Sex-dependent effects of ultra-low-dose-THC preventive treatment on neuroinflammation and cognitive decline in 5xFAD mice methods
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Main text
Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat some of the cognitive and emotional symptoms. In the past two to three years, new drugs have been approved for Alzheimer's disease, including symptomatic treatments like Benzgalantamine (a cholinesterase inhibitor, FDA-approved in July 2024) and disease-modifying therapies like Lecanemab and Donanemab (anti-amyloid monoclonal antibodies, FDA-approved in July 2023 and July 2024, respectively) [ ]. While these therapies mark significant progress, they do not prevent the disease. The distinction between slowing progression and true prevention is crucial, as no existing treatment can halt the pathological processes before they begin. Thus, while there have been advances in treating AD, there is still no way to...
Main text
In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hindered by the harmful effects of high doses of chronic THC use [ ]. One strategy that has been suggested to tackle this issue is to use an ultra-low dose of THC (ULD-THC) [ - ]. Indeed, we have recently shown that a single dose of ULD-THC has a beneficial effect on age-related cognitive decline in old female mice [ ] and alleviated AD-related cognitive impairments in the 5xFAD AD mice model [ ]. However, the potential of cannabinoids in the prevention of AD is still unexplored. The primary objective of this study was to evaluate the effects of preventive treatment of...
Main text
Building on prior findings of memory improvement following ULD-THC treatment after disease onset [ ], this study employs a preventive regimen to explore sex-specific molecular and behavioral responses to ULD-THC administered at earlier stages of AD pathology. We investigate here the preventive effects of ULD-THC on neuroinflammation and cognitive deficits in 5xFAD male and female mice and explore the molecular changes in both the hippocampus and PFC. 5xFAD mice start to exhibit early brain pathologies between 1.5 and 3 months, with behavioural phenotype staring at 6 months [, ]. Give our previous experiment indicate a beneficial effect of ULD-THC for up to 3-4 weeks after the single treatment, we employed a monthly ULD-THC treatments from 2 to 5 months of age were followed by behavioral assessments and molecular analyses at 6 months.
Study design
To determine the prevention efficacy of ULD-THC in the 5xFAD mice model, 80 3-month-old male and female 5xFAD mice and their WT littermates were treated with three i.p. injections of either ULD-THC or vehicle (10 per group) once a month for 3 months from the age of 3 to 5 months. At the age of 6 months, 3 weeks after the last treatment, mice were tested for both short-term and long-term spatial memory. The mice underwent one test per day for 2 weeks, after which they were sacrificed for biochemical examination. All male and female mice were subjected to the same behavioral and biochemical tests following an identical timeline.
Treatments
Δ9-THC (donated by Prof. Mechoulam, the Hebrew University, Jerusalem and by NIDA, USA) was dissolved from a stock solution in ethanol into a vehicle solution consisting of 1:1:18 ethanol: cremophor (Sigma-Aldrich): saline and was administered i.p at a dose of 0.002 mg/kg.
Treatments
Mice were treated with a monthly i.p. injection of either ULD-THC or vehicle, starting before disease onset at the age of 3-5 months. Thus, each mouse was treated 3 times - once at the age of 3 months, once at the age of 4 months and once at the age of 5 months. At the age of 6 months, 3 weeks after the last treatment, the animals were assessed for cognitive deficits.
Behavioural tests
Learning ability and spatial memory were evaluated using MWM. This classical spatial learning assay was performed as previously described [ ]. Briefly, the mouse was introduced into a round pool at different starting points, spatial cues (such as a black cross on the white wall, etc.) were visible for the mice, and was allowed 60 s to find the platform. The water was kept at a temperature of 23 degrees Celsius. Each mouse swam four times per day for three consecutive days. The time and route required for the mouse to find the immersed platform was recorded. On day 4 of the assay, the platform was removed, and the mouse was allowed to swim for 60 s ("probe test"). The time the mice spent in the platform zone (the location of the missing platform) was recorded.
