Main Research Findings

1) In a mouse model of Alzheimer’s disease, ACP-105 was able to mediate amyloid-beta levels and cognitive deficits.

2) ACP-105 prevents age-related cognitive decline by enhancing rotarod performance and regulating MAP-2 immunoreactivity levels.

 

Selected Data

1) In order to investigate the effects of ACP-105 on cognitive deficits and amyloid-beta content, the research team of George et. Al utilized colonies of male homozygous 3xTg-AD and wild-type mice. The test subjects were established at Lund University in the Department of Experimental Medicine; all mice were provided food and water ad libitum and were maintained on a 12 hour light, 12 hour dark cycle. Once the mice reached 3 months of age, they were anesthetized using isoflurane gas and underwent gonadectomy to deplete endogenous testosterone levels. 10 mg/kg of ACP-105 was intraperitoneally injected either by itself or in combination with 10 mg/kg of the selective estrogen receptor beta1 agonist, AC-186 with 0.1 mg/day of DHT or a vehicle. Treatment took place 4 days a week over an experimental period of either 4 or 7 months [1].

The mice completed a Morris water maze assessment. The water maze consisted of a circular tank filled with water and opacified by painting the interior of the container white. Movement of the mice was recorded and analyzed with EthoVision 3.1 video-tracking software. The assessment was taken a step further and a hidden platform task was included. Both transgenic and wild-type test subjects were placed in the pool at one of four starting positions: NE, SE, SW, and NW, and were then required to locate the submerged platform in the NW quadrant. Each mouse was subject to 24 trials in total. Intervals between trials averaged approximately 24 minutes while the maximum trial length was 90 seconds; the mice were guided to the hidden platform if they did not find it after 90 seconds [1].

The time to reach the platform was recorded for each mouse. 6 days after the cue trials the platform was removed and the test subjects were subject to a recall task. On this day, all mice were placed in the NE quadrant of the pool while the researchers recorded the amount of time the subjects spent in the NW quadrant where the platform was previously located. Next, working memory was measured using a reversal learning protocol that included placing the mice in the water maze where the hidden platform had been re-installed in a new location. Additionally, vision tests took place by placing the platforms in a position visible above the water line, from there the mice were placed in the four corners and timed on how long it took to identify the newly raised platform.

Spontaneous alternation and spontaneous locomotor activity were the next variables assessed by the research team. Alternation was measured by recording the arm choices of the mice entering the arm. The subjects were then allowed to explore the maze for 8 minutes while their movement was monitored. Mice that made fewer than 10 arm choices were excluded from this portion of the study. The score of spontaneous alternation was calculated as the proportion of alternation to the total number of opportunities for alternation. Spontaneous locomotor behavior was assessed through an open field test. Individual test subjects were placed in a 42 cm x 42 cm box and the research team monitored behavior and activity for 10 minutes and analyzed using EthoVision 3.1 video-tracking software [1].

Anxiety in the mice was investigated by placing the mice in the middle of an opaque maze raised 30 cm off the floor; the maze included two open and two closed arms, each measuring 5 cm wide by 28.5 cm long and 20 cm high. The total session duration was 5 minutes for each mouse and the number of entries into each arm was recorded by the research team. Additionally, when examining rotarod activity, the mice were placed on a 4.5 cm rod facing the opposite direction to that of the rotation. Pretraining consisted of walking for 30 seconds on the rod turning at a speed of 5 RPM. Over a 4 minute period, four trials were conducted and the rotation speed gradually increased to 40 RPM. Total time spent on the rotarod was recorded and averaged by the researchers for each mouse [1].

After the specified treatment period had passed, the animals were euthanized and their brains were extracted and the hemispheres were dissected in half. The tissue was then subjected to Western blotting and ELISA analysis. Protein levels were also determined through the use of bicinchoninic acid assay. The resulting homogenates were mixed with an SDS-PAGE sample buffer, heated for 5 minutes, and electrophoresed. The tissue membranes were incubated with primary antibodies as well as HRP-conjugated secondary antibodies while signals were detected through the use of chemiluminescence. ELISA assays were also used to determine the levels of amyloid-beta 1-40 as well as amyloid-beta 1-42. Samples of the mouse brain hemispheres were homogenized in ice-cold PBS; all homogenates were then centrifuged for 20 minutes prior to quantification with ELISA.

2) The study conducted by Dayger et. Al utilized two-month-old C56B1/6J female mice in order to assess how ACP-105 affects rotarod functioning and cued fear conditioning. The mice were provided food and water ad libitum and were kept on a strict 12 hour light/12 hour dark schedule with the lights first coming on at 6 am. The test subjects were then anesthetized with ketamine and xylazine, followed by sham-irradiation or irradiation using a dose of 10 Gy in a Mark 1 Cesium Irradiator. Lead was used to shield the eyes, body, and cerebellum of the mice. 24 hours after irradiation or sham-irradiation, the mice were implanted with mini-pumps full of ACP-105 (1 mg/kg/day) or a vehicle treatment (1.09 mg/200 uL).

