Decressac...Bjorklund, 2013, Sweden, Denmark
"This study shows that neurodegenerative changes induced by alpha-synuclein (a-syn) in midbrain dopamine neurons in vivo can be blocked through activation of the autophagy-lysosome pathway. Using an adeno-associated virus model of Parkinson disease to overexpress a-syn in the substantia nigra we show that genetic [transcription factor EB (TFEB) and Beclin-1 overexpression] or pharmacological (rapalog) manipulations that enhance autophagy protect nigral neurons (substantia nigra) from a-syn toxicity, BUT inhibiting autophagy exacerbates a-syn toxicity. The results provide a mechanistic link between a-syn toxicity and impaired TFEB function, and identify TFEB as a target for therapies aimed at neuroprotection and disease modification in Parkinson disease."
ASG: TFEB is master regulator of the autophagy-lysosome pathway. The secondary malfunction of this pathway appears to be a major final common pathways in PD. When increase or decrease this pathway protect or exacerbate PD. Rapalogs (rapamycin) enhance TFEB pathway and thus can provide neuroprotection in PD.
"The aggregation of a-syn plays a major role in Parkinson disease (PD) pathogenesis"
[ASG: aggregation of a-syn are oligomers and oligomers then form Lewy body, the pathologic hallmark of PD]
"Recent evidence suggests that defects in the autophagy-mediated clearance of a-syn contribute to the progressive loss of nigral dopamine neurons"
[ASG: Loss of nigral neurons is the cause of PD]
"Using an in vivo model of a-syn toxicity, we show that the PD-like neurodegenerative changes induced by excess cellular levels of a-syn in nigral dopamine neurons are closely linked to progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of TFEB, a major transcriptional regulator of the autophagy-lysosome pathway".
[ASG: TFEB needs to get into nucleus to work; but a-syn keeps TFEB in cytoplasm.]
"The changes in lysosomal function, observed in the rat model as well as in human PD midbrain, were reversed by overexpression of TFEB, which affored robust neuroprotection via clearance of the a-syn oligomers, and were aggravated by ...repression of TFEB..."
"Delayed activation of TFEB function through inhibition mTOR blocked a-syn induced neurodegeneration and further disease progression."
[ASG: "activation: by mTOR means phosphorylation which inhibits function of TFEB]
"The results provide a mechanistic link between a-syn toxicity and impaired TFEB function and highlight TFEB as a key player in the induction of a-syn-inducd toxicity and PD pathogenesis, thus identifying TFEB as a promising target for therapies aimed at neuroprotection and disease modification in PD."
[ASG: translation: This paper explains why and how lowering mTOR with rapamycin to increase activity of TFEB will ameliorate PD.]
"A major hallmark of PD that contributes to the progressive loss of nigral dopamine (DA) neuros is a alpha-synucleinopathy..In human PD and related Lewy Body disease, the presence of a-syn aggregates is associated with accululation of autophagosomes and reduction of lysosomal markers in affected DA neurons, suggesting a defect in lysosome-mediated clearance of a-syn-aggregates."
"How dysfunction of the autophagy-lysosome pathway (ALP) contributes to the pathogensis of PD remains unclear. (subject of this paper).
Under physiological conditions, a-syn is degraded by the ubiquitin-proteasome system and the ALP, including macroautophagy and chaperone-mediated autophagy. In cases of a-syn overload, however, misfolded or mutated a-syn fails to be processed and a-syn clearance by chaperone-mediated autophagy is blocked. In this situation, processing of excess a-syn, or toxic a-syn species, will depend on functional integrity of macroautophagy pathway..."
"The role of autophagy in the elimination of toxic protein species has generated interest in the development of therapeutic strategies for neurological diseases characterized by protein misfolding and aggregation...Pharmacologic activation function by inhibition of mTOR has produced ..protective effects in animal models of PD."
Recently TFEB has been identified as a "master regulator of the ALP, controlled by mTOR signaling"
"Enhancement of TFEB function has been shown to stimulate ALP function and promote protein clearance and neuropotection...Here we provide evidence that mechanistically links a-syn-mediated toxicity to impaired TFEB function both in a rodent PD model and in human PD.
"We have used an in vivo model of a-syn toxicity and report that TFEB function, as reflected by its translocation to the nucleus, is impaired in a-syn overexpressing midbrain DA neurons, and that the reduced nuclear expression of TFEB is seen also in markers of lysosome function, accumulation of a-syn oligomers, and development of DA neuron pathology and cell death."