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Evidence quotes (7)
Although the mechanism of AD has been investigated for more than 50 years, currently, there are only a few available drugs for AD that can slow Aβ accumulation and treat some of the cognitive and emotional symptoms. In the past two to three years, new drugs have been approved for Alzheimer's disease, including symptomatic treatments like Benzgalantamine (a cholinesterase inhibitor, FDA-approved in July 2024) and disease-modifying therapies like Lecanemab and Donanemab (anti-amyloid monoclonal antibodies, FDA-approved in July 2023 and July 2024, respectively) [ ]. While these therapies mark significant progress, they do not prevent the disease. The distinction between slowing progression and true prevention is crucial, as no existing treatment can halt the pathological processes before they begin. Thus, while there have been advances in treating AD, there is still no way to prevent the disease [ ].
In the last decade, studies have found many changes in the endocannabinoid system in AD [ - ], yielding several clinical [ - ] and pre-clinical [ - ] studies on the benefits of cannabis and its main compound - 9Δ-tetrahydrocannabinol (THC). However, the use of THC in patients is hindered by the harmful effects of high doses of chronic THC use [ ]. One strategy that has been suggested to tackle this issue is to use an ultra-low dose of THC (ULD-THC) [ - ]. Indeed, we have recently shown that a single dose of ULD-THC has a beneficial effect on age-related cognitive decline in old female mice [ ] and alleviated AD-related cognitive impairments in the 5xFAD AD mice model [ ]. However, the potential of cannabinoids in the prevention of AD is still unexplored. The primary objective of this study was to evaluate the effects of preventive treatment of ULD-THC on neuroinflammation and cognitive deficits in male and female 5xFAD AD mice.
Building on prior findings of memory improvement following ULD-THC treatment after disease onset [ ], this study employs a preventive regimen to explore sex-specific molecular and behavioral responses to ULD-THC administered at earlier stages of AD pathology. We investigate here the preventive effects of ULD-THC on neuroinflammation and cognitive deficits in 5xFAD male and female mice and explore the molecular changes in both the hippocampus and PFC. 5xFAD mice start to exhibit early brain pathologies between 1.5 and 3 months, with behavioural phenotype staring at 6 months [, ]. Give our previous experiment indicate a beneficial effect of ULD-THC for up to 3-4 weeks after the single treatment, we employed a monthly ULD-THC treatments from 2 to 5 months of age were followed by behavioral assessments and molecular analyses at 6 months.
To determine the prevention efficacy of ULD-THC in the 5xFAD mice model, 80 3-month-old male and female 5xFAD mice and their WT littermates were treated with three i.p. injections of either ULD-THC or vehicle (10 per group) once a month for 3 months from the age of 3 to 5 months. At the age of 6 months, 3 weeks after the last treatment, mice were tested for both short-term and long-term spatial memory. The mice underwent one test per day for 2 weeks, after which they were sacrificed for biochemical examination. All male and female mice were subjected to the same behavioral and biochemical tests following an identical timeline.
Δ9-THC (donated by Prof. Mechoulam, the Hebrew University, Jerusalem and by NIDA, USA) was dissolved from a stock solution in ethanol into a vehicle solution consisting of 1:1:18 ethanol: cremophor (Sigma-Aldrich): saline and was administered i.p at a dose of 0.002 mg/kg.
Mice were treated with a monthly i.p. injection of either ULD-THC or vehicle, starting before disease onset at the age of 3-5 months. Thus, each mouse was treated 3 times - once at the age of 3 months, once at the age of 4 months and once at the age of 5 months. At the age of 6 months, 3 weeks after the last treatment, the animals were assessed for cognitive deficits.
Learning ability and spatial memory were evaluated using MWM. This classical spatial learning assay was performed as previously described [ ]. Briefly, the mouse was introduced into a round pool at different starting points, spatial cues (such as a black cross on the white wall, etc.) were visible for the mice, and was allowed 60 s to find the platform. The water was kept at a temperature of 23 degrees Celsius. Each mouse swam four times per day for three consecutive days. The time and route required for the mouse to find the immersed platform was recorded. On day 4 of the assay, the platform was removed, and the mouse was allowed to swim for 60 s ("probe test"). The time the mice spent in the platform zone (the location of the missing platform) was recorded.
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