Figure 1, Various factors including aging and diabetes can cause increase in mTOR activity. Decreased clearance and increased production can cause accumulation of AB. In positive feedback increase in mTOR activity causes increase in AB and increase in AB causes increase in mTOR activity. Both increase AB and increase mTOR activity cause increase production and accumulation and hyperphosphorylation of TAU. Both increased Tau and increased AB cause cognitive deficits and neurodegeneration.(24)
In a healthy neuron, mTOR activity is tightly regulated and basal autophagy levels are sufficient to remove AB and tau.
During early stages AD, an increase in soluble AB levels leads to mTOR hyperactivity, which in turn will reduce autophagy induction (represented in diagram by a reduction in autophagosomes). Lower autophagy function will eventually lead to an increase in steady-state levels of AB and tau. Notably, high AB levels will further increase mTOR activity thus creating a vicious cycle that ultimately will promote higher AB levels.
During late stages of AD, autophagosomes fail to fuse with lysosomes. It is anticipated increasing autophagy induction in late stage of AD may further clog cells generating more autophagosomes that will not be cleared.
In 1906, Alois Alzheimer, a German Neuropathologist, was the first to describe the neurodegenerative disease, now called Alzheimer's disease. Using special histopathological staining techniques he identified amyloid plaques in the white matter of the cortex and neurofibrillary tangles within the nerves. Alzheimer's description of amyloid plaques and neurofibrillary tangles remain the microscopic hallmarks of AD. Amyloid plaques are composed of a peptide, called Amyloid-Beta. Neurofibrillary tangles are composed of a protein called Tau. For the next 100 years, the two protein inclusion first identified by Alzheimer, Amyloid -Beta and Tau have remained the main focus of research.
The normal function of AB is not well understood. Some animal models have shown the absence of AB does not lead to significant loss of physiologic function; while others have shown some function of AB. However, AD is clearly related to increased amount of AB and not a loss of function of AB.
The starting protein for AB is Amyloid Precursor Protein (APP). The action of B-secretase (BACE) liberates a C-terminal fragment which is immediately cleaved by proteolytic enzyme, gamma secretase to release peptides, AB. These peptides are generally 40 or 42 amino acids in length, called AB(40) and AB(42). The peptide AB(42) has higher propensity to aggregate than AB(40). 30% of AB fragments are rapidly secreted into the extracellular space and 70% of AB fragments remain in cell and are degraded by proteasome system.
Mutations related to gamma-secretase, called presenilin mutations, lead to increase ratio of AB(42) to AB(40) and are the cause of early onset AD, which account for less than 5% of cases of AD.
AB peptides are produced as soluble monomers. However, at sufficiently high concentration they begin to undergo dramatic conformational change. First the monomers aggregate to form oligomers which then further aggregate to form amyloid fibrils which then further aggregate to form dense formations called intracellular plaques.
The various aberrant species of AB are toxic. Large amounts of soluble AB are toxic to neurons.
The plaques in extracellular space can provoke intense inflammatory response to glia cells which can be be toxic to neurons.
Cerebral amyloid angiopathy is caused by large amount of AB in extracellular space. AB coats the outside of the vessels. When large vessels are involved this can lead to large vessel bleeds within the brain. Involvement of small vessels and capillaries can contribute to ischemia.
AD has been, to some degree, identified as a PROTEIN MISFOLDING disease. AB fibrils can act as a template to cause AB peptides to become misfolded. In this way, AD can act as a kind of non-infectious Prion disease.
Amyloid-beta deposits are most prominent in the hippocampal region, an area intimately associated with memory. For this reason, loss of hippocampal function, characterized by memory decline, is one of the earliest and most constant features of AD.
In the pathogenesis of AD, the ability of increased levels of AB to cause increase activity of mTOR and increased amounts of TAU is one of the major harmful effects of increase AB.
Increased amounts of AB and increased activity of mTOR result in increased levels of hyperphosphorylated Tau. The reverse is not true and Tau does not cause increased AB and increased mTOR. Hyperphosphorylated Tau plays a very major role in neurodegeneration.
Tauopathy belongs to a class of neurodegenerative diseases associated with the pathological accumulation of tau protein in neurofibrillary tangles in the human brain. These tangles are formed by hyperphosphorylation of a microtubule associated protein known as tau, causing it to aggregate in insoluble form. Hyperphosphorylated tau was originally identified as paired helical filaments.