[ASG: When a-syn is increased instead of ALP removal function increasing as needed, function is decreased, setting in motion a vicious cycle leading to progressive cell death]
"These degenerative changes are efficiently blocked by overexpression of TFEB, resulting in almost complete protection of nigral DA neurons and elimination of a-syn oligomers."
"Finally, we showed that delayed pharmacological activation of TFEB function, inhibition of mTOR, is highly efficient in blocking a-syn-induced degenerative changes and further disease progression in the affected DA neurons.
"Taken together, these data provide evidence for a central role of TFEB in the cellular defense against a-syn toxicity and as a key player in PD pathogenesis."
1. a-Syn overexpression causes dynamic changes in the ALP in DA neurons.
[ASG: The autophagosome-lysosome removal process is two steps. Mechanistically, first put toxic substance to be removed in a barrel (autophagosome); the second step is add acid to the barrel (lysosome). The two step process (put in barrel and add acid is called ALP.
In study caused overexpression of human wild-type a-syn by using a special virus called AAV (adeno-associated virus) in rat midbrain. This technique had previously been shown to induce PD-like neuropathology in nigral DA neurons.
At presymptomatic stage (10 days after AAV viral injection), increased a-syn is developing, but before neuron cell death; autophagy and lysosomal markers are BOTH up.
At 3 weeks when cell death starts to occur, autophagy markers are up; but lysosomal markers are down, suggesting accumulation of autophagosomes.
[ASG: The toxic stuff is being put in the barrel, but no acid is being added.]
Figure 1: a-syn impairs autophagy by SEQUESTRATING TFEB
"Failure of ALP was associate with a progressive accumulation of a-syn oliomers that was previously shown to be toxic for nigral neurons."
At early stage where ALP working, TFEB was translocated into the nuclear fraction. At 3 weeks when ALP not working, TFEB in cytoplasmic part cell in contrast to being in nuclear fraction.
[ASG: Translocation of TFEB into nuclear fraction is under control of mTOR, which causes phosphorylation of TFEB and thus keep TFEB out of nuclear portion.]
"Recent studies have shown that mTOR-mediated phosphorylation of TFEB leads to its sequestration by 14-3-3 proteins in the cytoplasmic compartment."
"Because a-syn shares structural an functional homology with the 14-3-3 proteins and can bind similar targets, we hypothesized that they (a-syn) may interact with TFEB"
"Strikingly, coimmunoprecipitation experiments showed that both human wild-type a-syn and 14-3-3 were able to bind to TFEB, suggesting that a-syn overexpression may contribute to sequestration of TFEB into the cytoplasm, thus hampering the ALP response and, thereby, its own clearance."
2. Changes in TFEB subcellular localization in PD.
"To further validate our observation made in rat model, we studied possible changes in postmortem human brains.
Fig 2: Control brain TFEB in both cytoplasmic and nuclear compartments.
In postmortem PD human brains, significant reduction TFEB in nuclear compartment"
Furthermore, TFEB is colocalized with a-syn in Lewy bodies.
3. Stimulalation of Autophagy by TFEB overexpression affords protection against a-syn induced toxicity.
4. Down-regulation of TFEB in DA Neurons aggrevated a-syn toxicity.
4. Pharmacological stimulation of TFEB function induces a disease-modifying effect.
"Studies reporting the protective effect of rapamycin in various models of neurodegenerative disease suggest that pharmacological stimulation of the ALP via mTOR inhibition may represent an interesting therapeutic strategy. In line with previous studies, mTOR inhibition may alleviate the repression of TFEB and facilitate its shuttling to the nucleus. Therefore, we tested whether...[temsirolimus, a rapalog), could afford neuroprotection in a more clinically relevant situation in animals with manifest a-synucleinopathy and ongoing dopaminergic degeneration , as seen at 3 weeks after AAV-a-syn injection.
The experiment in rats:
In intact animals, ..3 injection 2 days apart of rapalog, "efficiently inhibited mTOR... and triggered a proautophagic response in the brain, including ventral midbrain as seen by the increased levels of ALP markers. This effect was asociated with the nuclear translocation of TFEB.
"Delayed drug treatment in the AAV-syn model, starting 3 weeks after vector injection, BLOCKED FURTHER PROGRESSION OF THE DISEASE."
"At 8 week treated animals did not develop motor impairments."
"Consistent with this finding, significant survival of nigral DA neurons, striatal terminals, and maintenance of strialtal DA content was observed in the temsrolimus-treated group compared with controls."
