Calbindin-D9k is a Novel Risk Gene for Neurodegenerative Disease
Eui-Man Junga Yeong-Min Yooa Seon Young Parka Changhwan Ahna
Bo-Hui Jeona Eui-Ju Hongb Woo-Yang Kimc Eui-Bae Jeunga
aLaboratory of Veterinary Biochemistry and Molecular Biology, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea, bCollege of Veterinary Medicine, Chungnam National University, Yuseong, Daejeon, Republic of Korea, cDepartment of Biological Sciences, Kent State University, Kent, OH, USA
Key Words
Calbindin-D9k • Neurodegenerative diseases • Alzheimer’s disease • Parkinson’s disease
Abstract
Background/Aims: Calcium homeostasis plays a crucial role in neuronal development and disease. Calbindin-D9k (CaBP-9k) acts as calcium modulators and sensors in various tissues. However, the neurobiological functions of CaBP-9k are unknown. Methods: We used CaBP-9k knockout (KO) mice to investigate the roles of these gene in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. We used anatomical and biochemical approaches to characterize functional abnormalities of the brain in the CaBP-9k KO mice. Results: We found that the brains of CaBP-9k KO mice have increased APP/β-amyloid, Tau, and α-synuclein accumulation and endoplasmic reticulum (ER) stress-induced apoptosis. Neurons deficient for these CaBP-9k had abnormal intracellular calcium levels and responses. ER stress inhibitor TUDCA reduced ER stress-induced apoptosis and restored ER stress- and apoptosis-related proteins expression to wild-type levels in CaBP-9k KO mice. Furthermore, treatment with TUDCA rescued the abnormal memory and motor behaviors exhibited by older CaBP-9k KO mice. Conclusion: Our results suggest that a loss of CaBP-9k may contribute to the onset and progression of neurodegenerative diseases.
Introduction
Calcium is a ubiquitous second messenger stored intracellularly in mitochondria and in the endoplasmic reticulum (ER) [1] in a diversity of cell types, including neurons, in which it regulates various functions such as synaptic transmission, plasticity, and survival. Calcium homeostasis in neurons involves tight regulation of cytosolic concentrations, the disruption of which can lead to synaptic failure, network dysfunction, and cognitive impairment as well as neuronal death and various neurodegenerative diseases [2, 3]. For example, altered calcium homeostasis in the ER is associated with the pathogenesis of Alzheimer’s disease [3], as high intracellular concentrations induce amyloid precursor protein (APP)/β-amyloid and Tau accumulation and neuronal cell death. Elevated calcium levels also disrupt mitochondrial and ER function contributing to the death of dopamine neurons in the substantia nigra pars compacta (SNc) that characterize Parkinson’s disease [4].
The homeostasis of intracellular calcium concentrations involves the binding of cytosolic calcium to members of the EF-hand family of proteins, including calbindin-D9K (CaBP-9k) and calbindin-D28K (CaBP-28k) [5, 6]. CaBP-9k is widely distributed and this protein is co-expressed in mature-, GABAergic, dopaminergic, and oxytocinergic neurons in the rat brain [7]. Futuremore, CaBP-28k is widely distributed throughout the central nervous system, where it is found in the somas, axons, and terminals of neurons. CaBP-28k plays roles in the differentiation and plasticity of hippocampal neurons [8], and may protect neurons from elevated intracellular calcium levels in Alzheimer’s disease [9, 10]. Its expression is decreased in temporal and parietal cortices of patients with dementia of other histopathological types [11]. CaBP-28k is also expressed in neurons of the SNc and ventral tegmental area (VTA) in rats and monkeys [12, 13], and CaBP-28k protects VTA dopamine neurons against oxidative stress, calcium toxicity, and α-synuclein accumulation in Parkinson’s disease [14]. Less is known about CaBP-9k, which along with CaBP-28k, suppresses prion protein expression and may be influenced by prion disease in mouse brains [15, 16]. CaBP-9k expression induced by melatonin also suppresses hydrogen peroxide-mediated cell death in rat pituitary GH3 cells [17, 18].
To provide a clearer understating of the roles of these CaBP-9k in the brain, we used CaBP-9k knockout (KO) mice. We found that CaBP-9k KO leads to neurodegeneration resembling Alzheimer’s and Parkinson’s diseases, with increases in APP/β-amyloid, Tau, and α-synuclein and ER stress-induced apoptosis in the brains of CaBP-9k KO mice. Furthermore, CaBP-9k ablation induced abnormal memory and motor behaviours, which were rescued by treatment with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA). The findings presented here establish a critical role for CaBP-9k in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases.