Primary tauopathies, i.e., those in which neurofibrillary tangles (NFT) are predominant include:
Primary age-related tauopathy (senile dementia with NFT similar to AD but without plaques.
Frontotemporal dementia (Pick's disease)
Chronic tramatic encephalopathy
Progressive supranuclear palsy
Neurofibrillary tangles are also part of Alzheimer's disease combined with amyloid plaques.
Tauopathies often overlap with synucleinopathies in Parkinsonian-dementia.
Tau was first identified in the brain in 1975 and named after the Greek letter. It was recognized as being part of a large family of microtubule-associated proteins which play a central role in binding to microtubules and providing neuron stability.
Microtubules are the major structural element of neurons without which the neuron could not maintain its exaggerated shape. Microtubules act as railways along which molecular proteins convey cargo. Microtubules are major part of axons and dendrites of neurons.
By electron microscopy it was determined that the NFT had the shape of paired helical filaments. In 1985 it was determined that Tau was the major antigenic component of the paired helical tangles that comprises NFT.
It was recognized that TAU in the NFT was phosphorylated at multiple sites and the amount of phosphorylation was abnormal and increased.
HYPERPHOSPHORYLATED TAU was then recognized as major factor in pathogenesis of AD.
Hyperphosphorylated tau is soluble, toxic, does not function and precipitates into insoluble NFT.
AD brain's pathology show increased amount of total tau and increased amount of hyperphosphorylated Tau.
JJ Pei, a world's leading expert on Tau, in paper "Mammalian Target of Rapamycin (mTOR) mediates Tau Protein Dyshomeostasis", 2013, (34) writes:
"The formation of tau inclusions (NFT) is widely thought to contribute to AD pathogenesis as NFT correlates with the duration and progression of AD. Both insoluble and soluble forms of abnormally hyperphosphorylated tau exist in AD brains, and they do not interact with tubulin. Furthermore, when the soluble form of abnormally hyperphosphorylared tau is present, it sequesters normal tau and microtubule-associated proteins accelerating disruption of the microtubule network."
"It was demonstrated in transgenic mouse brains that the abnormal hyperphosphorylation of tau precedes the formation of NFTs and neuronal loss. The expression of tau pseudophosphorylated...trigers apoptosis, which is accompanied by tau aggregation and breakdown of the microtubule network. On the other hand, expression of wild type tau in vivo leads to synaptic loss, whereas deletion of tau rescues B-amyloid-induced toxicity at the synapse."
"This evidence suggests that dysregulated production, phosphorylation, and aggregation of tau might be the KEY events that trigger neuronal degeneration in AD."
The above statement that hyperphosphorylated tau sequesters normal tau is also very important. It means hyperphosphorylated tau stops function of normal tau and normal tau is essential to neurons
[this discussion from Galvan 2015 paper, "mTOR At the Crossroads", cited above] (24)
In Hit Two, major factor in pathogenesis of AD major is the crosstalk between mTOR , AB and Tau.
Hyperactive mTOR increases both AB production and Tau production. This results in increased production and accumulation of AB and increased production and accumulation of Tau.
AB and mTOR activity is a TWO WAY street. Increased AB through positive feedback on to mTOR further increases mTOR activity.
Increased AB directly causes increased Tau and indirectly causes increased Tau by increasing activity of mTOR which then Increases Tau.
Increased Tau and increased AB then both cause cognitive deficits and neurodegenertive.
Increased AB <------------------->Increased mTOR activity
Increased AB -----------------------> Tau
Increased mTOR activity -------> Tau
[from "The role of mTOR signaling in Alzheimer's disease", Salvatore Oddo, 2012, section 7, mTOR and Autophagy and Figure 1.] (35)
Autophagy plays a major role in AD.
The autophagy system is an intracellular system for the degradation of long-lived proteins. It is a very important house-cleaning system in which removed proteins are recycled.
The material to be degraded is first put in a bag, forming something called an autophagic vacuole and then the bag (vacuole) is fused with lysosomes for protein/organelle degradation. This is autophagy/lysosome system.
mTOR negatively regulates autophagy. Elevated levels of mTOR causes less autophagy.
