CLSPCOL rescues Alzheimer’s disease mouse models

2.1 Mouse CLSPCOL

The amino acid sequence of the mouse CLSPCOL (mCLSPCOL: MSHGFTKEEVAEFQAAFNRFDKNKDGHISVEELGDVMKQLGKNLPEKDLKALISKLDTDGDGHPGHNGTPGRDGRDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEP) is composed of the N-terminal 61 amino acids of the mouse CLSP-1, CLSP(1–61), and the 60-amino-acid-long collagen-homologous region (48–107 amino acids) of the mouse adiponectin that is directly fused. The mouse CLSPCOL was chemically synthesized by BioSynthesis (Lewisville, TX, USA). Its purity was more than 85%. Mass spectrometry analysis using MALDI-TOF was performed to confirm the molecular weight of the product. It was dissolved in sterile Milli-Q water (Millipore, Bedford, MA, USA) for use.

2.2 Mouse handling and the administration of scopolamine and CLSP for memory behavior tests

All mice used in the current study were male. The number of mice and the age of mice used in the experiment are presented in each figure legend.

APP/PS1 mice (B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax, Cat# 005864, RRID:MMRRC_037568-JAX) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). They were bred and maintained at the Pre-clinical Research Center of Tokyo Medical University under specific pathogen-free conditions. They were housed in an air-conditioned room with a 12/12-h dark/light cycle and the start of light cycle at 8:00 am. After weaning at 1 month of age, the mice were genotyped using tail specimens and identified using ear-punched IDs. Only male mice were used for the experiments. Six mice of the same genotype were bred in cages. The same reagents were administered to all mice in each cage. The experimenters were not blinded to the genotypes of mice which they were handling. Mouse CLSPCOL in saline or saline without CLSPCOL was subcutaneously injected into the male APP/PS1 mice aged 12 months. As a control group, saline without CLSPCOL was subcutaneously injected into the male littermate wild-type (WT) mice aged 12 months. Five nanomol of mCLSPCOL were injected every other day for the first 13 days and every day for the last 16 days. The schedule for the subcutaneous injection of mouse CLSPCOL is illustrated in Figure S1.

For the treatment with scopolamine hydrobromide, male Jcl:ICR mice aged 10 weeks were purchased from Japan SLC Inc. (Shizuoka, Japan). Scopolamine hydrobromide (Cat. # S0929) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Scopolamine (1 mg/kg mouse) with 5 nanomol of CLSPCOL or saline was subcutaneously injected into each mouse at 1 h before the Y-maze test.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical University (No. H290026) and were conducted according to the Society’s Policies on the Use of Animals in Neuroscience Research.

2.3 A schedule of memory tests for APP/PS1 mice

Sample sizes were determined according to the previous study (Figure S1) [7]. Male APP/PS1 mice and their littermate WT mice at an age of 12 months were automatically allocated to groups by their identification numbers that were assigned when their genotype was determined. Experimenters were not blinded to mouse groups because they are involved in the handling of all breeding steps. The mice were subjected to the forward training session of the modified Morris water maze test on the 11th–13th days and the reverse training session on the 17th and 18th days [7]. After the water maze test was finished, spatial working memory performance was evaluated by monitoring spontaneous alternation (SA) behavior in the Y-maze test. The Y-maze test was carried out as described previously [11,12], on the 22nd–24th days. The modified fear conditioning test was performed on the 25th–30th days.

2.4 Modified Morris water maze test

Mice were placed in a water bath and trained to escape onto the platform in the pool for three consecutive days (11th–13th days; 18 male mice per each group) for the forward training session. The pool was 100 cm in diameter with opaque water at 22°C. The top surface of the hidden platform (10 cm in diameter) was placed 1 cm below the water surface. The time spent to find and escape onto the hidden platform was measured. The first trial was started at 9:30 am and three trial sessions per day were performed with an interval of about 30 min. In each training session, mice were allowed to swim until they found the platform or until 60 s had elapsed. Mice that failed to find the platform were guided to the platform and then allowed to remain there for 30 s. The platform was moved to a position opposite to that in the training period, and the reversal training was monitored for 2 days (17th and 18th days). The behavior of mice was recorded with a CCD camera (O’Hara & Co., Tokyo, Japan).

