 3 times after 3^rd injection, and 10 times after 4^th injection. OCR was calculated based on the average of all hippocampal sections during each measurement. Tissue sections contained basal respiration below 50 pmol/min or above 200 pmol/min were unhealthy and excluded from the study.

Immunohistochemistry and histopathology

For detailed methods on histopathology of hippocampal slices, please see Lee et al., 2018 (R. H. Lee et al. 2019). To evaluate neuronal cell death in the CA1 region of the hippocampus, whole animal perfusion and fixation were performed 7 days after ACA due to the prevalence of delayed neuronal cell death that occurs 3–7 days after ACA (Lin et al. 2014b; R. H. Lee et al. 2017; R. H. Lee et al. 2019; Della-Morte et al. 2011; Dave et al. 2009). Rats were perfused with physiological saline (0.9 %) for 2 mins at a constant pressure of 110–120 mmHg, followed by a mixture of 36% formaldehyde, glacial acetic acid, and methanol (1:1:8) for 15 min. The head was immediately removed and immersed in a formaldehyde, glacial acetic acid, and methanol mixture and stored at 4 °C for at least 24 hours. The brain was then dissected from the skull, processed in a tissue processor (Leica ASP300S, Wetzlar, Germany) and embedded in paraffin (Leica EG1150, Wetzlar, Germany). Coronal sections (6 μm thickness) were cut by a microtome blade (VWR, Radnor, PA) on a rotary microtome Leica RM2245 (Leica Biosystems, Wetzlar, Germany). Brain slices were collected at intervals of 100 μm and attached on a glass slide M1000W (StatLab, McKinney, TX). To remove excess paraffin, the slides were incubated overnight in an oven at 60 °C. The slides were stained with hematoxylin and eosin (H&E) (ThermoFisher, Waltham, MA) (Raval et al. 2005) or 0.0001% Fluoro-Jade C (FJC) (AG325, Millipore, Burlington, MA) plus DAPI (TR-100-FJT, Biosensis, Thebarton, Australia) (R. H. Lee et al. 2019). For H&E staining, the slides were photographed under 40X magnification via microscope (Leica aperio versa, Wetzlar, Germany). Our assessment of dead (via H&E) and degenerating (via FJC) neurons in the CA1 region of the hippocampus was determined by visual inspection of this particular region.

Oxygen Glucose Deprivation (OGD)

For detailed methods on organotypic hippocampal slices culture, please see Lee et al., 2018 (R. H. Lee et al. 2019). Briefly, organotypic hippocampal slices (postnatal day 9–12) were maintained for 12–14 days in vitro before the experiments described in previous sections (Neumann et al. 2015). Transverse slices (400 μm) of the hippocampus were dissected from the hippocampi and placed in the ice-cold (4°C) Grey’s balanced salt solution with 6.5 mg/ml glucose. Tissue slices from two hippocampi were mounted onto one membrane insert (30 mm in diameter) (Millicell-CM, Millipore) and then each insert was placed into a six-well culture dish containing 1 ml/well of culture medium (50% MEM, 25% HBSS, 25% heat-inactivated horse serum with 6.5 mg/mL glucose, and 1 mM glutamine). The tissue cultures were maintained at 37ºC with 5% CO₂ in the humidified culture chamber. To induce ischemia, hippocampal slices were exposed to 45 mins of oxygen and glucose deprivation (OGD) as previously described (Neumann et al. 2015). Hippocampal slices were washed three times with aglycemic Hank’s Balanced Salt Solution (in mM: 1.26 CaCl₂•2H₂O; 5.37 KCl; 0.44 KH₂PO₄; 0.49 MgCl₂; 0.41 MgSO₄•7H₂O; 136.9 NaCl; 4.17 NaHCO₃; 0.34 Na₂HPO₄•7H₂O; 15 sucrose; pH 7.4), followed by exposure to an oxygen free environment (90% nitrogen, 5% hydrogen, and 5% CO₂, 37°C) for 45 mins via a Biospherix C-Chamber (Parish, NY, USA). Vehicle (ddH₂O) and PYY_3–36 (500 nM) were administered immediately after OGD for 1 hr. In a separate set of experiments, slices were subjected to a 1-hour treatment of N-methyl-D-aspartate (NMDA; 500 μM) without OGD to induce maximal neuronal cell death. 24 hrs after OGD or NMDA procedures, hippocampal slices were incubated with propidium iodide (PI) at 2 μg/ml for 1 hr prior to determine neuronal cell death. Fluorescence images were captured via a 1.4-megapixel Peltier cooled fluorescent CCD camera and were digitized via IS Capture software (AM Scope, Irvine, California, USA). Neuronal cell death was analyzed by Image-J (NIH) and expressed as percentage of relative optical intensity. Neuronal cell death in the organotypic hippocampal slices was further normalized with NMDA-induced maximum neuronal cell death.

