Ex-vivo analysis platforms for amyloid precursor protein cleavage

Yuji Kamikubo (  ykamiku@juntendo.ac.jp ) Juntendo University: Juntendo Daigaku https://orcid.org/0000-0003-1985-5212 Hao Jin Juntendo University Hongo Campus: Juntendo Daigaku Kazue Niisato Juntendo University Hongo Campus: Juntendo Daigaku Yoshie Hashimoto Juntendo University Hongo Campus: Juntendo Daigaku Nobumasa Takasugi Juntendo University Hongo Campus: Juntendo Daigaku Takashi Sakurai Juntendo Daigaku Hongo Campus: Juntendo Daigaku


Introduction
Organotypic brain culture, including slice culture are used in central nervous system research because they have advantages over both in vivo and in vitro platforms [1,2]. Cultured brain slices preserve tissue structure, neural circuit, and extracellular environment, and replicate the native status of the in vivo context. The slice cultures from hippocampi are widely used to investigate the effect of pharmacological, physiological, and genetic manipulation on neurons and glia [3,4]. Three typical methods are used to maintain brain slices in vitro, including roller-tube, membrane-interface, and matrix embedded culture [5].
Membrane-interface neural tissue culture was improved for practical use by Stoppini et al. [6]. Since lter membrane cup is designed to easily change the medium, this apparatus as an interface is convenient for long-term maintenance of the explant tissue. The cultured hippocampal slices on the lter membrane cup are amenable for completely replacing of culture medium. Previously, we reported about neural circuit, synaptic plasticity, development, and Alzheimer's disease (AD) using cultured hippocampal slices [7][8][9].
AD is a chronic neurodegenerative brain disorder that usually starts slowly and worsens over a long period of time, and the most common cause of dementia in the elderly. Numerous genetic and biochemical studies support the hypothesis that an excessive accumulation of amyloid β peptide (Aβ) results in aggregation and amyloid deposition in the brains of AD patients (amyloid hypothesis) [10]. Aβ derive from the amyloid precursor protein (APP), which an integral type I membrane protein expressed in many tissues and concentrated in the synapses. APP is commonly cleaved by membrane protease in the secretase family, α-, β-and γ-secretase. Aβ is generated by sequential cleavage by β-secretase (BACE1) [11] and γ-secretase, whereas alternative cleavage by α-secretase precludes Aβ production [12]. Neuronal activity is known as an important regulator of β cleavage of APP [13,14]. Regulation of cleavage, accumulation and elimination of Aβ are explored as a target for therapy and prevention of Alzheimer's disease (AD).
Large amount of data from cell line model-based assay revealed the molecular mechanism of Aβ secretion to understand AD [10]. Although cell line models are powerful tool for molecular biological studies, discordances are observed between cell line and animal models. Because neuronal and glial cell are morphologically and functionally specialized, dissociated primary cultures of neuronal tissue have an advantage to reveal the pathological processes and molecular mechanisms of neuropsychiatric disorder. Although experiments using cultured neuron provide important information, they have lost the tissue architecture of the brain. Neural circuits, glial network, and the extracellular environment are important for neurodevelopment and neurodegenerative disorders, including AD, schizophrenia, and autism spectrum disorder [11,[15][16][17]. The animal model, however, have disadvantages; they need lengthy animal experiments and substantial cost, and laborious monitoring of multiple parameters following manipulations. Furthermore, genetic engineered animals might be affected by compensatory alterations and developmental changes [18].
Organotypic culture is a platform amenable to simultaneous and continuous analysis of Aβ secretion and neural function.
observation and experimental manipulation is required.
For the above reason, the hippocampal slice culture may be a potential technique linking between animal model and neural cell.
The hippocampus is thought to be one of the rst regions of the brain to suffer damage in AD [19]. In this report, we describe methods of organotypic hippocampal slice culture and consecutive analysis of Aβ and related products, which plays a central role in the pathogenesis of AD. Continuous collection and analysis of Aβ is a powerful approach to elucidate the cellular and molecular mechanism underlying AD. To develop and evaluate therapeutic agents and approach for AD, long term and consecutive analysis are also required. Here we provide the standard experimental conditions for analyzing of Aβ production.