Materials and Methods
CaBP-9k knockout mice and TUDCA treatment
CaBP-9k KO mice were generated as previously described [19, 20]. The genotypes of offspring were determined by PCR analysis, as described previously [19]. The Mice were housed in a cage with 12:12h light-dark cycle. No more than 5 mice were housed in a cage. Mice were handled according to a protocol approved by the Ethics Committee of the Chungbuk National University. TUDCA-treated mice were fed a diet of standard laboratory chow (Purina Mills, USA) supplemented with either 0.4 % (wt/wt) TUDCA (sodium salt; Matrix Scientific, USA). Treatment was started when the mice were 5 months old and continued for 8 months. Behavioral testing started at 7 months of age, where CaBP-9k KO mice start to display memory deficits and lasted for a month. Weight was measured at the beginning of the treatment TUDCA and general activity was monitored during experiments.
Real-time reverse transcription PCR
Quantitative real-time PCR was performed as described previously [21]. Total RNA was extracted from wild-type and CaBP-9k mutant brains using Trizol reagent (Invitrogen). First-strand complementary DNA (cDNA) was prepared by reverse transcription using the MMLV cDNA synthesis kit (Thermo Fisher Scientific). To determine the conditions for the logarithmic phase during PCR amplification with target mRNA, aliquots (1 µg) were amplified using different numbers of cycles. A linear relationship between PCR product band visibility and the number of amplification cycles was observed for target mRNAs. Real-time PCR was performed with 1 µl of the cDNA template added to 10 ml of 2x SYBR Premix Ex Taq (TaKaRa Bio) and specific primers (10 pM each). Primer sequences are presented in Supplementary Table 1 (for all supplemental material see www.cellphysiolbiochem.com). Real-time PCR (Applied Biosystems) was carried out for 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 15 sec. Target gene expression was quantified relative to that of an internal control gene (Gapgh) based on the comparison of the threshold cycle (CT) at constant fluorescence intensity. The amount of transcript was inversely related to the observed CT and the CT was expected to increase by 1 for every two-fold dilution of the transcript. Relative expression (R) was calculated using the equation R = 2-[ΔCT sample-ΔCT control]. All data were normalized relative to Gapgh as well as to the respective controls.
Western blot analysis
Western blotting was performed as described previously [21, 22]. Brain lysates were extracted with RIPA buffer (Invitrogen). Proteins (40 μg/lane) were separated on 10~15% SDS-PAGE gel and transferred to an immobilon-P membrane (Millipore) by a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). The resulting blot was blocked in TBS (tris-buffed saline) containing 5% BSA for 60 minutes, then incubated with a primary antibody rabbit anti-calbindin-D9k (Invitrogen, PA5-68289, 1:1000), rabbit anti-BACE (Cell Signaling Technology, #5606, 1:1000), rabbit anti-Tau (Cell Signaling Technology, #4019, 1:1000), p-SAPK/JNK (Cell Signaling Technology, #9251, 1:1000), mouse anti-Bax (Santa Cruz, sc-7480, 1:500), mouse anti-Bcl-2 (Santa Cruz, sc-7382, 1:500), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, #9664, 1:1000), rabbit anti-cleaved caspase-9 (Cell Signaling Technology, #7237, 1:1000), PARP (Cell Signaling Technology, #9532, 1:1000), mouse anti-GADD153/CHOP (Cell Signaling Technology, #2895, 1:1000), rabbit anti-p-PERK (Cell Signaling Technology, #3179, 1:1000), rabbit anti-PERK (Cell Signaling Technology, #3192, 1:1000), rabbit anti-p-IRE1α (Thermo Fisher Scientific, PA1-16927, 1:1000), rabbit anti-IRE1α (Cell Signaling Technology, #3294, 1:1000), rabbit anti-p-eIF2α (Cell Signaling Technology, #3597, 1:1000), rabbit anti-caspase-12 (Cell Signaling Technology, #2202, 1:1000) and GAPDH (Santa Cruz, sc-25778, 1:500) for overnight at 4°C. After washing in TBS containing 0.1% Tween 20, the membrane was incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (1:2500, Cell Signaling Technology) for 1 hour at room temperature. After washing, the membrane was developed by using ECL Western-blotting reagents (Pierce Biotechnology). Immunoreactive proteins were visualized by exposure to Agpa CP-BU X-ray film (Agfa-Gevaert NV). Protein bands were visualized by image-scanning, and optical density was measured by using ImageJ analysis software 1.37 after the data were corrected by background subtraction
Immunostaining
Immunostaining of brain sections or dissociated cells was performed as described previously [19, 21, 22]. Primary antibodies used were mouse anti-APP/β-Amyroid (Cell Signaling Technology, #2450, 1:500), mouse anti-Tau (Cell Signaling Technology, #4019, 1:300), rabbit anti-PARP (Cell Signaling Technology, #9532, 1:500), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, #9664, 1:500), mouse anti-BrdU (BD Biosciences, #555627, 1:800), rabbit anti-Ki67 (Cell Signaling Technology, #9129, 1:500), mouse anti-α-synuclein (Cell Signaling Technology, #2647, 1:500), chicken anti-tyrosine hydroxylase (AVES, TYH, 1:800), chicken anti-GFP (Invitrogen, A10262, 1:800), rabbit anti-calbindin-D9k (Invitrogen, PA5-68289, 1:800) antibodies. Appropriate secondary antibodies conjugated with Alexa dyes (Invitrogen) were used to detect primary antibodies. DAPI (Sigma-Aldrich) was used to stain nuclei.