Behavioral testing began two weeks after irradiation, the mice were housed singly 3 days before testing was conducted in order to assess sensorimotor function of the rotarod during week 1. Living alone in the cage was not shown to affect any physiological indexes of stress or emotional behavior. The rotarod apparatus initially rotated at 4 RPM; every 15 seconds the rod accelerated by 15 RPM. Fall latency was recorded by a timer that stopped when the photobeams at the bottom of the chamber were broken by the mouse. Each test subject underwent three trials a day for three subsequent days [2].

Fear conditioning was tested during week 2 of the study. In order to stimulate fear conditioning the mice learned to associate the environment with an accompanying foot shock. On experimental day 1 the mice were placed in a conditioning chamber and allowed to habituate and explore for 2 minutes, followed by a 30 second tone and a 2 second foot shock. Two minutes later a second food shock was administered.

The next day the mice were placed in the same chamber without a loud tone or foot shock while hippocampus-dependent contextual freezing was monitored for 3 minutes. One hour later the test subjects were placed in a brand new conditional chamber with a different shape and odor. The mice were exposed to the fear conditioning tone for 3 minutes in order for the research team to assess hippocampus-independent cued fear conditioning [2].

After behavioral testing occurred the mice were euthanized while the brains were extracted and allowed to equilibrate in 30% sucrose overnight. Samples were then properly stored in order for future procession of MAP-2 and synaptophysin immunohistochemistry. Coronal brain sections were serially mounted on microscope slides, allowed to dry for 10 minutes and were then placed on a slide warmer for 45 minutes to promote adhesion to the slide. The slides were rehydrated and washed twice with PBS for 10 minutes at room temperature followed by a third wash with PBS containing 0.2% BSA + 0.2% Triton X-100 (PBT) for 15 minutes at room temperature.

Nonspecific binding was blocked with the application of 5% normal donkey serum and 1% fish skin gelatin. The slides were then incubated with mouse anti-synaptophysin or an anti-MAP-2 antibody overnight in a humidifying chamber. Slides were then washed and incubated with PBT several times followed by treatment with 10 mM CuSO4 in a 50 mM NH4OAc buffer and more washing with PBS. Anti-fade mounting media was then added to slides that were then sealed with enamel [2].

For each mouse 8 sections of the hippocampus were used while every 7th section was analyzed using an unbiased stereological approach. Immunoreactivity was assessed with an Olympus spinning disk confocal microscope in order to observe the dentate gyrus, CA1 and CA3 regions of the hippocampus, and the sensorimotor and entorhinal cortices. Each image was generated using a Z-stack which was then collapsed further into a single projection. The images were then analyzed for area occupied by MAP-2 and synaptophysin immunoreactivity through the use of Slidebook software [2].

 

Discussion

1) The results of the study conducted by George et. Al reported that gonadectomized 3xTg-AD mice treated with ACP-105 for 4 months displayed improved results in the Morris water maze, indicating that the SARM can enhance long-term spatial memory. The experimental and the control groups of mice were trained to find a hidden platform over the course of 6 days. There were no significant differences between the mice treated with ACP-105 versus the vehicle-treated animals. However, it is important to note that all mice learned the task by the sixth day. On day 7 the hidden platform was removed. The gonadectomized male 3xTg-AD treated with ACP-105 displayed increased frequency in platform crossings as well as more time spent in the platform zone [1].

In order to test working memory, the mice underwent a reversal learning trial where the mice were placed in the water maze after the hidden platform was moved to a new location. The mice were treated with ACP-105 or a vehicle and trained to identify the platform in its new location. No significant differences were seen between the SARM-treated and vehicle-treated animals. The swim speed was also analyzed to further control for possible confounds in movement sores, again, no significant differences were noticed between the experimental and control groups.

Figure: E) frequency of platform crossing in response to different treatment, F) time in platform zone in response to different treatments

This study also assessed the anxiety-like behavior in the male gonadectomized 3xTg-AD treated with ACP-105 through the use of an elevated plus maze. In the subjects treated with ACP-105 alone, anxious behavior was significantly reduced, however, there was no significant difference between the experimental and control groups in terms of time spent in the open arms of the maze. In the mice treated with both ACP-105 and AC-186, there was a significant increase in the percentage of total number of open arm entries compared to total arm entries [1].

Anxious behavior was also measured by assessing the frequency of entries into the closed arms of the elevated plus maze. Following treatment with ACP-105 anxious behavior was significantly reduced. Additionally, there was no dramatic difference between the treatment groups in terms of the total number of arms visited. This suggests that there was similar locomotor behavior between the treatment groups. Instances of stretching postures were correlated to stress and anxiety in the test subjects, following treatment, stretching behavior in the elevated maze was analyzed. However, results reported that there were no significant differences between treatment and control groups [1].

Figure: changes in A) time spent in open arms, B) % open entries/total entries, C) frequency in closed arms, D) total number of arm visits, E) stretching frequency in the center of the maze, in response to different treatments

Spontaneous locomotor activity was also linked to anxiety-like behavior in the test subjects. Locomotor activity was analyzed over the course of 10 minutes and did not exhibit significant changes between the treatment groups. Locomotor activity was increased over the first 2 minutes of the assessment and gradually decreasing suggesting habituation to the environment. To further assess anxiety in an open field, stretching frequency was analyzed and revealed a dramatic decrease in the mice treated with ACP-105. The amount of time the mice spent on the rotarod apparatus further confirmed that there were no locomotor impairments identified in any of the treatment groups. Overall, the results of the study found combination treatment with ACP-105 and AC-186 led to reduced anxiety, this claim was further supported by the observed decrease in stretch frequency [1].