Hippocampal slice culture method
The experiments in this study were approved by the committees on animal experiments of Juntendo University. Animals were purchased from Nihon SLC (Hamamatsu, Japan) and treated according to our institutional guidelines. Rat or mouse hippocampal slice cultures were prepared following protocol. Whole brains were excised from anaesthetized Sprague-Dawley rats or ICR mice at postnatal day 7-8 and the hippocampi were isolated. Slices of 400-µm thickness were obtained from the central region of the hippocampi, using a tissue chopper (McIllwain). The slices were placed on a polytetra uoroethylene membrane lter (Millicell-CM, Millipore,), and culture medium (see below for composition) was added up to the bottom surface of the lter. These prepared cultures were maintained at 37˚C with 5% CO 2 -entiched humidi ed atmosphere. The slice culture medium (SCM) contained 50% minimal essential medium based on Hanks' salts (Nacalai), 25% Hank's balanced salt solution (Nacalai), and 25% heat-inactivated horse serum (Life Technologies, Gaithersburg, MD). The culture medium was replaced twice a week with fresh medium during the entire culture period. 3. Prepare the tissue chopper placing on clean bench and mounting a sterilized blade, and placing autoclaved overhead projector (OHP) sheet, cut into 5 cm squares, on cutting chamber. 4. Prepare six well plates. Add 1 mL slice culture media (SCM) per well and place culture inserts in each well. Make sure the lter membranes are thoroughly wet with no bubbles underneath. Place the plates with lter membrane cups maintained at 37˚C with 5% CO 2 -entiched humidi ed atmosphere until needed. 5. The rodent pups are deeply anaesthetized and then killed by decapitation. Cut the scalp and expose the skull. Remove the skull by cutting along the sagittal suture and then from rostral to caudal side. Scoop out the brain quickly with a micro medicine spoon and place it in ice-cold oxygenated GBSS. Place the brain on autoclaved lter paper immersed in ice-cold oxygenated GBSS on 100mm dish. Use a scalpel to separate the hemispheres leaving out the midbrain.
6. Under the stereomicroscope; after the gently scoop the thalamus, the hippocampi are then exposed on each hemisphere in ice-cold oxygenated GBSS. Then gently scoop the hippocampus out with cortex using micro spoon (Fig. 1C). 7. Transfer the hippocampus to the OHP sheet on the tissue chopper chamber. Align the hippocampi perpendicular to the blade to obtain coronal sections and drain excess of GBSS (Fig. 1D).
8. Slice the hippocampi every 400 µm using tissue chopper. 9. Transfer sliced hippocampi from the OHP sheet to 60 mm dish lled with ice-cold oxygenated GBSS.
Since tissue chopper and hippocampi are placed at room temperature, quick operation is required in steps 6 to 8. 10. Under the stereomicroscope (Olympus, SZX7); separate well de ned and undamaged slices from damaged slices, and pinch the cortex with precision tweezers and divide the hippocampal slices one by one. And then split into cortex and hippocampus by scalpels (Fig. 1E). It is important to complete the steps 5 to 10 within 45 minutes. 11. Incubate separated hippocampi in new ice-cold oxygenated GBSS for 30-60 min.
12. Transfer individual slices on to the lter membrane in the six-well plate with SCM. Place 1-6 slices per membrane, not to place the slices either close to the insert wall or close to each other (Fig. 1F).
13. Move dish back to incubator and prepared slices maintained at 37˚C with 5% CO 2 -entiched humidi ed atmosphere.
14. The culture medium was replaced twice a week with fresh medium during the entire culture period. Aspirate the medium in dish, and add 750µL of fresh pre-warmed medium per well. Approximate 250µL of medium would be remained in the culture dish and the lter membrane.