Cell culture
For evaluation of CaBP-9k knockdown by shRNA constructs, Neuro2a cells maintained in DMEM/10% FBS were transiently transfected using Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. Transfections were allowed to proceed for 4–5 hours, and then cells were cultured in 10% FBS/DMEM for 24 hours.
The primary neuronal cells were isolated from E14.5-16.5 CaBP-9k KO mice. Meninges were removed, and primary neuronal cells were dissociated with trituration after trypsin/EDTA treatment. The cells were plated onto poly-D-lysine/laminin-coated coverslips and cultured in the medium containing Neurobasal medium (Invitrogen), 2 mM glutamine, 2% (v/v) B27 supplement (Invitrogen), 1% (v/v) N2 supplement (Invitrogen), and 50 U/mL penicillin/streptomycin (Invitrogen).
TUNEL assay
As described previously [19, 21], tissue sections and cell cultures were fixed with 4% PFA, and in situ detection of cells with DNA-strand breaks was performed using In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer's instruction.
Measurement of intracellular calcium
The primary neuronal cells were plated in 96 well plates. DIV7 neuronal cells were incubated with calcium dye, Rhod-4 (Abcam), for 1h at 37°C. The plates were then placed into a Lionheart™ FX Automated Microscope (BioTeK) for the calcium response to monitor cell fluorescence (Rhod-4: Excitation = 540 nm and Emission = 590 nm). Images were acquired using a 4x objective at a rate of 3 frames per second. In-line injectors were used to dispense 20 μL of glutamate (30 µM, Sigma-Aldrich) into the wells, and cells were imaged for an additional 60 seconds to monitor response.
Behavioral assays
All behavioral assays were done during the light cycle. Health conditions including weights, activity and feeding were checked prior to assays. We used male and female mice for most behavioral assays. All behavioral assays were done blind to genotypes with age-matched littermates of mice.
Novel object recognition test. As described previously [21], a test mouse was first habituated to an open field arena (60 × 60 cm) for 5 min. Following habituation, the test mouse was removed from the arena and two identical objects with size (10.5 × 4.5 × 2.5 cm) were placed in the opposite corners of the arena, 15 cm from the side walls. Then the test mouse was reintroduced into the center of the arena and allowed to explore the arena including the two novel objects for 10 min. After 6 h, one object was replaced with another novel object, which was of similar size but different shape and color than the previous object. The same test mouse was placed in the arena to explore the arena and the two objects. The movement of mice was recorded by a camera for 10 min and further analyzed by the video tracking EthoVision XT 14 software (Noldus).
Passive avoidance test. Mice were individually habituated to the lighted compartment before a test. During the training session, each mouse was placed into the lighted compartment and the latency to enter the dark compartment was recorded. When the mouse entered the dark compartment with all four paws, a foot shock (2 mA, 3 sec) was delivered. During retention session 24 h later, each mouse was placed into lighted compartment again and the latency to enter the dark compartment was recorded.
Morris water maze test. Mice were introduced into the perimeter of a circular water-filled tank 90 cm in diameter and 42 cm in depth with visual cues that were present on the tank walls as spatial references. The tank was divided into four equal quadrants (Q1-4) by lines drawn on the floor. A circular plexiglass platform was submerged 1 cm deep in Q2 and as such hidden from the mice. The mice started the task from one of three quadrants Q1, Q3 and Q4, varied by day of testing. Four trials were performed per mouse per day for ten days. Each trial lasted 1 minute and ended when the mouse climbed onto and remained on the hidden platform for ten seconds. The mouse was given 20 seconds to rest on the platform between trials. The time taken by the mouse to reach the platform was recorded as its latency. The time for four trials was averaged and recorded as a result for each mouse. On day 10, the mice were subjected to a single 60-second probe trial without a platform to test memory retention. The mice started the trial from Q4, the number of annulus crossings was counted, and the swimming path was recorded and analyzed using the Ethovision XT 14 tracking software (Noldus).
Cylinder test. Each mouse was placed in a transparent acrylic cylinder (10 cm in diameter and 14 cm high). The number of wall contacts with each forelimb when rearing in at least 15 rearing cycles was computed. Animals that did not meet this criterion were excluded from this assay. The cylinder test score was determined as follows: (use of the affected forepaw (contralateral) – intact forepaw (ipsilateral)/total (contralateral + ipsilateral + both).