Y-maze for short-term (working) memory assessment

The Y-maze spontaneous alternation test was performed 3 days after ACA surgery due to the prevalence of neuronal cell death that occurs 3–7 days afterwards. The Y-maze is made of 3D printed black polylactic acid filament that consists of three closed arms [20 (L) x 4.5 (W) x 11.5 (H) inch] and one central partition in the middle to separate three arms in a “Y” shape (120° from the adjacent). Rats were transferred into the test room 10 mins before the trial to allow for acclimation. To eliminate scent bias, the maze floor was covered by a thin layer of cage bedding (1 cm thick) and replaced between trials. The rats were then placed into the central partition (starting zone) of the maze and were allowed to freely explore the maze for 10 mins. Spontaneous alternation was recorded by an overhead camera and calculated as the ratio of the number of alternations (the rat selected three different arms consecutively) to the total number of arms entered minus 2 (total possible alternations). 70% ethanol and distilled water were utilized to clean the Y-maze thoroughly at the end of experiments.

Statistical analysis

All animals in this study were randomized to each group. The collected data and analyzed results were blinded to treatment allocation. Power calculation was based on histopathology from our previous publications(R. H. Lee et al. 2017; R. H. Lee et al. 2019). We used this functional readout to analyze for power because it is one of the main endpoints and conclusion of this research. This yielded a standard error (SM) of the group means of 388, a SD of 90 within the groups, and an effect size (SM/SD) of 4.3. For each aim, the proposed sample of 8 subjects per group will allow 99% power to an effect size (SM/SD) of 0.6 for an F test and 91% power to detect a mean difference of 285, using the Dunnett’s multiple comparison test at a 0.05 significance level. Power calculation was performed with SPASS (NCSS, Kaysville, Utah). Results are expressed as mean ± S.E.M. Statistical analysis was evaluated by independent t-test (for CBF measurements), one-way ANOVA (Tukey’s post-hoc test), and two-way ANOVA (for Iba-1 and protein chip assay) (Tukey’s post-hoc test) as appropriate with SPSS (Chicago, IL). p≤0.05 level of probability is accepted as significant.

Results

mRNA and protein levels of NPY were enhanced, while post-treatment with PYY_3–36 reduced NPY levels 24 hours post-ACA

Reverse transcription real-time PCR and ELISA were first utilized to investigate the impact of ACA on plasma and hippocampal levels of two major sympathetic neurotransmitters (NE and NPY). Results from ELISA indicate plasma NE levels were enhanced 10 mins after ACA (9.40±1.48) but quickly returned to baseline 24 hrs after ACA (2.73±0.56) (Figure. 1A). In addition, rats subjected to ACA surgery had similar NE levels in the hippocampus 24 hrs after ACA as compared to rats in the control group (4.00±0.54 vs 3.11±0.15) ( Figure. 1B). NE levels in the plasma and hippocampus before and 24 hrs after ACA was not significantly different (Figures. 1A and 1B). We further investigated how ACA affects mRNA and protein levels of NPY in the hippocampus, indicating that NPY mRNA (1.95±0.06) and peptide levels (4.29 ±0.23) in the hippocampus were enhanced 24 hrs after ACA v. control rats but attenuated to normal levels with post-treatment with PYY_3–36 (40 μg/Kg) (1.28±0.10) (Figures. 1C and 1D). Overall, NPY levels were enhanced 24 hrs after ACA.

Figure. 1. NPY but not NE levels were enhanced 24 hrs. after ACA, while post-treatment with PYY_3–36 decreased NPY levels in the brain.