Immunohistochemical staining
For the immunohistochemical staining of presynaptic structures, the cultured slices were xed with 4% paraformaldehyde in phosphate-buffered saline for 1 h at 4˚C. The xed preparations were rinsed with phosphate-buffered saline (PBS) and then treated with PBS containing 0.1% Triton X-100 and 5% fetal bovine or horse serum at 24˚C for 30 min. The treated slices were incubated with a primary antibody against NeuN The primary antibody treated slices were incubated with a secondary antibody conjugated with Alexa Fluor 488 or 594 at 24˚C for 2-2.5 h. The slices were examined using a Leica SP5/TCS confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) or an Olympus IX71 (Olympus, Tokyo, Japan) with Orca-ER cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan).

Electrophoresis and Immunoblotting
Acute or cultured hippocampal slices were solubilized in ice-cold RIPA buffer with a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany). Samples were resolved by SDS-PAGE and transferred onto polyvinylidene uoride membranes (Immobilon-P, Millipore). Membranes were incubated in 5% skim milk in TBST at 25 ˚C for 30-60 min to block nonspeci c binding. Membranes were incubated at 4 ˚C overnight or 25 ˚C for 2 h with primary antibodies against GFAP (BioLegend), Iba1 (Wako Pure Chemical Industries), anti-galectin-3 (mouse monoclonal, A3A12, Santa Cruz, CA, USA), or anti-β-actin (a loading control, mouse monoclonal; Wako Pure Chemical Industries). After four changes of 1× TBST and three 5-min washes at 25 ˚C, membranes were incubated in horseradish peroxidase-conjugated secondary antibodies. After four washes at 25 ˚C, membranes were incubated with ECL solution (Thermo Fisher Scienti c, MA, USA). For quanti cation, chemiluminescence light signals in Super Signal Dura substrate (Thermo Fisher Scienti c) were captured by a cooled charge-coupled device camera system (LAS-3000plus; Fuji Photo Film Company, Kanagawa, Japan) that ensured wide ranges of linearity. Densitometric quanti cation of synaptic protein expression normalized to GAPDH or β-actin. The protein content was estimated using bicinchoninic acid (BCA) reagent (Thermo Fisher Scienti c).
The levels of Aβ 40 were measured using a solid phase by sandwich ELISA kit following the supplier's information (IBL, Gunma, Japan). The test samples and standards were added to the 96 well plate (precoated with anti-human Aβ (35−40) (1A10) mouse IgG) and incubated overnight at 4˚C. After several washes with EIA wash buffer, 100 µL of HRP-labeled anti-Aβ (1−16) rabbit IgG solution was added to each well (except the wells corresponding to reagent blanks) and incubated at 4˚C for 1 h. After a thorough wash, 100 µL of tetramethylbenzidine solution was added to each well and incubated in the dark at 24˚C for 30 min. The reaction was stopped by adding 100 µL of 1N sulfuric acid, and the readings were taken at 450 nm. The levels of Aβ in the test samples were calculated by incorporating the unknown values into the standard curve obtained in the assay. The levels of soluble amyloid precursor protein β (Aβ 42 ) were measured using the IBL assay kit (IBL, 96 well plate precoated with anti-Aβ (38−42) rabbit IgG, HRP-labeled anti-Aβ (1−16) rabbit IgG Fab) as described above. The levels of soluble APPα and APPβ were measured using the assay kit (IBL, 96 well plate precoated with anti-mouse APP(599) rabbit IgG, HRP-labeled antimouse N-APP rabbit IgG Fab) or (IBL, 96 well plate precoated with anti-mouse sAPPβ-w rabbit IgG, HRPlabeled anti-mN-APP rabbit IgG Fab) as described above.