"Several neurodegenerative disorders are characterized by the abnormal accumulation of aggregated proteins."
AD is characterized by accumulation of amyloid-Beta and Tau.
"Autophagy decreases with age and age is the major risk factor for AD and other neurodegenerative disorders, suggesting that the age dependent decrease in autophagy function may contribute to chronic buildup of aggregates in neurons".
"Genetically reducing autophagy leads to profound neurodegeneration" associated with accumulation of inclusions. "Consequently, it has been proposed that inducing autophagy may have beneficial effects in a variety of neurodegenerative disorders."
Previously discussed mouse model of AD show that reducing mTOR and increasing autophagy rescues AD mice from cognitive deficits and AD-like neuropathology and AB accumulation.
A study cited by Paul Greengard's group showed that inducing autophagy by rapamycin led to reduction in AmyloidBeta levels by 80%. While studies done in early stages of AD showed a beneficial effect, studies by Nixon group in late AD showed accumulation of autophagic vacuoles and lack of beneficial effect.
Oddo's Figure 1 shown above is a schematic representation of the involvement of mTOR in AD.
"In a healthy neuron, mTOR activity is tightly regulated and basal autophagy levels are sufficient to remove AB and tau.
During early stages of AD, an increase in soluble AB levels leads to mTOR hyperactivity, which in turn will reduce autophagy induction (represented in the diagram by a reduction in autophagosomes). Lower autophagy function will eventually lead to an increase in the steady-state levels of AB and tau. Notably, high AB levels will further increase mTOR activity thus creating a vicious cycle that will ultimately promote higher AB levels. Increasing autophagy induction in early AD may represent a valid therapeutic approach as it will facilitate autophagosome formation and thus remove AB and tau.
During late stages AD, there is evidence that autophagosomes fail to fuse with lysosomes. It is anticipated that increasing autophagy induction in late stages AD may further clog cells generating more autophagosomes that will not be cleared."
That failure of autophagy in late AD may provide part of explanation of 2011 mouse study noted above. When rapamycin was started late in AD mouse models of AD at stage when they had established plaques and tangles, even though mTOR was decrease and there was increase in autophagy, mice had no benefit
There is very strong evidence from many laboratories that have consistently shown that hyperactive mTOR contributes to tau pathology.
In postmortem human AD brains, hyperactive mTOR signaling was found in neurons that were predicted to develop tau pathology. "Up-regulation of Phosphorylated/Activated p70 S6 Kinase and its Relationship to Neurofibrillary Pathology in Alzheimer's Disease"; JJ Pei, 2003, The American Journal of Pathology.(36)
"Phosphorylated/Activated p70 S6 Kinase" is the immediate downstream product of mTOR and "Neurofibrillary" is hyperphosphorylated Tau.
They looked at AD brains that span the pre-clinical, symptomatic and late stages corresponding to Braak's stages of AD pathology. They examined various areas of the brain, especially the hippocampus and temporal cortex areas of brain most involved in AD. The brains were studied with confocal microscopy and special stains. The findings were summarized:
"We found that activated p70 S6 kinase (activated mTOR) is co-distributed with the neurofibrillary pathology (Tau) in a predictable sequence from the entorhinal cortex, to the hippocampal CA1 and layers III and V of the temporal cortex. Activated (mTOR) was obviously increased in neurons before developing NFTs. Only levels of (activated mTOR) showed a dependent correlation with total tau and hyperphosphorylated tau."
They found that a significant amount (@ 60%) of normal tau remained in supernatant of AD brains. They hypothesized that to keep the cell functioning, neurons enduring hyperphosphorylated tau which fails to function would compensate by making more tau. The problem being as made more Tau that would be converted to hyperphosphorylated tau; so that attempt to compensate just made pathology worse.
In an animal model of tau pathology, "Hyperactive TOR in Drosophilia (fly) facilitates the development of tau pathology and the associated neurodegeneration." [Oddo, 2015, mTOR at Crossroads].(24)
"mTOR Regulates Tau Phosphorylation and Degradation: Implications for Alzheimer's disease and other Tauopathies", Caccamo...Oddo, 2013. (7)
Oddo, who was the lead researcher in 2013 study and also authored 2015 review article, "mTOR at crossroads" (23) states:
"Mice with hyperactive mTOR also have increased brain levels of total and phosphorylated tau. Conversely, reducing mTOR has beneficial effects on tau pathology. To this end, reducing mTOR with rapamycin in a transgenic mouse expressing mutant human tau decreased tau pathology and improved the associated motor deficits."