Blood samples were collected from the femoral artery 10 mins and 24 hrs. after ACA, while total protein from the entire hippocampus was collected 24 hrs. after sham/ACA surgery. NE concentration was measured via ELISA, while mRNA and peptide levels of NPY were measured via reverse transcription real-time PCR and ELISA, respectively. (A) Plasma NE concentration (via ELISA) was significantly increased 10 mins after ACA but returned to normal levels 24 hrs. after ACA. (B) Rats subjected to ACA surgeries had similar NE protein levels in the hippocampus 24 hrs after ACA as compared to control rats. mRNA (C) and peptide (D) levels of NPY in the hippocampus were increased 24 hrs after ACA, while post-treatment with PYY_3–36 reduced NPY levels. n indicate the number of animals used per group. Results were expressed as mean ± S.E.M. *P<0.05 indicates significantly different from before and 24hrs after ACA. #<0.05 indicates significantly different from control. &<0.05 indicates significantly different from ACA (1 day), evaluated by one-way ANOVA with Tukey’s post-hoc test.

PYY_3–36 can enhance cortical CBF to alleviate hypoperfusion 24 hrs after ACA

Since ACA-induced hypoperfusion is one of the major causes of neuronal cell death and neurological deficits, we performed in vivo laser speckle contrast imaging to investigate the impact of NPY on cortical CBF after ACA. Due to the prevalent occurrence of hypoperfusion, cortical CBF was measured 24 hrs after the initial onset of ACA (R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2014a; Lin et al. 2014b). Cortical CBF decreased in rats subjected to ACA only surgery, but increased (33.5±12.9) 24 hrs after ACA in the rats receiving post-treatment with PYY_3–36 (Figure. 2), suggesting inhibition of NPY release in the brain can alleviate hypoperfusion following ACA.

Figure. 2. Inhibition of NPY via post-treatment with PYY_3–36 enhanced CBF after ACA.

Cortical CBF was measured for 5 mins via intra-vital laser speckle contrast imaging in the anesthetized rat 30 mins before and 24 hrs after ACA surgery. Body temperatures were maintained at 37°C during the recording. (A) Representative flux images of cortical vasculature before and after ACA. The dashed ovals indicate the region of interest where cortical blood flow was measured via the cranial window. Changes in CBF were presented as percent change in flow from baseline CBF (30 min before ACA) and summarized in panel B. Results were expressed as mean ± S.E.M. *p<0.05 indicates significantly different from ACA-only rats, evaluated by independent t-test. n indicates number of experiments.

PYY_3–36 can reduce neuroinflammatory cytokines 3 and 7 days after ACA

Hypoperfusion following cerebral ischemia subsequently leads to neuroinflammation (Fowler et al. 2018; Back et al. 2017; K. M. Lee et al. 2015; Yamada et al. 2011). Post-treatment with PYY_3–36 alleviated ACA-induced hypoperfusion (Figure. 2) to suggest PYY_3–36’s neuroprotective role against neuroinflammation. We utilized a capillary-based immunoassay to determine if inhibition of NPY release via post-treatment with PYY_3–36 (40 μg/kg) can alleviate neuroinflammation after ACA. Since activation of microglia is essential for the process of neuroinflammation (Karve et al. 2016), we initially measured protein levels of specific markers for microglial activation (Ionized calcium binding adaptor molecule 1, IBa1) (Cerbai et al. 2012; Lian et al. 2016) in the hippocampus 1, 3, and 7 days after ACA in the presence or absence of PYY_3–36. IBa1 protein levels in the hippocampus were significantly enhanced 3 and 7 days after ACA (2.17±0.15 and 2.50±0.31) v. control rats, but levels were inhibited by post-treatment with PYY_3–36 (40 μg/kg) (1.76±0.06) (Figures. 3A and 3B). We further utilize protein chip assay to screen 10 inflammatory cytokines responsible for 1) the recruitment and activation of various immune cells [i.e. interleukin (IL)-1α/β, IL-2, IL-4 IL-6, IL-10, IL-13, and interferon (INF)-γ], 2) inflammatory mediators [tumor necrosis factor (TNF)-α] in the hippocampus (Figure. 3C). We found that post-treatment with NPY-2R agonist (PYY_3–36) at 40 μg/kg (Figures. 3D–3I) reduced inflammatory cytokines indicating that NPY is essential for neuroinflammation.

Figure. 3. Post-treatment with PYY_3–36 inhibited microglial activation and neuroinflammation after ACA.