Statistical analysis
All quantitative data were presented as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical analysis was performed using either Student's t-test (two-group comparison) or ANOVA (more than two groups) followed by post hoc comparison. Statistical analyses were performed with Prism 5.0 (GraphPad Software, Inc., CA, USA). The level of signi cance was indicated by asterisks: *P < 0.05, **P < 0.01,***P < 0.001 3. Results

Chronological analysis of cultured hippocampal slices
Previously, we have reported that cultured rat hippocampal slices retained neuronal activity for several weeks [7,20]. Furthermore, we recently showed reorganization and maturation of synapses occurred in cultured hippocampal slices [9]. In this report, we investigated morphological feature of neuronal and glial cell in cultured hippocampal slice ( Fig. 2A-C). Glial brillary acidic protein (GFAP) is the hallmark intermediate lament protein in astrocytes [21]. Ionized calcium-binding adaptor protein-1 (Iba1) is a microglial and macrophage-speci c protein [22]. Next, to quantitatively evaluate the existence of neural cells, we assessed total protein contained in organotypic hippocampal slice cultures by Micro BCA Protein Assay Kit. These data indicate that neural cells remained in cultured hippocampal slices for over 12 weeks (Fig. 2D). Furthermore, we assessed the amount of astrocytes and microglia using immunoblotting ( Fig. 2E-H). Astrocytes and microglia increased 1 day after plating on lter membrane cups. Galectin-3, which required for microglia activation in injured brain [23], was elevated for 1 week after preparation and then declined.

Alternative cleavage of APP
Previously, reorganization and maturation of neuron were reported in cultured hippocampal slices [24,25].We evaluated the expression level of BACE1, and APP by immunoblotting assays for several weeks. Our data indicated that expression of BACE1 and APP were stable after two weeks (Fig. 3A-C).
APP is alternatively cleaved by the members of the α or β secretase family. The α-secretases are members of the ADAM family, and considered to be a part of the non-amyloidogenic pathway in APP processing [26,27]. We assessed enzyme dependencies on APP metabolism in hippocampal slice culture, using each secretase inhibitor. We cultured hippocampal slices with fresh SCM containing vehicle (DMSO) or inhibitors after washing three times with 1 mL fresh SCM for 24 h. In accordance with previous reports [28], treatment of secretase inhibitors increased immature and mature APP as a result of reduction of APP cleavage. Treatment with both inhibitors blocked almost all the rst step of APP cleavage, further increasing the amount of APP full-length APP (Fig. 3D-G).

Biochemical analysis of production of Aβ
Since organotypic cultures are arti cial environments, we are able to control extracellular conditions including temperature, oxygen content, nutritional factor, and neural activity. To elucidate the optimal condition for analysis of Aβ secretion, we examined the effect of the number of slices on lter membrane cup and incubation period. Because reducing the number of slices in a cup allows for more e cient experiments, we plated on 1 to 6 slices on a lter membrane cup for culture, and cultured slices were incubated with 1mL fresh SCM for 24h. We measured Aβ in SCM from cultured hippocampal slices with a two-site ELISA assay (see materials and methods). For quantitative analysis, we collected SCM and performed Aβ 40 assay. In accordance with previous reports, single hippocampal slice secreted 75-120 pg Aβ 40 for 24h (Fig. 4A). Further analysis showed that Aβ 40 secretion per total protein amount is 1.7 to 2.8 pg/µg for 24h (Fig. 4B, C). For assessing incubation time for Aβ 40 assay, we cultured four hippocampal slices for 1 to 90 h with fresh SCM after washing three times with 1mL fresh SCM. Our time-lapse analysis of Aβ secretion showed that the secretion amount increased linearly up to 90h, and single hippocampal slice secreted 2.68 ± 0.82 pg for 1h (Fig. 4D, E).

Chronological analysis of production of Aβ
We investigated whether or not the amount of Aβ secretion changed depending on the culture period. To quantify Aβ secretion levels, we collected SCM cultured hippocampal slices (1-58 DIV) for 24 h after washing three times with 1 mL fresh SCM. We previously reported that secretion of sAPPβ was between 3 to 6 ng/mL, and secretion of Aβ 40 was between 200 to 300 pg/mL during the monitoring period [9]. The Aβ 40 and Aβ 42 are the most common isoform of Aβ in brain and the main component of the amyloid plaques found in the brains of AD. Here we measure the Aβ 42 in same SCM preparation using a two-site ELISA assay. Secretion of Aβ 42 was 23 to 50 pg, and ratio of Aβ 42 to Aβ 40 was 10 to 17 % during the monitoring period (Fig. 4F, S1).