Rotarod test. Using an accelerating rotarod apparati (PanLab) the rotarod test was performed by placing mice on rotating drums (2.5 cm diameter) and measuring retention time on the rod. The speed of the rotarod accelerated from 4 to 40 rpm over a 5 min period. At least 20 min recovery time was allowed between trials.
Pole test. Mice were placed head-up on top of a metal pole (50 cm high and 1 cm wide) that has been wrapped in wooden wire. The base of the pole was placed in the home cage. When placed on the pole, mice orient themselves downward and descend the length of the pole back into their home cage. The time to orient downward (t-turn) and the total time to descend (t-total) were measured.
Nesting behavior. Each animal was provided with a Nestlet (5 × 5 cm2 piece of cotton; Ancare). After 24h, the next morning remaining Nestlets were scored on a scale from 1-5. (1) nesting material unmodified; (2) flat nest with partially shredded nesting material; (3) shallow nest with shredded material but lacking fully formed walls; (4) nest with well-developed walls; and (5) nest in a shape of a cocoon with a partial or complete roof. Pictures were taken of all nests.
Open field test. A mouse was placed near the wall-side of a 60 × 60 cm open-field arena, and the movement of the mouse was recorded by a camera for 5 min. The recorded video file was further analyzed using EthoVision XT 14 software (Noldus). The number of entries into and the overall time spent in the center of the arena (30 × 30 cm imaginary square) were measured.
Tail suspension test. A mouse was suspended from the hook of a tail suspension test box, 60 cm above the surface of a table using adhesive tape placed 1 cm away from the tip of the tail. After 1 min acclimatization, immobility duration was recorded by a camera for 5 min. Mice were considered immobile only when they hung passively and were completely motionless.
Statistical analysis
Normal distribution was tested using the Kolmogorov-Smirnov test and variance was compared. Unless otherwise stated, statistical significance was determined using two-tailed unpaired Student’s t-tests for two population comparison followed by the Bonferroni's post hoc test for multiple comparisons. Data were analyzed using the GraphPad Prism and presented as means ± SEM. P values for each comparison were described in the legends or supplementary information section. To determine and confirm sample sizes (n), we performed a power analysis. The values for the power (1-β) and the type I error rate (α) were 0.8 and 0.05 (or 0.01), respectively. Each experiment in this study was performed blind and randomized. Mice were assigned randomly to the various experimental groups, and data were collected and processed randomly. The allocation, treatment, and handling of mice were the same across study groups. Control mice were selected from the same litter as the test group. The individuals conducting the experiments were blinded to group allocation and the allocation sequence. Exclusion criteria for mice were based on abnormal health conditions including weights below 15g at 6 weeks and noticeably reduced activity or feeding as used in previous studies [23, 24]. Statistical data and n numbers for all behavioral assays were described in the legends.
Results
Ablation of CaBP-9k induces signs indicative of Alzheimer’s disease in mice
We first measured the expression of CaBP-9k in mouse brain extracts by Western blotting and real-time PCR. CaBP-9k mRNA and protein levels were undetectable in CaBP-9k KO mice. Moreover, CaBP-28k mRNA and protein levels in CaBP-9k KO mice were not different from those in wild-type mice (Supplementary Fig. 1). The localization of CaBP-9k was examined by immunostaining, which revealed a wide distribution of CaBP-9k-positive cells throughout the brain, including the olfactory bulb, cerebral cortex, hippocampus, thalamus, hypothalamus, paraventricular nucleus, SNc, VTA, cerebellum, and brain stem. Double immunostaining revealed that CaBP-9k was expressed by mature (NeuN-positive) and dopaminergic (tyrosine hydroxylase [TH]-positive) neurons in the hippocampus and in the SNc and VTA, respectively (Supplementary Fig. 2). Immunostaining for microtubule associated protein 2 (MAP2) revealed that CaBP-9k KO did not alter the lengths or numbers of primary and secondary neurites (Supplementary Fig. 3).