The hippocampus was collected 1, 3, and 7 days after ACA surgery. Relative protein levels of Iba1 in the hippocampus were measured by capillary-based immunoassay. (A) Synthetic bands of Iba1 bands at 17 kDa. Results from the capillary-based immunoassay were summarized in panel B. Relative Iba1 protein levels were normalized to the internal control (β-actin) and expressed as the ratio of Iba1 levels in control animals. Upregulation of Iba1 protein expression (red bars) can be observed in rats treated with ACA, while post-treatment with PYY_3–36 (40 μg/kg) reduced Iba1 protein expression (green bars) after ACA. (C) Heatmap of protein expression of the selected cytokines involved in neuroinflammation. Values were normalized with control animals and expressed as log₂ (fold change). Relative cytokine levels in the hippocampus were measured via protein chip assay. Results from protein chip assay were summarized in panels D-M. Results were presented as % changes from the baseline (dashed line, control animals). Protein expression of inflammatory cytokines in the hippocampus was enhanced after ACA (red squares), while post-treatment with PYY_3–36 (40 μg/kg) significantly reduced inflammatory cytokine levels (green triangles). n indicates number of experiments. Results were expressed as mean ± S.E.M. *p≤0.05 indicates significantly different from control animals. #p≤0.05 indicates significantly different from the respective days after ACA (red squares), &p<0.05 indicates overall significantly different from ACA-treated rats (red squares), evaluated by two-way ANOVA with Tukey’s post hoc test.

PYY_3–36 can alleviate mitochondrial dysfunction 7 days after ACA

In addition to neuroinflammation, hypoperfusion following ACA can also cause mitochondrial dysfunction and subsequently neuronal cell death (Schon and Manfredi 2003; Fiskum 2000; Niizuma et al. 2009). Based on the data obtained from laser speckle contrast imaging, PYY_3–36 alleviated ACA-induced hypoperfusion, which suggests PYY_3–36 can improve mitochondrial function after ACA. Since delayed neuronal cell death occurs 3 to 7 days after ACA, the hippocampal slices were utilized to investigate the effects of PYY_3–36 on mitochondrial function 7 days after ACA. Mitochondria-mediated Adenosine triphosphate (ATP) production (38.9±3.40 v. 42.7±10.3), coupling efficiency (36.9±3.00 v. 36.2±5.40), and various types of respiration, including ATP-linked (57.7±6.22 v. 59.9±10.6), proton leak-linked (47.9±5.00 v. 57.8±13.2), basal (105.6±9.044 v. 107.3±18.73), and maximal respiration (92.0±12.1 v. 126.7 ±17.43) did not change 7 days after ACA v. control (Figure. 4). Interestingly, rats subjected to ACA-only surgery had significantly lower mitochondrial respiratory reserve capacity (an indicator of oxidative stress-mediated mitochondrial dysfunction) 7 days after ACA compared to control rats (−3.53±4.45 vs. 18.3±4.88) (Figures. 4A and 4F), to suggest ACA-induced mitochondrial dysfunction. Post-treatment with PYY_3–36 at 40 μg/kg enhanced reverse capacity after ACA (15.3±0.99) (Figures. 4A and 4F), to suggest that inhibition of NPY with PYY_3–36 can protect mitochondria in the post-ischemic hippocampus.

Figure. 4. PYY_3–36 maintained hippocampal mitochondrial function from ischemia following ACA.

Oxygen consumption rate (OCR) was measured in the hippocampal slices (200 μm, diameter) 7 days after ACA via Seahorse XF analyzer. (A) Changes in mitochondrial respiration were manipulated by injection of 20 μg/ml oligomycin, 1 mM pyruvate and 80 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 20 μM antimycin A. Results were summarized in panels B-H. Although ACA alone (red line and bar) did not affect basal respiration and ATP production, and proton leak in the mitochondria (A), mitochondrial reserve capacity was reduced 7 days after ACA but reversed with post-treatment with PYY_3–36 (green line and triangle) (A, F) to suggest that inhibition of NPY via NPY2R is important to maintain mitochondrial function. n indicates number of experiments. Results were expressed as mean ± S.E.M. *p≤0.05 indicates significantly different from control (white circle) and ACA+PYY-treated rats (green triangle), evaluated by one-way ANOVA with Tukey’s post-hoc test.