Comparison of species differences in APP cleavage
In order to apply hippocampal slice culture to various research elds, we assessed the amount of Aβ secretion by comparing rat and mouse. The amount of Aβ 40 secretion form for cultured mice hippocampal slice was 160 ± 15 pg/mL (Fig. 5A). Although Aβ secreted from mouse hippocampus slices were less than that of rats (350 ± 65 pg/mL), there was no signi cant difference in Aβ secretion compared to the total protein amount of slices (mice: 1.75 ± 0.3 pg/µg, rats: 1.86 ± 0.3 pg/µg, Fig. 5B, C). APP is commonly cleaved by α-or β-secretase both remove and release nearly the entire extracellular domain which called soluble APP (sAPPα, and sAPPβ). This alternative cleavage is critical for revealing the molecular mechanism of AD pathology. We revealed that both sAPPα and sAPPβ are secreted from hippocampal slice cultures by sAPP assay. The amount of secretion of sAPPα and sAPPβ from four cultured rat hippocampal slices were 1.30 ± 0.21 ng and 0.63 ± 0.18 ng. The amount of secretion of sAPPα and sAPPβ from four cultured mouse hippocampal slices were 0.69 ± 0.09 ng and 0.44 ± 0.03 ng (Fig. 5D, E). These results indicated that α cleavage was dominant as in the model animal.