As an accumulation of APP/β-amyloid [25, 26], Tau protein [27, 28], and α-synuclein [29] in the hippocampus are hallmarks of Alzheimer’s disease, we examined these proteins in CaBP-9k KO mice. Immunostaining for APP/β-amyloid revealed an increase in plaque numbers with age when comparing young (<2 months) and old (>8 months) CaBP-9k KO mice. Furthermore, the numbers of hippocampal plaques were significantly higher in CaBP-9k KO than in wild-type mice at an old age (Immunofluorescence: wild-type, 0.43 ± 0.32 cells; CaBP-9k KO, 12.29 ± 2.65 cells/1.78 mm2; Immunohistochemistry: wild-type, 0.33 ± 0.33 cells; CaBP-9k KO, 25.67 ± 1.86 cells/1.78 mm2) (Fig. 1A-D). In young mice, hippocampal APP/β-amyloid plaques were undetectable in CaBP-9k KO mice (Supplementary Fig. 4A). Similarly, the densities of Tau-immunopositive neurons were significantly higher in CaBP-9k KO than in wild-type mice at an old age (CaBP-9k KO, 11.49 ± 1.61 cells/0.02 mm2) (Fig. 1E, F). Additionally, the densities of hippocampal α-synuclein aggregates were increased in CaBP-9k KO at an old age (CaBP-9k KO, 10.75 ± 0.85 cells/1.78 mm2) (Fig. 1G, H). In young mice, α-synuclein aggregate were undetectable in CaBP-9k KO mice (Supplementary Fig. 5A). The expression levels of beta-secretase (BACE) and another biomarker for Alzheimer’s disease, Tau, were assessed by Western blotting. The levels of BACE and Tau were significantly increased in old CaBP-9k KO mice (relative expression for BACE: wild-type, 100 ± 4.64%; CaBP-9k KO, 173.66 ± 13.68%; for Tau: wild-type, 100 ± 2.72%; CaBP-9k KO, 122.39 ± 4.19%), compared with brain lysates from wild-type mice at an old age (Fig. 1I, J). These results suggest that ablation of CaBP-9k may contribute to the development of Alzheimer’s disease.
Ablation of CaBP-9k induces signs indicative of Parkinson’s disease in mice
To determine if CaBP-9k KO results in a loss of dopaminergic cells in the SNc and VTA, which characterize Parkinson’s disease [30, 31]. First, we assessed the intensity of α-synuclein immunofluorescence in TH-positive dopaminergic cells in SNc and VTA of young and old CaBP-9k KO mice and found increases of 56.65% in old CaBP-9k KO mice (relative intensity: wild-type, 100 ± 5.13%; CaBP-9k KO, 156.65 ± 12.11%) (Fig. 2A, B), and increases of 39.83% in young CaBP-9k KO mice (relative intensity: wild- type, 100 ± 5.13%; CaBP-9k KO, 156.65 ± 12.11%) (Supplementary Fig. 5B, C). Next, we examined TH immunostaining. We found that the numbers of TH-positive cells significantly decreased by 35.64% in CaBP-9k KO mice (wild-type, 715.71 ± 56.86 cells; CaBP-9k KO, 460.67 ± 58.55 cells/0.42 mm2), compared with that in the SNc and VTA of wild-type mice (Fig. 2C, D). Also, the decreases in the numbers of TH-positive cells suggest that the CaBP-9k KO mice may have abnormal expression of dopamine-related genes, such as those encoding Th, dopamine active transporter (Dat), dopadecarboxylase (Ddc), and glial cell line-derived neurotrophic factor (Gdnf). Real-time PCR analyses revealed that Th and Dat mRNA expression levels were decreased in the brains of CaBP-9k KO (relative expression for Th: wild-type, 100 ± 4.09%; CaBP-9k KO, 73.55 ± 6.96%; for Dat: wild-type, 100 ± 12.38%; CaBP-9k KO, 65.61 ± 4.78%). However, mRNA levels for genes encoding Ddc, Gdnf, brain-derived neurotrophic factor (Bdnf), dopamine receptor D1a (Drd1a) and dopamine receptor D2 (Drd2) were not altered by CaBP-9k KO (Fig. 2E). These results suggest that ablation of CaBP-9k may contribute to the development of Parkinson’s disease.
Ablation of CaBP-9k induces loss of neurons by ER stress-induced apoptosis and suppressed proliferation
CaBP-9k act as intracellular calcium buffers and calcium sensors [32]. Therefore, the ablation of CaBP-9k may induce ER stress in neurons, resulting in apoptosis. We found that the protein levels of GADD153/CHOP and cleaved caspase-12 and the phosphorylation of PERK, IRE1α and eLF2α were significantly higher in brain lysates from old CaBP-9k KO than in those from wild-type mice (Fig. 3A, B). Levels of apoptosis markers, namely, Bax, cleaved caspase-3 and -9 and PARP, and SAPK/JNK phosphorylation, were significantly increased in brain lysates from CaBP-9k KO compared with in those from wild-type mice, whereas the levels of Bcl-2 were significantly decreased (Fig. 3C, D). Moreover, greater numbers of hippocampal cells immunopositive for cleaved caspase-3 were observed in young and old CaBP-9k KO mice than in their wild-type counterparts (young: wild-type, 6.67 ± 0.88 cells; CaBP-9k KO, 32.33 ± 1.76 cells/0.42 mm2; old: wild-type, 8.50 ± 0.65 cells; CaBP-9k KO, 36.00 ± 1.78 cells/0.42 mm2) (Fig. 3E, F and Supplementary Fig. 4B, C). Consistent with these results, TUNEL staining showed more cells with DNA fragmentation in young and old CaBP-9k KO than in their wild-type counterparts (young: wild-type, 12.00 ± 1.32 cells; CaBP-9k KO, 33.33 ± 2.08 cells/0.42 mm2; old: wild-type, 18.75 ± 1.31 cells; CaBP-9k KO, 72.25 ± 3.68 cells/0.42 mm2) (Fig. 3G, H and Supplementary Fig. 4D, E).
