APOE4 transgenic Mouse, Rapamycin, AD

Intro to APOE4 Mouse Studies

This is the leading study suggesting rapamycin can prevent AD in APOE4 carriers. Many prior studies starting in 2010 showed rapamycin could prevent AD in mouse models of AD, primarily by reduction of activity of mTOR and reducing amyloid accumulation in the brain. This is first study to use transgenic APOE4 mice and focus is on the cerebral blood flow. The study concluded, "As rapamycin is FDA-approved...this study may have "TREMENDOUS TRANSLATIONAL POTENTIAL". [Translational potential is going from basic science to clinical medicine. ]


The paper is presented in detail as this paper is of crucial impotance to my decision to provide rapamycin "off-label" to prevent AD in APOE4 carriers.


"Rapamycin rescues vascular, metabolic and learning deficits in apolipoprotein E4 transgenic mice with pre-symptomatic Alzheimer's disease", Ai-Ling Lin, Veronica Galvan, Arlan Richardson, et.al. 2015. (11)


Introduction: "The Apolipoprotein E e4 allele (APOE4) is the major genetic risk factor for Alzheimer's disease (AD). Individuals that possess one or two APOE4 alleles have a 4- to 8-fold increased risk for developing AD, with an age of onset occurring 7-15 years earlier when compared to non-carriers.   


"Cross-sectional studies in healthy young APOE4 carriers, who have intact memory and are free from amyloid beta (AB) or tau pathology, have reported reductions in cerebral metabolic rate of glucose (CMR,Glu) in brain areas later vulnerable to AD decades before the possible onset of symptoms. Longitudinal studies further showed that regional cerebral blood flow (CBF) is reduced in an accelerated manner in cognitively healthy APOE4 carriers. Collectively, these finding indicate that brain physiology is altered in APOE4 carriers years before clinical markers such as AB, tau pathology and memory deficits appear."


"Cerebrovascular impairments were proposed to be an initiating event that leads to neuronal activity alteration, proinflammatory cytokine production, AB/tau deposition, and memory loss. therefore, preserving cerebrovascular function early in life in APOE4 carriers may be critical for preventing the onset of AD."


"The therapeutic and preventive potential of preserving cerebrovascular function was highlighted by our recent studies. We showed that rapamycin, a drug that extends lifespan by delaying aging, restored cerebrovascular functions, including CBF and vascular density in mice modeling AD. We also showed that the vascular restoration was associated with reduced AB and improving spatial learning and memory in AD transgenic mice. These results suggested that rapamycin could be an effective treatment to restore cerebrovascular function and block or attenuate the progression of established AD-like deficits in mice modeling AD."


"In this study, our goal was to determine whether rapamycin administered early in disease progression would restore vascular and metabolic function in the young mice expressing human APOE4 genes. Particularly, we wanted to identify if we could rescue these functions in AD pre-symptomatic mice. We hypothesized that declines of CBF and CMR,Glu, in addition to increased blood-brain barrier (BBB) leakage, will precede cognitive impairments, and rapamycin can rescue the vascular and metabolic deficits in the young APOE4 carriers."

Methods, Study timeline

Female mice were used. They were fed low dose oral rapamycin, same as used in Harrison 2009 study which extended lifespan. In previous studies reported these mice begin to show vascular deficits at 2 weeks of age, followed by neuronal dysfunction at 4 months of age and memory decline at 12 months. 

Rapamycin was started at 1 month of age, and continued for 6 months. Behavior tests and CBF, CMR,glu and BBB evaluated at 7 months.

Results

Rapamycin restored brain vascular functions in APOE4 mice:

At 1 month of age APOE4 mice had 20% reduction in global CBF. At 7 months APOE4 mice had dramatically decreased CBF (-33% cortex, -25% hippocampus, -38% temporal lobe. The rapa treated mice had no difference from wild type controls.

BBB integrity at 7 months showed an impaired/normal ratio of 0.31. Rapamycin significantly improved BBB integrity.

Rapamycin restored metabolic functions of APOE4 mice.

APOE4 control mice had dramatic reduction of CMR, glucose in cortex, hippocampus and temporal lobe. Rapa treated mice had no significant difference from WT-control mice.

Rapamycin attenuated incipient spatial learning deficits of APOE4 mice

Rapa treated mice were indistinguishable from from WT control mice.