Post-treatment with PYY_3–36 promoted neuronal survival in the CA1 region of the hippocampus 7 days after ACA

We utilized histopathological analysis (H&E and FJC) to determine dead and neurodegenerative neurons in the CA1 region of the hippocampus 7 days after ACA. Dead/injured neurons in the CA1 region of the hippocampus dramatically increased in ACA-treated rats compared to control rats (Figure. 5A), suggesting delayed neuronal cell death 7 days after ACA. Rats post-treated with PYY_3–36 (40 μg/kg) exhibited less dead/injured neurons as compared to rats subjected to ACA-only surgery (Figure. 5A). Similar results were further confirmed with FJC staining, showing an increase in FJC-positive (neurodegenerative) neurons (Figure. 5B) 7 days after ACA. Since PYY_3–36 at 40 μg/kg reduced neurodegenerative neurons in the CA1 region of the hippocampus (Figure. 5B), this suggests inhibiting NPY release via PYY_3–36 promotes neuronal survival against ACA.

Figure. 5. Post-treatment with PYY_3–36 alleviated neuronal cell death in the CA1 region of the hippocampus 7 days after cerebral ischemia.

Whole animal perfusion and fixation were performed 7 days after ACA for brain histopathology of the hippocampus. Cortical brain sections from control, ACA only, and ACA+PYY_3–36-treated rats were stained with H&E (blue/pink) (A), and fluoro-jade C (FJC, green) (B), and DAPI nuclear counterstain (blue). Control (no ACA, no drug administration) group was served as an internal control. Representative images of H&E in panel A indicate that healthy neurons contain a lightly stained nucleus with a dark-stained nucleolus and a red-stained cytoplasm. On the contrary, dead/injured neurons exhibit shrunken cytoplasm and pyknotic nuclei. Representative images of H&E and FJC staining in panel A and B indicate that degenerative neurons can be observed in the CA1 region of the hippocampus 7 days after ACA, while post-treatment with PYY_3–36 (40 μg/kg) inhibited neuronal degeneration. Red arrows in panel A indicate neuronal cell death. Red arrowheads in panel B indicate degenerative neurons. In a separate set of experiments, neuronal cell death following 45 mins of OGD was evaluated via propidium iodide (PI) fluorescence. (C) Representative propidium iodide fluorescence images of hippocampal organotypic slices following OGD + Vehicle (ddH₂O), OGD+PYY_3–36 (500 nM), Vehicle only, and PYY_3–36 (500 nM) only treatments. NMDA at 500 μM was utilized to induce maximal neuronal cell death at the end of experiments. Neuronal cell death with/without OGD in the present/absent of PYY_3–36 was normalized with NMDA-induced maximum cell death and expressed in the bar graph in panel D. 45 mins of OGD resulted in neuronal cell death in the CA1 region of the hippocampus, while PYY_3–36 at 500 nM reduced OGD-induced cell death. n indicates number of animals used per group. Scale bars in panel C indicate 500 μm. Results were expressed as mean ± S.E.M. *p≤0.05 indicates significantly different from vehicle only, PYY_3–36 only, and OGD+PYY_3–36 groups via one-way ANOVA with Tukey’s post-hoc.

PYY_3–36 mitigated neuronal cell death following in vitro model of cerebral ischemia

Previous studies suggest NPY2R’s angiogenic, proliferative, and differentiated properties in wound healing and bone formation (Baldock et al. 2002; Ekstrand et al. 2003; Kuo et al. 2007; Parker and Balasubramaniam 2008; Yulyaningsih et al. 2011). In addition to neuroprotection directly related to CBF modulation, we investigated the possibility of additional neuroprotection from PYY_3–36 independent of CBF. We utilized organotypic hippocampal slices coupled with OGD, an in vitro model of cerebral ischemia without CBF involvement. Hippocampal slices were treated with either PYY_3–36 (500 nM) or vehicle (ddH₂O) after OGD for 1 hr. Hippocampal neuronal cell death (white or bright signal) was observed in the vehicle-treated slices 24 hrs. after OGD (49.3±3.05%), while post-treatment with PYY_3–36 (500 nM) alleviated neuronal cell death (26.8±4.66%) (Figures. 5C and 5D). These results suggest PYY_3–36 has innate neuroprotective properties (irrespective to CBF) against cerebral ischemia.