Next, we then examined the proliferation of neural progenitors in the dentate gyrus, which is the source of neurons in the adult brain. The numbers of Ki67-immunostained proliferating cells were significantly lower in old CaBP-9k KO than in the wild types (wild-type, 8.50 ± 1.19 cells; CaBP-9k KO, 0.25 ± 0.25 cells/0.41 mm2) (Fig. 3I, J). In primary neuronal cultures, the percentages of Ki67- and BrdU-positive cells after a 24 h BrdU pulse were lower in cultures from CaBP-9k KO than in those from wild-type mice (% positive relative to wild-type for Ki67: wild-type, 100 ± 17.93%; CaBP-9k KO, 34.53 ± 10.54%; for BrdU: wild-type, 100 ± 15.94%; CaBP-9k KO, 35.32 ± 8.35%) (Fig. 3K-M). The effect of CaBP-9k ablation on neuronal proliferation was confirmed in cells transfected with pSuper_GFP/shCaBP-9k constructs (Supplementary Fig. 6). The numbers of proliferating shRNA-transfected cells (i.e., double-positive for GFP and BrdU) were reduced 92.91% for GFP/shCaBP-9k in comparison with control transfected cells (pSuper_GFP, 67.14 ± 9.38%; pSuper_GFP/shCaBP-9k, 4.76 ± 4.76%/0.09 mm2) (Fig. 3N, O). Conclusion, these data show that the loss of CaBP-9k results in a loss of neurons from ER stress-induced apoptosis and reduced proliferation.
CaBP-9k ablation disrupts calcium levels
The loss of CaBP-9k in neurons may result in abnormal calcium responses and calcium levels. We assessed calcium influx in primary neuronal cultures from CaBP-9k knockout mouse via Rhod-4 staining. Peak calcium concentrations in response to glutamate treatment were significantly higher in neurons from CaBP-9k KO mice (Fig. 4A, B). However, the mRNA levels of glutamate receptors were not different between wild-type and CaBP-9k KO brains (Supplementary Fig. 7D). Real-time PCR analyses revealed that transcript levels of calcium channel genes were mostly downregulated in the brains of old (Supplementary Fig. 7B), but not young (Supplementary Fig. 7A), CaBP-9k KO mice. These data suggest that CaBP-9k ablation impairs the regulation of intracellular calcium in neurons via abnormal calcium channel gene expression. We also found that knockdown of CaBP-9k induce an intracellular calcium concentration in Neuro2a cells (Fig. 4D, E). Conclusion, these results indicate that CaBP-9k are an important role in the regulation of intracellular calcium levels.
Abnormal memory and motor behaviour in CaBP-9k KO mice
We tested CaBP-9k KO mice for memory and motor behaviors that model clinical features of Alzheimer’s and Parkinson’s diseases [33]. In assessments of memory, old wild-type mice spent 19% more time approaching and remaining in the proximity of a novel object than the familiar object, whereas the CaBP-9k KO mice did not show any preference (Fig. 5A). However, the preference for a novel object was observed in young CaBP-9k KO mice (Supplementary Fig. 8A). In a passive avoidance conditioning test in old mice, the latency to enter a dark compartment was shorter for CaBP-9k KO mice than for wild-type mice (Fig. 5B), whereas there were no differences among young CaBP-9k KO mice (Supplementary Fig. 8B). During the acquisition phase of the Morris water maze test, old CaBP-9k KO mice exhibited longer escape latencies than wild-type mice. A difference between CaBP-9k KO mice emerged after 6 days of training (Fig. 5C). Escape latencies among young CaBP-9k KO mice did not differ in the training process (Supplementary Fig. 8C). In probe trials of the Morris water maze test, old CaBP-9k KO mice crossed the platform fewer times than the wild-types (Fig. 5D-F), whereas young CaBP-9k KO were no differences (Supplementary Fig. 8D). In assessments of motor function using cylinder test, rotarod and pole test, old CaBP-9k KO mice showed significant impairments in contralateral forelimb use and motor learning and increased turning time on a pole, respectively (Fig. 5G-I), but no other motor impairments were observed in young CaBP-9k KO mice (Supplementary Fig. 8G-I). Additional testing was performed to assess other behaviours. For example, old CaBP-9k KO mice showed deficits in nest building, a social behaviour that provides shelter essential for heat conservation and reproduction, which was assessed 24 h after the nesting material was placed in their home cages (Supplementary Fig. 9C, D). However, this deficit was not displayed when young CaBP-9 KO mice were tested (Supplementary Fig. 9A, B). In the open field and tail suspension tests for the evaluation of anxiety and depression, respectively, no differences between young and old CaBP-9k KO and wild-type mice were observed (Supplementary Fig. 9G-J, M and N). Conclusion, these results demonstrate that CaBP-9k ablation produces memory and motor impairments in mice at an old age.