Rapamycin restored CypA levels in cerebro-vasculature in APOE4 mice. 


[Note: CypA is of critical impotance to pathogenesis of AD in APOE4 carriers. APOE4 has weak binding to very important transport protein, low-density lipoprotein 1, in contrast to APOE3 and APOE2. CypA is supposed to remain in cortical tissue, not vascular tissue. Failure of APOE4 to properly transport CypA results in increased levels of CypA in vasculature where it cause increased levels of nuclear factor -kB, which results in inflammation and subsequent BBB breakdown.]


In study, measured CypA levels in cortical and cerebrovascular components in WT, AD control and AD-rapa treated mice. "We found that the total CypA levels (in cortical tissue and vasculature) among the three groups were similar. However, the APOE4-control group had significantly lower CypA levels in brain tissue, but dramatically higher CypA levels in vasculature compared to the WT-control group, suggesting redistribution of CypA, and an increased inflammation in vasculature of mice expressing APOE4 gene. Notably cerebrovascular CypA levels, were restored to WT levels in APOE4-Rapa animals. 

A similar trend was observed in NF-kB levels."

Discussion

"We demonstrated that cerebrovascular deficits occur early in mice with the APOE4 genotype. At one month of age, the APOE4 mice already showed significant CBF reductions in the whole brain and in regions involved in higher order of cognitive functions, including hippocampus and temporal lobe. BBB deficit was also found in the temporal lobe. This is consistent with human studies which suggest AD pathology evolves early in temporal lobe. We also found significantly decreased glucose metabolism in the APOE4 mice. This is in good agreement with ample body of literature demonstrating that APOE4 carriers exhibit mitochondrial dysfunction and an overall reduction of glucose metabolism in the brain...Collectively, consistent with human studies, we found that vascular and metabolic defects occur early in life and long before irreversible memory loss in APOE4 mice".

"We further demonstrated that rapamycin  was sufficient to restore brain vascular function in young APOE4 mice. The APOE4-Rapa group had restored CBF and BBB integrity after 6 months of treatment."

"These vascular restorations were associated with the reduced CypA levels in the vasculature. CypA is a proinflammatory cytokine and that has previously been shown to have deleterious effects on the vascular system in mice lacking murine APOE with aortic aneurysms and atherosclerosis. CypA causes BBB breakdown by activating the NF-kB-matrix-metalloproteinase-9 (MMP9) pathway. We also observed a trend toward increase vascular NF-kB."...

"Our results suggest that down-regulating CypA activity in cerebro-vasculature alone has significant protective effects on vascular integrity and that these changes may be sufficient to improve cognitive outcomes."

"Our finding indicate that rapamycin was able to inhibit endothelial CypA and restore its distribution between brain and the vasculature."

'In addition to endothelial CypA inhibition, we previously showed that rapamycin restores vascular function via mTOR pathway. In a symptomatic mouse model of AD mTOR inhibition with rapamycin increased the activity of endothelial nitric oxide synthase and led to release NO, a vasodilator. The subsequent increase in CBF was dependent on NOS (nitric oxide synthase) activity. "In that study, we found the restoration of CBF and cerebrovascular density were associated with improved cognitive function and lower brain AB levels." ...That APOE is intricately linked to AB clearance only furthers our suggestion that mTOR-mediated regulation of cerebral vasculature may be a crucial target in AD-like cognitive deficits."

"Cerebrovascular integrity is highly coupled with brain glucose metabolism. In line with this, we observed restored CMR,glu in the APOE4-Rapa mice. Glucose utilization plays a critical role in sustaining neurotransmission and memory formation. As such, rapamycin-mediated restoration of glucose metabolism may play a role in the restoration of the observed incipient learning and memory deficits in the APOE4 mice." 

Previous studies showed that cognition is highly associated with CBF and CMR.glc levels and preserving brain hemodynamics and metabolism are critical for optimizing brain health. consistent with the literature, we have found that preserving cognition was reflected by the preservation of both CBF and CMR.glc in the APOE4-Rapa mice. 

"Taken together, our results suggest that rapamycin may delay or even block early events in the progression of physiological and cognitive deficits in APOE4 mice."