PYY_3–36 improved short-term (working) memory 3 days after ACA

Since PYY_3–36 has the capacity to protect CA1 neurons from ACA-induced lethal ischemic attacks (Figure. 5), we further utilized the Y-maze spontaneous alternation test to further evaluate neuroprotection with PYY_3–36 against ACA-induced short-term memory deficits. As a result, rats subjected to ACA-only surgery had a lower spontaneous alternation ratio (0.49±0.07) 3 days after ACA as compared to the control rat group (0.74±0.04) (Figure. 6). These findings suggest short-term memory is impaired after ACA. Inhibiting NPY levels in the brain with post-treatment with PYY_3–36 (40 μg/kg) increased the spontaneous alternation ratio (0.64±0.02) after ACA (Figure. 6), indicating that this treatment is beneficial to counteract ACA-induced learning and memory deficits.

Figure. 6. Post-treatment with PYY₃–₃₆ improved ACA-induced neurocognitive/memory.

3 days after ACA surgery, the Y-maze spontaneous alternation test were utilized to examine the effects of NPY inhibition on hippocampal-related working/short-term memory after ACA. Spontaneous alternation ratio was decreased in rats subjected to ACA only surgery as compared to control, while post-treatment with PYY_3–36 at 40 μg/kg increased alternation rate. n indicates number of animals used per group. Results were expressed as mean ± S.E.M. *p<0.05 indicates significantly different from control rats, # p<0.05 indicates significantly different from ACA only group via one-way ANOVA with Tukey’s post-hoc.

Discussion

A major challenge of post-resuscitative care is CBF derangements, which leads to brain injury and neurological deficits (Harukuni and Bhardwaj 2006; Sabri et al. 2013; Manole et al. 2009; R. H. Lee et al. 2020). CA-induced CBF derangements refers to two stages of blood flow/perfusion abnormality in the brain: stage I begins with a short-term hyperemia (increase in CBF), which occurs within 5 to 10 mins after initial resuscitation (Kunz and Iadecola 2009; Lin et al. 2010; Todd et al. 1986). Following hyperemia, stage II is characterized by a prolonged hypoperfusion (decrease in CBF) that can last from hours to days after resuscitation (R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2010; Lin et al. 2014b; R. H. Lee et al. 2020). CA-mediated delayed neuronal cell death and neurological deficits (learning/memory) are believed to occur during the long-lasting hypoperfusion 3 to 7 days after the initial onset of CA (Harukuni and Bhardwaj 2006; Manole et al. 2009; R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2010; Lin et al. 2014b). Since cerebral blood vessels are controlled by a combination of vasoconstriction and dilation factors, identifying innovative agents that can modulate vasoconstriction/dilation factors to alleviate hypoperfusion in the context of CA will provide immense benefits to neuronal survival and functional recovery (R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2010; Lin et al. 2014b).

We previously discovered that ACA-induced hypoperfusion is derived from the overstimulation of the sympathetic nervous system (Figure. 7) (R. H. Lee et al. 2017) by surgical interruption of sympathetic projections (via bilateral decentralization) from the sympathetic chain to the cerebral arteries lead to more favorable outcomes after ACA (R. H. Lee et al. 2017; Guth and Bernstein 1961). Since this resulted in a decrease of neurotransmitters/vasoconstrictors released from presynaptic nerve terminals, we sought to clarify the role of two major sympathetic neurotransmitters, NE and NPY, as they relate to CA-induced hypoperfusion and brain injury.

Figure. 7. Cartoon illustrating the role of NPY in ACA-induced brain injury.

Excessive NPY release (yellow circles) from perivascular sympathetic nerve terminals following ACA results in (I) persistent hypoperfusion, ultimately causing neuronal cell death and neurological deficits (blue arrows). (II) NPY may directly influence CA1 neurons via independent modulation of vascular perfusion to cause neuronal cell death and learning/memory deficits. The use of PYY₃–₃₆ (blue triangles) to activate presynaptic NPY2 receptors can inhibit NPY release after ACA to provide neuroprotection against ACA-induced hypoperfusion, neuronal cell death, and neurological deficits (green arrows).

We first used an enzyme-linked immunosorbent assay (ELISA) to determine NE levels in the plasma and brain region vulnerable to ischemia (i.e. hippocampus), which suggest that NE does not contribute to ACA-induced hypoperfusion. Hypoperfusion is prevalent 24 hrs after ACA (R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2010; Lin et al. 2014b) (Figure. 2), while plasma and hippocampal NE levels returned to baseline levels 24 hrs after ACA (Figures. 1A and 1B). We further investigated the possibility of NPY as a modulator of hypoperfusion and brain injury after ACA via reverse transcription real-time PCR and ELISA. We found NPY levels in the hippocampus were enhanced 24 hrs. after ACA, which was well-correlated with hypoperfusion, highlighting the importance of NPY in the pathogenesis of ACA (Figures. 1C and 1D).