2.5 Fear conditioning test

This test consisted of two sessions: the conditioning and contextual sessions. During the conditioning session, the mice were placed in a chamber. They were supplied with a conditioned auditory stimulus and an unconditioned electric foot shock for several times. Freezing behavior emerges as fear to the insult. The freezing time during the conditioning session represents the levels of acquisition of fear-conditioned memory.

The conditioning chamber (length, 14 cm; width, 16 cm; height, 12 cm; O’Hara & Co., Tokyo) with a stainless-steel grid floor was installed in a soundproof box (200 lx, 55 dB white noise as a background noise). During the conditioning session, the mice were placed in the conditioning chamber and allowed to explore freely for 2 min. A tone of 65 dB white noise was sounded as the conditioned stimulus for 30 s, followed by a 2 s mild electric foot-shock (0.3 mA) as the unconditioned stimulus (a tone-shock pair). Two more tone-shock pairs were given at 2 min intervals, and their behaviors were monitored for 8 min (a conditioning session; Figure S2). Twenty-four hours after the conditioning session, the mice were placed again in the conditioning chamber and monitored for 5 min as a contextual session (Figure S2). During this session, they were exposed to the background noise without the unconditioned or conditioned stimulus. Photographs were taken every half second using a CCD camera. Freezing movements were automatically judged to occur by a recording and analyzing software (O’Hara & Co.), based on the movements of a mouse in two consecutive pictures.

2.6 Y-maze test

Briefly, the Y-maze consisted of three intersecting arms made of black acrylic panels. Each arm was 40 cm long, with a trapezoidal cross section consisting of a 10 cm top, a 3 cm bottom, and a 12 cm height. Each mouse was placed at the end of one arm and allowed to explore the Y-maze freely for 8 min, and the series of arm entries were recorded to evaluate short-term spatial working memory based on spontanous alteration (SA) behavior. The mice were considered to have chosen an arm when their entire torsos had entered the arm completely. SA behavior is based on the behavior of animals in preferring to choose newer locations. When the mice chose a third arm different from the previous two among the three consecutive arm choices in the Y-maze, the mice were considered to have carried out actual alternation. SA (%) was calculated as the percentage of actual alternations out of the maximum alternations that were defined as the total number of arm entries minus two. For example, if a mouse made 10 entries in an order such as arm A → B → C → B → A → B → C → B → A → C, the actual and maximum alternations would be 5 and 8, respectively, and alternation behavior would be 62.5%. The arm entries were also counted.

2.7 Inclusion and exclusion criteria

To exclude the effect of the sexual cycle on the results, only male mice were used for the experiments. If the mice did not move at all, they were excluded from analysis in the Morris water maze test. During the Y-maze test, if the number of the arm entries of a mouse was less than six, it was excluded from the analysis.

2.8 Preparation of interstitial fluid (ISF)-containing brain samples after the subcutaneous injection of CLSPCOL to mice

Some APP/PS1 mice aged 16 months received subcutaneous injection of 5 nanomol of CLSPCOL or saline every day for 14 days. Some Jcl:ICR mice aged 8 weeks received single subcutaneous injection of 5 nanomol of CLSPCOL. At times after the last injection of CLSPCOL, they were anesthetized with isoflurane (Wako Pure Chemicals, Tokyo, Japan). Blood was then aspirated from hearts and centrifuged at 4,000×g for 10 min at 4°C. The vascular space of the brain was washed free of blood by the perfusing 20 mL of ice-cold lactated-Ringer’s solution (Otsuka Pharmaceutical, Tokyo, Japan) through the left ventricle of the heart. Subsequently, the mice were decapitated and the brains were removed. The whole brain was once washed with the lactated-Ringer’s solution, to clear the contamination of CSF. The cortex was then separated by homogenizing with two-fold weight of saline. After the homogenate was centrifuged at 4,000×g for 10 min at 4°C, the supernatant was collected as the three-times diluted ISF-containing brain sample. The ISF-containing samples and sera were subjected to ELISA analysis.