Discussion
In this report, we showed a method for preparation of hippocampal slice culture and consecutive analysis of Aβ. The cultured hippocampal slice preserved tissue architecture, neural circuits, and synaptic dynamics [6, 24,25]. Here, we showed that the existence of microglia, astrocyte, and neuron in a cultured slice for several weeks or potentially several months (Fig. 2). Our chronological immunoblotting analysis indicated that glial cells have proliferated for 1 to 2 weeks, and then be maintained for several weeks. Besides, galectin-3, which required for microglia activation and proliferation in injured brain [23], was elevated for1 week after preparation and then declined (Fig. 2H). The microglia were activated for scavenging dead or apoptotic cells. Quantitative immunoblotting analyses suggested that expression levels of BACE1 and APP were not changed signi cantly after 2 weeks. The expression levels of synapse marker proteins, synaptophysin and PSD95 decreased from 1 to 4 days, and increased after 7 days [9]. These results provide that slices cultured after 2 or 3 weeks are suitable for analysis of neural functions.
Aβ is formed after sequential cleavage of the APP by β-and γ-secretase, accumulates in the central nervous system and subsequently initiates the neural dysfunction. An excessive accumulation of Aβ results in aggregation and amyloid deposition in the brains of AD patients [29]. The γ secretase cleaves within the transmembrane region of APP and can generate a number of isoforms of 30-51 amino acid residues in length [30]. The Aβ 40 and Aβ 42 are the most common isoform of Aβ in brain and the main component of the amyloid plaques found in the brains of AD. The Aβ 40 form is the more common of the two, but Aβ 42 is the more brillogenic and is thus associated with disease states [31].
Since the pathological changes of AD are progressing slowly, a model for continuously analyzing central nervous function for a long period is required. In this report, we evaluated the method to investigate Aβ formation continuously using cultured hippocampal slices. To estimate Aβ production, we measured Aβ 40 and Aβ 42 secreted from hippocampal slices using a two-site ELISA assay. In culture medium, four cultured hippocampal slices secreted 200-400 pg Aβ 40 and 23-50 pg for 24 h [9]. These data indicated that 2.0-4.0 pg Aβ 40 and 0.2-0.5 pg Aβ 42 were secreted by a cultured hippocampal slice in 1 h. In accordance with previous reports [13], the ratio of Aβ 42 to Aβ 40 was between 10 to 17 % (Fig. 4). As a result of examining the differences between species, there was no difference between the amount of Aβ 40 and sAPP secretion in mouse and rat hippocampal slices (Fig. 5).
Here we tried to optimize the incubation period and the amount of slices for analysis of Aβ. We placed 1 to 6 hippocampal slices on a membrane cup for one week culture, and incubate these slices for 24h with fresh SCM for Aβ assay. The concentration of Aβ linearly increases corresponding to the number of slices (Fig. 4). For time-lapse analysis of Aβ secretion, we cultured four hippocampal slices and incubate for 1 to 90 h with fresh SCM after washing three times with 1 mL fresh SCM. Our results indicate that single hippocampal slice secreted 2.68 ± 0.82 pg for 1h (Fig. 4B, C). The dispersion of Aβ assay indicates that the carryover medium signi cantly impacts the measurement results with short term incubation. When many slices are placed on a membrane cup, it is easy to analysis of Aβ production, but proper placement of slices is di cult. We suggest that it is appropriate to incubate two to six hippocampal slices in a membrane cup.
The α-secretase family alternatively cleaves the APP within the Aβ sequence. Thus, α-cleavage precludes Aβ formation and is considered to be part of the non-amyloidogenic pathway in APP processing [32]. In cultured hippocampal slice, alternative cleavage of APP is conserved (Fig. 3), and thus application of the α-secretase inhibitor increased secretion of Aβ [9].
Synaptic plasticity is activity dependent changes in synaptic function that is thought to play a crucial role in learning and memory [33]. Two forms of long-term plasticity, long-term depression (LTD) and long-term potentiation (LTP) are involved in long term memory and induce in acute brain slice. In cultured hippocampal slice, the synaptic plasticity was occurred [25], and synapse formation or elimination is induced by repetitive induction of LTP or LTD [7,8]. Because synaptic plasticity is disturbed in neuropsychiatric disorder model including AD [34,35], rodent hippocampal slice culture is a good in vitro experimental model for pathological changes in the central nervous system. Here we showed no difference in Aβ secretion between mouse and rat hippocampal slice culture (Fig. 5).
Because of individual differences and di culty of manipulation in animal models, comparative analyses before and after administration in same preparation are not easy. To evaluate drug e cacy, however, this comparative analysis between before and after administration in same cultured preparation is meaningful. Because primary dissociated neural cultures are vulnerable to drug washout during media changes, comparative analysis using the same preparation is extremely di cult in actuality. Filter membrane cups allow easy handling of preparations; therefore, we were able to collect and change culture media without damage and perform comparative analyses using the same preparations (Fig. 4).
Because the membrane cup is designed for disposable use, slice culture is a costly method. Using selfmade devices and lter membranes can reduce costs [36].
Slice culture are usually derived from early postnatal or embryonic rodents. Because essential cytoarchitecture are already established but neural circuits are still immature, early postnatal periods (day 0 to 10) are ideally suited for culturing. Some attempts have been made to culture adult tissue of model animal for elucidating age-related neurodegenerative diseases [37].
Slice culture have been used for wide research eld ranging from physiology, pharmacology, endocrinology, biochemistry and development to pathology. We anticipate that hippocampal slice culture will be used to reveal molecular and cellular basis of neuropsychiatric disorder as ex vivo model.

Conclusions
We provide a method of consecutive analysis protein secretion from brain slices to study neurodegenerative disease. Hippocampal slice culture is an experimental system that effectively keeps the in vivo neuronal network and seems to be a convenient method to study the cellular and molecular mechanism underlying the neuropsychiatric disorders including AD, and to evaluate therapeutic approach for such diseases.

Declarations
Ethics approval and consent to participate: All the biosafety and approvals for the ethical and humane use of animals were obtained prior to the start of the study. All procedures performed in this study were in accordance with the ethical standards of Juntendo University. This study did not involve human participants.  The ratio of Aβ40 to total slice protein.