"This study may have tremendous translational potential since rapamycin has been approved by the FDA...and has been applied for various uses in humans since 1999 and as such the toxicity profiles are well characterized. ..

"Mannick et al. reported than low doses of rapamycin significantly improved immune functions in the elderly with minimal side effects."

"In summary, our finding indicate that abnormal vascular and metabolic functions precede irreversible cognitive decline in APOE4 carriers...the studies herein suggest rapamycin may be effective at mitigating the early vascular deficits associated with AD. Using neuroimaging, we were further able to non-invasively detect the vascular and metabolic changes that precede cognitive dysfunction in young APOE4 mice and were able to restore those physiological and cognitive functions through mTOR inhibition. As rapamycin is FDA-approved and neuroimaging is readily used in humans, this study may have tremendous translational potential". 

Comment

This paper was presented in detail because the authors of this paper are the world's leading researchers on the potential for prevention of AD in APOE4 carriers by inhibition of mTOR with rapamycin. It presents the evidence and cites many references which form the backbone of this website. These references will be further reviewed in detail.

This paper develops the crux of the theory underlying the treatment plan for prevention of AD in APOE4 carriers. The theory is the TWO HIT THEORY. The first hit is years of deterioration of the microvascular circulation and the second hit is the development of Amyloid-Beta and Tau pathology.

2017 Ai-Ling Lin: Rapamycin and APOE4 mice

In Aril 2017, Ai--Ling Lin published the following paper, "Rapamycin as a Novel Therapeutic for Alzheimer's Disease: Prevention assesses through Neuroimaging." (14)


This study, like 2015 study, consisted of giving rapamycin to APOE4 transgenic mice. What was new was assessing prevention through brain scans.


Abstract: "Data strongly supported the initial hypothesis that with the introduction of Rapa, APOE4 mice will show improved cognition, memory, and neurological health. Cerebral blood flow and crucial brain metabolites were  restored in Raps mice: Rapa mice exhibited cogent memory in comparison to their counterparts in cognitive tests: Rapa reduced neuroinflammation--- all with significance."


"Thus vascular neuroimaging is a promising early AD detection technique and Rapa holds high potential in preventing AD development."


Ai-Ling Lin is the world's leading expert on use of rapamycin as treatment for ApoE4 carriers. Her extraordinary work  is large part of scientific basis for the  plan to prevent AD in APOE4 carriers with rapamycin.  


10/6/2017 Neuroscience news announced that Ai-Ling Lin, professor at Saunders-Brown at University of Kentucky was starting new research study, with 3 million dollar grant from NIH. The study will give low dose rapamycin to ApoE4 mice. What is novel about study is they will follow the mice with multimodal MRI (mMRI). This is MRI combined with CT scans and PET scans. In this way will be able to detect changes in mice cerebral blood flow and glucose metabolism and other measures to provide early changes indicating development of dementia. The results will be validated with further biological and behavioral testing to confirm mMRI's accuracy. In this way mMRI could act as surrogate in human studies. Studies to determine if rapamycin prevented development of AD in human carriers of ApoE4 would not have to wait 20 years for results; but mMRI could yield surrogate results. 


Article states, "It will be a while before Rapamycin as a therapeutic target for Alzheimer's will be ready for human clinical trials, but the concept of mMRI as a surrogate for other testing methods has the potential for speedy adoption...as available to be used in humans. This was hailed as another important step in Saunder's-Brown race against Alzheimer's.

"As many as one in five people carry the Azheimer's gene APOE4, so the idea that a readily-available drug already demonstrated to be SAFE IN HUMANS might prevent what is arguably the most devastating and expensive disease in modern history is exciting."


 After the current Lin study, my estimation is it would take three consecutive human studies to obtain FDA approved label expansion for use of rapamycin to prevent AD in APOE4 carriers. 


First: A phase 1 type study to determine the proper dose and time interval for using rapamycin to prevent AD. 


 Second: A small phase II human study. 


 Third:  a large phase III study to gain FDA approval.


 My ball park estimate is these human studies would cost around 100 million dollars and take an additional 10-15 years. 


The studies reported in Basic Science section confirm the results of the 2015 study discussed above.


The reason for off-label use is that while these results are extremely encouraging that rapamycin will prevent AD in APOE4 carriers; many people don't have 10-15 years to wait.