TUDCA reverses brain pathology in CaBP-9k KO mice
TUDCA is a pharmacological ER stress inhibitor that protects cells from ER stress-related apoptosis induced by abnormal calcium levels [34]. We fed presymptomatic 5 months CaBP-9k KO mice a diet of standard laboratory chow supplemented with 0.4% TUDCA for 3 months to determine if we could eliminate the brain pathologies induced by CaBP-9k ablation. First, we assessed the accumulation of APP/β-amyloid and α-synuclein in the hippocampus. Interestingly, APP/β-amyloid immunofluorescence was not detected in the hippocampi of TUDCA-treated CaBP-9k KO mice (Fig. 6A). Moreover, no α-synuclein staining was observed in any of the TUDCA-treated CaBP-9k KO mice (Fig. 6B). Protein levels of BACE and Tau did not differ between wild-type and CaBP-9k KO mice treated with TUDCA (Fig. 6C, D). Also, intensity of α-synuclein immunofluorescence in dopaminergic neurons in SNc and VTA did not differ between TUDCA-treated CaBP-9k KO and wild-type mice (Fig. 6E, F). Western blotting analyses revealed that treatment of CaBP-9k KO mice with TUDCA completely restored their expression of ER stress- and apoptosis-related proteins to wild-type levels, whereas the levels of cleaved caspase-9 were significantly decreased (Fig. 6K-N). Additionally, treatment with TUDCA restored or increased the expression of genes encoding calcium channels in CaBP-9k KO mice (Supplementary Fig. 7C). These data suggest that TUDCA suppresses ER stress and eliminates signs of pathology in the brains of CaBP-9k KO mice.
TUDCA rescues abnormal behaviors in CaBP-9k KO mice
We next examined whether TUDCA treatment can also rescue the abnormal behaviors observed in CaBP-9k KO mice. In the novel object recognition test, TUDCA-treated wild-type and CaBP-9k KO mice spent more time investigating the novel object than the familiar one (Fig. 7A). In the passive avoidance conditioning test, the latencies to enter the dark compartment were now similar among CaBP-9k KO mice (Fig. 7B). In addition, TUDCA treatment eliminated differences in escape latencies between wild-type and CaBP-9k KO mice in the Morris water maze test, such that the groups were not different after 6 days of training (Fig. 7C). CaBP-9k KO mice treated with TUDCA also performed as well as wild-type mice on the probe trials of the Morris water maze task, with no differences in the platform crossing times or escape latencies (Fig. 7D-F). With regard to motor behaviours, TUDCA treatment reversed the effects of CaBP-9k ablation on contralateral forelimb use and on motor learning in the rotarod test. In the pole test, CaBP-9k KO mice treated with TUDCA exhibited similar turning times (Fig. 7G-I). TUDCA treatment also improved the nesting behaviour in CaBP-9k KO mice (Supplementary Fig. 9E, F), and no differences in the times spent in the centre area of an open field were seen among the mice (Supplementary Fig. 9K, L). Conclusion, these results show that TUDCA rescues impaired memory and motor behaviors in CaBP-9k KO mice.
Discussion
Calcium is involved in various developmental processes, such as cell differentiation, proliferation, and apoptosis [35-37] as well as neurotransmitter release and neuronal membrane excitability [38-40]. Essential factors for calcium homeostasis include CaBP-9k [41], which buffer increases in calcium levels [32], such as increases from 10-7 M in the resting cell to 10-5 M in the activated cell [42-44], and the ER, which regulates the uptake, storage, and mobilization of intracellular calcium [45]. Despite the link between altered calcium homeostasis and neurodegenerative disease, the contributions by calcium-binding proteins are not well understood. Here, we report that CaBP-9k KO mice results in Alzheimer’s and Parkinson’s disease-like pathologies and that associated effects and behavioural impairments are rescued by the inhibition of ER stress, and this study will provide critical information regarding the pathogenic mechanisms of neurodegenerative diseases including Alzheimer’s disease and Parkinson's disease.