The study consisted of two parts. The first part used a genetically modified mouse heterozygous for TSC gene. TSC inhibits mTOR so missing one gene would reduce inhibition of mTOR and result in increased expression of mTOR. These mice were called TSC+/-.
First: Measure activity of mTOR in hippocampus of the TSC+/- mice. mTOR activity was elevated showing hyperactive mTOR.
Second: Measured Tau in hippocampus of mouse brain. Tau was elevated 1.5 fold.
Third: Measured the degree of phosphorylation of Tau. Tau was significantly hyperphosphorylated.
Fourth: Determined that GSK3B was elevated. This is kinase enzyme determined to be responsible for increased phosphorylation of Tau.
Fifth: mRNA was not elevated showing there was not increased production of Tau.
Sixth: Two major forms of protein degradation are protesome and autophagy. Protesome activity was normal. Autophagy related enzymes were decreased. This showed autophagy was decreased.
The results showed that hyperactive mTOR increased level of Tau by increased pathologic phosphorylation combined with decreased autophagy.
Second part of study used a pharmacologic approach to determine the relationship between Tau and mTOR. They used a transgenic mouse that harbors a mutant human gene which causes mice to develop tau pathology. The mice developed age-dependent accumulation of tau inclusions starting at 4-5 months. As the mice age the tau pathology becomes worse with neuronal loss at 12 months. Mice with the abnormal gene were called TG mice and controls called non-TG mice.
At two months of age TG mice were divided into group fed rapamycin and group fed regular chow. The rapamycin preparation was same low dose preparation Harrison used to extend lifespan in mice in 2009 study.
TG mice were treated with rapamycin chow or regular chow for 6 months and then tested at 8 months of age.
TG mice fed regular chow had significant motor impairment. TG mice fed rapamycin had no motor impairment and performed at same level as control mice.
TG mice showed elevated activity of mTOR in brain.
TG mice had robust tau accumulation and phosphorylation in brain. Rapamycin treated mice had same brain findings as normal controls.
TG mice showed elevated activity of GSK3B kinase, responsible for hyperphosphorylation.
TG mice had reduced autophagy and rapamycin treated mice showed induction of autophagy.
They concluded reduction of autophagy was partly responsible for elevated Tau.
They state in discussion: " Here we offer first evidence in mammals of a direct link between mTOR signaling and tau accumulation. Notably, not only did we show that genetically increasing mTOR signaling increases tau levels and phosphorylation; but we also showed that reducing mTOR signaling with rapamycin ameliorated tau pathology and rescues motor deficits in a mouse model of tauopathies.
Summary: "Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles...Here, we used multiple animal models and complimentary genetic and pharmacologic approaches to show mTOR regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamyin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau.
"In summary, we show that increasing mTOR facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence showing that reducing mTOR signaling increases lifespan and health span, the data presented here may have profound clinical implications for aging and taupathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide pre-clinical data indicating that reducing mTOR signaling may be a VALID THERAPEUTIC APPROACH FOR TAUOPATHIES."
Oddo in 2015 paper, "at the crossroads" continued: " ((24) Similar to these observations, chronic treatment with a rapamycin ...(rapalog) in mutant tau mice, decreased mTOR signaling, stimulated autophagy, reduced tau levels and neurofibrillary tangle density, which led to attenuation of motor deficits."
Oddo continued: "The mechanism underlying these observations is likely multifactorial. For example hyperactive mTOR signaling decreased autophagy turnover, which is known degradation pathway for tau. mTOR can also regulate tau levels by increasing translation of it's mRNA. Indeed, direct evidence from primary hippocampal neurons showed that inhibition of mTOR suppress tau translation, while constitutively active mTOR signaling increased tau translation
An excellent analysis of the abnormal metabolism of Tau in AD is presented by JJ Pei, a leading expert on Tau in paper, "Mammalian target of rapamycin (mTOR) mediates tau protein dyshomeostasis: implications for Alzheimer disease", 2013. (34)
Five molecular events can be described in regard to abnormal Tau metabolism in AD. All five of these steps are mediated and controlled by mTOR.