3.1 Statistical analysis

All data were analyzed using Prism8 for Mac OSX (GraphPad, San Diego, USA). The data are shown as mean ± standard error of the mean (SEM). The statistical analysis was performed using two-way ANOVA, followed by post hoc Tukey’s multiple comparison tests for water maze and fear conditioning tests. For analysis of the Y-maze test non-parametric Kruskal–Wallis test, followed by Dunn analysis as a post-hoc test, was used. If the p-value was less than 0.05, it was considered statistically significant.

4.1 CLSPCOL injection corrects the impaired fear-conditioned memory

Using APP/PS1 mice aged 12 months, we first performed the modified Morris water maze test, according to the previous report [7]. Memory performance was already impaired in the APP/PS1 mice at this age (Figure 1 and Figure S3). Although the treatment of CLSPCOL showed the inclination to improve the memory performance, it did not improve it in a statistically significant fashion. We then performed the Y-maze test. The results indicated that the short-term working memory did not deteriorate in the APP/PS1 mice significantly (Figure S4). Consequently, we were unable to test the effect of the CLSPCOL treatment on the short-term working memory, although it tended to improve the memory performance in APP/PS1 mice (Figure S4).

We next examined the effect of the CLSPCOL treatment on the fear-conditioned memory, using a modified fear conditioning test. During the contextual session, the fear memory is recalled based on the conditioning environment. The APP/PS1 mice at this age had impaired fear-conditioned memory during both the conditioning and contextual sessions (Figure 2). The CLSPCOL treatment corrected the acquisition of fear-conditioned memory during the conditioning stage almost completely. Although it showed the inclination to correct the impairment of memory recalling during the contextual session, it did not correct it in a statistically significant manner (Figure 2).

Figure 1

4.2 A single subcutaneous injection of CLSPCOL rescues scopolamine-induced short-term working memory

Scopolamine is an M2 muscarinic acetylcholine receptor antagonist. A single injection of scopolamine causes memory impairment in mice by disturbing cholinergic neurosignals. Previous studies showed that an intracerebroventricular or intraperitoneal injection of humanin or CLSP rescued scopolamine-induced memory impairment [11,12]. It is likely that CLSP antagonizes scopolamine by potentiating cholinergic neurotransmission via the htHNR [12,13]. In the current study, we found that a single subcutaneous injection of 5 nanomol of CLSPCOL suppressed scopolamine-induced impairment of short-term working memory, using the Y-maze test (Figure 3).

Figure 2

4.3 Measurements of CLSPCOL concentrations in ISFs and sera

Using ELISA assay for CLSPCOL concentrations, we first measured the steady-state trough CLSPCOL concentrations in ISFs and sera of APP/PS1 mice aged 16 months. They received the subcutaneous injection of 5 nanomol of CLSPCOL every day for 14 days. The mean trough CLSPCOL concentration (3.067 nM) in ISFs of the mice was far more than the concentrations (0.1 nM) that are required for the complete inhibition of AD-related neuronal cell death in vitro (Figure 4a).

Figure 3

We also examined the time course of the ISF and serum concentrations of CLSPCOL after the single subcutaneous injection of 5 nanomol of CLSPCOL to Jcl:ICR mice aged 8 weeks. The mean CLSPCOL concentrations in ISFs of the mice within 24 h after the single subcutaneous injection were more than the concentrations (0.1 nM) that are required for the complete inhibition of AD-related neuronal cell death in vitro (Figure 4b).