NPY (a 36 amino-acid neuropeptide and vasoconstrictor) is stored in the perivascular nerve fibers of cerebral arteries to regulate cerebral microcirculation (Hakanson et al. 1986; Schebesch et al. 2013) and there are currently 5 known NPY receptors (NPY1R, NPY2R, NPY4R, NPY5R, and NPY6R) identified in the human brain (Yulyaningsih et al. 2011). NPY causes sustained and prolonged vasoconstriction 100-fold more potent than NE by binding exclusively to postsynaptic NPY1 receptors (G protein-coupled receptor) on vascular smooth muscle cells. Activation of NPY1 receptors further enhances cyclic adenosine monophosphate/phospholipase C as a result of enhanced intracellular Ca²⁺, vasoconstriction, and decreased CBF (Franco-Cereceda and Liska 1998). We investigated whether NPY caused hypoperfusion after ACA using presynaptic NPY2R agonist (PYY_3–36) via in vivo laser speckle contrast imaging. Cortical hypoperfusion was observed 24 hrs. after ACA (R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2014a; Lin et al. 2014b) (Figure. 2), but alleviated with PYY_3–36 (40 μg/kg) (Figure. 2) which suggests NPY is the major contributor to hypoperfusion after ACA (Figure. 7).

In recent years, hypoperfusion has been reported to cause neuroinflammation aggravating neuronal cell death after ACA (Fowler et al. 2018; Back et al. 2017; K. M. Lee et al. 2015). The role of NPY in modulating inflammation was first reported in late 1980s in peripheral nerves (Chandrasekharan et al. 2013; Chiu et al. 2012). Activation of NPY1R and NPY2R by NPY modulates neutrophil chemotaxis, T helper cell differentiation, activity of natural killer cell, and antigen-presenting cells, to potentiate the autoimmune and allergic responses (Chandrasekharan et al. 2013). In contrast to NPY’s pro-inflammation effects, administration of NPY increased the release of anti-inflammatory cytokines (i.e. transforming growth factor-β1) (Zhou et al. 2008) in the human macrophage cell line. The number of macrophages and their release of cytokines was suppressed in mice treated with NPY recombinant peptide (Macia et al. 2012). Moreover, blocking NPY signaling via specific NPYR antagonists promotes dendritic cell maturation and gene expression of inflammatory cytokines (i.e. IL-6, TNF-α, and nitric oxide synthase 2) in vitro (Singer et al. 2013). These discrepancies in the literature led us to further delineate the role of NPY in neuroinflammation following ACA.

From the present study, excessive release of NPY in the brain is actually detrimental during ACA as it can potentiate neuroinflammation, leading to neuronal cell death and neurological deficits. These conflicting results may be attributed to the fact that 1) different methodologies used to induce ischemia (peripheral tissue vs. central nervous system; in vitro vs. in vivo) and/or 2) most of the aforementioned studies regarding NPY and inflammation mainly focused on the direct interaction of NPY with immune cells unrelated to cerebral ischemia and neurodegeneration, while our research is the first study to delineate the consequences among CBF derangements, NPY, and neuroinflammation.

In addition to neuroinflammation, mitochondrial dysfunction following hypoperfusion can lead to neuronal cell death (Schon and Manfredi 2003; Fiskum 2000; Niizuma et al. 2009). Prior studies of cultured rat cardiomyocytes show NPY alone can impair mitochondrial structure and membrane potential through calcium/calmodulin-dependent protein kinase (Hu et al. 2017; Luo et al. 2015). We studied the impact of NPY on mitochondrial stress in the hippocampus after ACA via Seahorse respirometry. Mitochondrial respiratory reserve capacity in the hippocampus, which refers to the capability of mitochondria to acclimate rising metabolic demands during stress, was dramatically reduced 7 days after ACA (Figure. 4A and 4F). This reduction after ischemia could expose neurons to oxidative stress and lead to their death (Figure. 5). Post-treatment with PYY_3–36, to inhibit NPY signaling, improved mitochondrial respiratory reserve capacity indicating NPY is detrimental to mitochondrial function and neuronal survival. We further evaluated the therapeutic potential of NPY inhibition via PYY_3–36 as a neuroprotective agent through brain histology. H&E and FJC staining were utilized to evaluate CA1 neuronal survival in the presence of PYY_3–36 after ACA (Figure. 5), highlighting PYY_3–36’s protective effects on CA1 neurons. Alternative potential mechanism of action independent of cerebral blood flow is the fact that activation of NPY2R has inherent neuroprotective properties against excitotoxicity in the hippocampus (Silva et al. 2003). This may be achieved by inhibiting glutamate release, thought to attenuate excitotoxicity, via the presynaptic NPY2R receptor (Silva et al. 2007) activated by PYY_3–36, affording overall neuronal survival (Figure 5).