1. Degradation: autophagy described above.
2. Translation (synthesis)
3. Phosphorylation, Kinases, adding
4. Phosphorylation, Phosphatase, removal
5. Aggregation (fibrillation)
Translation, Phosphorylation and Aggregation are controlled by both mTORC1 and mTORC2 and taken together constitute "Dyshomeostasis. By this is meant an imbalance of tau homeostasis, a condition required for neurons to maintain physiologic function. The result is both a loss of function and toxic function.
Translation: There is increase in tau synthesis. This is caused by up-regulated mTORC1 activity.
This is adding phosphate groups. Tau becomes hyperphosphorylated, adding too many phosphate groups. This is mediated by both mTORC1 and mTORC2 increased activity. A critical factor is location of where phosphate groups are added. mTORC2, acting through phosphatase GSK-3B adds phosphate groups to flanking region. This inhibits microtubule binding capacity and results in loss of function and converts tau into toxic molecule.
Phosphorylation: phosphatase. This is removing phosphate groups. Main phosphatase is protein phosphatase 2A (PP2A). This activity is impaired by mTORC1 and mTORC2. Activity is restored by rapamycin by lowering activity of mTOR.
[Activity is also restored by metformin. (see paper "Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling"; Kickstein, 2010].(38)
The balance between Kinase action (adding phosphate groups) and phosphatase action (pruning phosphatase groups) is critical to maintain proper level of phosphorylation. Increased phosphorylation is caused by increased activity mTOR.
Aggregation (fibrillation). This is process by which soluble tau becomes insoluble fibrillates. The process is mediated through mTORC2 and includes apoptosis.
The pathogenesis of TAU abnormal translation, hyperphosphorylation and aggregation causes neurodegenerative tauopathies.
Oddo in 2015 paper concludes: (24)
"One startling implication of these observations is that long-term exposure to hyperactive mTOR might increase tau translation and decrease its degradation/turnover, while concomitantly increasing tau phosphorylation."
"Collectively, these studies highlight multiple pathways by which mTOR signaling contributes to tau pathology."
The relationship betweem AB and mTOR were summarized above.
Studies show an increase in mTOR directly related to increase in soluble AB. Furthermore, an increase in mTOR resulted in increase in AB.
Studies showed an increase in mTOR signaling of 3 fold in the medial temporal cortex of AD cases. This is consistent with mTOR activity within cortex and hippocampus in AD mice. Furthermore in AD mice, reduction in AB pharmacologically or genetically resulted in decrease in mTOR activity
Confocal microscopy data showed a direct interaction between intraneuronal AB(42) and mTOR.
In AD mice, rapamycin-mediated reduction in mTOR signaling ameliorated AD-like pathology and cognitive deficits.
In 2015 Oddo paper noted above, the section on AB and TOR concluded:
"Genetic studies strength the link between mTOR and AD pathogenesis. To this end, genetically and selectively reducing mTOR signaling in the brains of Tg2576 (AD) mice was sufficient to rescue memory deficits. This rescue of cognitive deficits was associated with reduced AB deposits and a change in the abnormal pattern of hippocampal gene expression of the Tg2576 mice to a more similar pattern found in wild-type control mice."
"Collectively, these studies suggest that hyperactive mTOR in AD contributes to the accumulation of Amyloid-Beta."(26)
BBB: Rapamycin downregulates action CypA, NF-kB, MMP9 pathway which preserves pericyte cells, endothelal cells and BBB integrity and vascular density.
Rapamycin increases activity of NO synthase which increases release Nitric Oxide which causes vasodilation and restores cerebral blood flow.
Amyloid-Beta: Rapamycin increases Autophagy which decreases AB.
Rapamycin preserves cerebral microcirculation which removes excess amyloid-B.
Tau: Rapamycin decreases TOR and TOR has all the following adverse effects:
Decrease degradation: autophagy
Increase translation: Synthesis
Increase Hyperphosphorylation by increase kinase action (adding phosphate groups) and decrease phosphatase action (removal phosphate groups