ER stress induces neuronal cell death in Alzheimer’s and Parkinson’s diseases [46, 47]. The ER stress response regulates neuronal apoptosis [48] via the activation of GADD153/CHOP, PERK, IRE1α, eIF2α and cleaved caspase-12 [49]. This process is suppressed by calbindins upregulation [19], whereas knockdown of calbindins increases cell death [50]. The induction of CaBP-28k expression occurs during the neuronal activation with elevated cytosolic calcium [51]. Furthermore, the intracellular calcium is significantly elevated in hippocampal neurons at old rats (12–16 months) compared with young rats (4–5 months), which means that neurons need a lot of calcium buffer function to comes with age [52]. We observed significantly higher expression of ER stress-related proteins in brains from old CaBP-9k KO mice, which were accompanied by increases in apoptosis-related proteins. We also found that the intracellular calcium is elevated in the CaBP-9k KO brain. Thus, CaBP-9k KO plays an important role in protecting the brain from ER stress-induced apoptosis and thus may represent a crucial target in treating neurodegenerative diseases.
Genetic mouse models of Alzheimer’s and Parkinson’s diseases exhibit pathological features and impaired memory and motor behaviors consistent with those observed in patients [53-56]. One study found that five gene mutations, including CaBP-28k KO, associated with familial Alzheimer’s disease aggravate the disease pathogenesis at 6 months of age [57]. The number of CaBP-28k positive neurons was reduced in Alzheimer’s disease, amyotrophic lateral sclerosis disease (ALS) and dementia patients [58, 59]. Other studies of CaBP-28k KO mice revealed signs of ataxia resulting from abnormal calcium transients in Purkinje dendrites [60, 61] and impaired fear memory and social behaviour as well as reduced anxiety-like behaviour in the elevated plus maze [62]. CaBP-28k expression has been shown to protect against pathological processes in Parkinson’s disease [63, 64] and against the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in dopaminergic cells [65]. Although the types of neurons expressing CaBP-9k and CaBP-28k are different, functions may be similar in the brain. Our results are similar to those of these studies, revealing abnormal memory and motor behaviors in CaBP-9k KO mice. We suggested that CaBP-9k also likely contributes to the pathogeneses of Alzheimer’s and Parkinson’s diseases.
TUDCA is a water-soluble bile acid, and it is the taurine conjugate of ursodeoxycholic acid [66, 67]. TUDCA reduces liver damage, oxidative stress, and scarring of tissue in mice [68]. TUDCA can cross the blood brain barrier [69], and alleviates some symptoms of neurodegenerative diseases. For example, TUDCA significantly reduces apoptosis caused by APP/β-amyloid plaque accumulation in mouse brain [70] and rescues memory function in a mouse model of Alzheimer’s disease [71]. TUDCA was also shown to prevent cell death in rodent models of Parkinson’s disease [72-75]. Moreover, patients with amyotrophic lateral sclerosis that were treated with TUDCA had improved functional scores on measures of disability [76]. As TUDCA inhibits ER and oxidative stress [77, 78], we hypothesized that it would be beneficial in protecting the CaBP-9k KO mice from neurodegenerative disease-like pathology. Our results revealed that TUDCA reversed the alterations in memory and motor behaviors in CaBP-9k KO mice. These data suggest that pharmacological manipulation of ER stress or calcium homeostasis, such as with TUDCA, represents a potential intervention for symptoms of neurodegenerative disease.
Conclusion
This study is the first to demonstrate the role of CaBP-9k in the development of pathologies related to Alzheimer’s and Parkinson’s diseases. Of note, CaBP-9k represents critical regulators of calcium homeostasis, and the CaBP-9k KO mice described here represent a valuable animal model of neurodegenerative diseases. Furthermore, treatment with TUDCA can potentially prevent or slow neurodegenerative disease progression. Further research will focus on therapeutic strategies directly targeting drugs or hormones that regulate CaBP-9K expression (Fig. 8).
Abbreviations
CaBP-9k (calbindin-D9K); CaBP-28k (calbindin-D28K); ER (endoplasmic reticulum); APP (amyloid precursor protein); TUDCA (tauroursodeoxycholic acid); TH (tyrosine hydroxylase); BACE (beta-secretase); SNc (substantia nigra pars compacta); VTA (ventral tegmental area); Dat (dopamine active transporter); Ddc (dopadecarboxylase); Gfap (glial cell line-derived neurotrophic factor); ALS (amyotrophic lateral sclerosis disease); MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).
Acknowledgements
Author contributions
E-M. J and E-B. J conceived the study, designed, performed and analyzed the experiments, and wrote the paper. S. P, B-H. J, C. Aand Y-M. Y performed the experiments. E-J. H and W-Y. K revised the manuscript. E-B. J supervised the study.
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant of Korean government (MEST) (2017R1A2B2005031) to E-B. J and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1A6A3A11043046) to E-M. J. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Disclosure Statement
The authors have no conflicts of interest to declare.
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