All NPY receptors have been identified as post-synaptic receptors, except for NPY2R which is the only one localized both pre and post-synaptically, highlighting NPY2R’s unique role in modulating physiological function (Parker and Balasubramaniam 2008; Stanic et al. 2011). It has been reported that NPY2R can promote proliferation/differentiation of adipocytes, angiogenesis of capillaries, wound healing, and bone formation (Baldock et al. 2002; Ekstrand et al. 2003; Kuo et al. 2007; Parker and Balasubramaniam 2008; Yulyaningsih et al. 2011). While we have observed that activation of NPY2R via post-treatment with PYY_3–36 following ACA can restore cortical CBF and provide benefits against ACA-induced neuronal cell death in the CA1 region of the hippocampus, it is unclear if activation of NPY2R via PYY_3–36 can afford neuroprotection via independent modulation of vascular perfusion. To investigate if activation of NPY2R by PYY_3–36 protects neurons from ischemia in a CBF-independent manner, organotypic hippocampal slice cultures coupled with oxygen-glucose deprivation (OGD), an in vitro ischemia-reperfusion injury model, and PI staining were used. Surprisingly, results from PI staining indicate that PYY_3–36 alone also reduced CA1 neuronal cell death after OGD challenge (Figures. 5C and 5D). Altogether, our results from laser speckle contrast imaging and OGD studies suggest that neuroprotective effects of PYY_3–36 is multifaceted, which can not only enhance post-resuscitative CBF but also directly promote neuronal survival following ischemia.

Since neurons in the CA1 region of the hippocampus are essential for learning/memory formation (Bahar et al. 2011), patients who survive the CA with severe CA1 neuronal cell death suffer devastating neurocognitive deficits from moderate learning/memory impairment to persistent vegetative state (Harukuni and Bhardwaj 2006; Nichol et al. 2008). Numerous pre-clinical studies mainly focus on therapies targeting a specific pathway network of ischemic injury, but have ultimately failed in clinical practice (Geocadin et al. 2008; Schulman et al. 2006; Wright and Geocadin 2006). Therefore, we utilized the Y maze to study if NPY inhibition via PYY_3–36 improved functional outcomes (i.e. protects working learning/memory) after ACA, which could provide high translational value to the present findings. The choice of using Y maze is based on our previous studies indicating that ACA-induced neurocognitive deficits are closely related to working (short-term) memory (Hayashida et al. 2014; Dember 1989; R. H. Lee et al. 2019; R. H. Lee et al. 2017). Rats subjected to ACA surgery had hippocampal damage and memory impairments 3 days after ACA due to delayed neuronal cell death (Dave et al. 2009; Della-Morte et al. 2011; R. H. Lee et al. 2017; R. H. Lee et al. 2019; Lin et al. 2014b). This resulted in a spontaneous alternation ratio lower than the control rats (Figure. 6) (R. H. Lee et al. 2017; R. H. Lee et al. 2019; C. Y. C. Wu et al. 2018) since they had difficulties locating earlier explored goal arms. Post-treatment with PYY_3–36 increased the spontaneous alternation ratio (Figure. 6) after ACA, indicating that activation of NPY2R can protect functional learning/memory after ACA. In the present study, we have established that the NPY system in the brain is actually detrimental during/after CA and can cause persistent hypoperfusion leading to neuronal cell death and functional learning/memory deficits (Figure. 7). The use of PYY_3–36 to inhibit NPY may provide novel therapeutic opportunities in the treatment against CBF-related neurodegenerative diseases including CA, stroke, and Alzheimer’s disease.