The results of this study have also reported that both treatment with ACP-105 alone, and in combination with AC-186 led to significant differences in the frequency of entries to the closed arms of the maze. While there was a large jump from baseline, there was no significant difference between the treatment groups in terms of frequency of entries. This allowed the researchers to conclude that SARM treatment results in reduced anxiety in the elevated plus maze and the open field utilized in this study [1].

In addition to its effects on anxiety-like behavior, the research team of George et. Al hypothesized that ACP-105 has the potential to regulate levels of amyloid-beta, the deposition of which is highly associated with the development of Alzheimer’s Disease. The results of the ELISA assay performed suggests that treatment with ACP-105 and AC-186 leads to decreased levels of amyloid-beta-40 and amyloid-beta-42. The ratio between the two compounds was also significantly decreased after 7 months of treatment with the SARM, the same results were not observed after only 4 months of treatment. Immunohistochemistry was used to analyze amyloid-beta levels after 4 or 7 months of treatment with ACP-105. The assay revealed a decrease in amyloid beta/c99 immunoreactivity [1].

Figure: Levels of amyloid-beta 42 after four or seven months of different treatments.

2) The test subjects included in the study conducted by Dayger et. Al were first assessed for their sensorimotor functioning abilities using the rotarod apparatus. Vehicle-treated irradiated mice had lower fall latencies than the sham-irradiated mice treated with the vehicle. There was an observed day x irradiation interaction and an effect of irradiation exhibited in the vehicle-treated animals, however, these results were not seen when the test subjects were treated with ACP-105 [2].

Figure: rotarod performance of sham-irradiated versus irradiated animals treated with either a vehicle or ACP-105.

Fear conditioning in the mice was tested next. Neither ACP-105 or irradiation did not affect hippocampus-dependent contextual fear conditioning. The research team calculated % freezing each group: sham-irradiated, vehicle-treated mice: 41.51 ± 2.70, n = 7; sham-irradiated ACP-105-treated mice: 41.15 ± 4.39; irradiated, vehicle-treated mice: 38.81 ± 1.88, n = 8 mice; irradiated ACP-105-treated mice: 43.71 ± 4.40, n = 7 ACP-105-treated mice. Hippocampus-independent cued fear conditioning was affected by irradiation. Additionally, ACP-105 had the ability to enhance instances of freezing in both sham-irradiated and irradiated mice [2].

Figure: Cued fear conditioning in sham-irradiated and irradiated mice treated with either a vehicle or ACP-105.

MAP-2 and synaptophysin immunoreactivity was assessed in the CA1 and CA3 regions of the hippocampus as well as the sensorimotor and the entorhinal cortices. Results reported that there was an interaction observed between the cortex of the sham-irradiated mice and ACP-105. ACP-105 was found to reduce MAP-2 immunoreactivity in the sensorimotor cortex while MAP-2 immunoreactivity was slightly increased in the entorhinal cortex. In irradiated mice there was no interaction between ACP-105 and examined brain areas, as well as no effect of ACP-105 on MAP-2 immunoreactivity in the irradiated cortex or the irradiated and sham-irradiated hippocampus [2].

Additionally there was no interaction between brain area and ACP-105 or effect of ACP-105 on synaptophysin immunoreactivity. This was observed in the sham-irradiated and the irradiated cortex as well as the sham-irradiated and the irradiated regions of the hippocampus. Overall, the researchers were able to conclude that irradiation impaired sensorimotor function in mice treated with a vehicle compound, but not in the subjects treated with ACP-105. On the other hand, irradiation reduced fear conditioning in both sham-irradiated and irradiated mice but not in mice treated with ACP-105. Treatment with SARM also led to enhanced rotarod performance. This was further associated with reduced MAP-2 immunoreactivity in sham-irradiated mice. These results suggest that SARMs are capable of enhancing brain function after radiation [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] George S, Petit GH, Gouras GK, Brundin P, Olsson R. Nonsteroidal selective androgen receptor modulators and selective estrogen receptor β agonists moderate cognitive deficits and amyloid-β levels in a mouse model of Alzheimer’s disease. ACS Chem Neurosci. 2013 Dec 18;4(12):1537-48. doi: 10.1021/cn400133s. Epub 2013 Sep 25. PMID: 24020966; PMCID: PMC3867967.

[2] Dayger C, Villasana L, Pfankuch T, Davis M, Raber J. Effects of the SARM ACP-105 on rotarod performance and cued fear conditioning in sham-irradiated and irradiated female mice. Brain Res. 2011 Mar 24;1381:134-40. doi: 10.1016/j.brainres.2010.12.088. Epub 2011 Jan 8. PMID: 21219889; PMCID: PMC3048897.

 

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