"Molecular analysis revelaed that the disease-modifying effect was mediated by the sustained reduction in phospho-mTOR level, thereby facilitating nuclear translocation of TFEB. As observed with the gene-transfer approach, survival of nigral DA neurons resulted from the reduced accumulation of toxic oligomeric a-syn."
"The results highlight TFEB as a central player in the protective response against excessive levels of a-syn in mibrain DA neurons, and provide evidence that stimulation of TFEB function, by gene transfer, or pharmacologically blockade of mTOR, can efficiently rescue nigral DA neurons from a a-syn toxicity."
"Under baseline conditions, a-syn will be eliminated by both the proteasome system and the ALP. However, in case of a-syn overload, it has been shown that processing by the ALP becomes the dominant pathway for a-syn removal. Here we show that overexpression of human a-syn in nigral DA neurons induces dynamic changes in ALP function that are well correlated with the cytoplasmic retention of a major regulator, TFEB.
"At an early presymtomatic stage, 10 days after vector injecton, when a-syn is well expressed and before any cell death has occurred, TFEB was translocated to the nucleus and the expression of lysosomal and autophagic markers was markedly increased. This finding suggest that at this stage TFEB is mobilized to activate the ALP in response to the elevated cytoplasmic levels of a-syn.
After 3 weeks when the pathological changes have become prominent and cell death starts to occur, TFEB was largely retained in the cytoplasmic fraction and accompanied by a decline in markers of lysosomal function. At this stage, a-syn was colocalized..." with autophagy substrate in punctate structures signifying an accumulation of a-syn in autophagosomes and blockade of lysosomal processing, resulting in the progressive accumuluation of a-syn oligomers."
"The work of Ballabio and colleagues has identified TFEB as a key regulator of ALP activity that is controlled by mTOR signaling."
The fact that a-syn shares both structural ad functional homology with 14-3-3 proteins and the recent finding that phosphorylated TFEB is sequestered in the cytoplasm by 14-3-3 proteins, suggests a possible pathogenic mechanism where increased cytoplasmic level of toxic a-syn could bind to the phosphorylated form of TFEB thus prevent it from getting access to the nucleus. This process, in turn, will result in impaired ALP function and defective clearance of toxic a-syn oligomers."
"Our observation in sections from human postmortem tissue show that TFEB is indeed expressed in human nigral DA neurons and is normally distributed in both the cytoplasm and nucleus. In contrast, in the PD cases TFEB expression in the nuclear compartment was much reduced, providing evidence that this mechanism may be operating also in human PD. Previous observation of postmortem material have shown that 14-3-3 proteins and ALP markers...are present in Lewy Bodies. The colocalization of TFEB with a-syn in Lewy Bodies is an intriguing and interesting finding, suggesting that TFEB may be sequestered--and thus inactivated--together with a-syn in the intracellular inclusions."
"Recent studies have shown that the nuclear translocation of TFEB is regulated by mTOR, as part of the mTORC1 complex."
"Here we show that inhibition of mTOR activity...with the rapamycin analog temsirolimus was efficient in blocking the development of a-syn-induced pathology in the AAV-a-syn treated rats..."In these animals we observed a sustained reduction in phospho-mTOR levels, coupled with an increased nuclear translocation of TFEB and a reduce formation of a-syn oligomers. These data suggests that stimulation of TFEB function through mTOR inhibition may be an efficient tool to block DA neurodegeneration in PD, and also to counteract more widespread a-syn aggregation and pathological changes associated with a-synucleinopathy."
"In summary, the results highlight TFEB as a key player in the development of a-syn-induced toxicity and PD-like pathogenesis, and identify this transcription factor as an interesting target for neuroprotective or disease-modifying therapies in PD."
"In the model proposed here, protein clearance by the ALP will initially be activated in response to elevated levels of a-syn, indeed by translocation of TFEB to the nucleus. When a-syn reaches toxic levels, more and more of TFEB will be bound to a-syn and sequestered in cytoplasmic inclusions, thus preventing its translocation to the nucleus. Our results show that therapeutic interventions using...pharmacologic tools aimed to stimulate nuclear translocation of endogenous TFEB (e.g. through inhibition of mTORC1 function) will be effective not only in blocking the development of a-syn-induced toxicity, but also as a disease-modifying intervention to block the progression of pathology in already affected neurons."
"Although current mTOR inhibitors, such as rapamycin and temsirolimus are FDA approved for use in patients, their pharmacokinetic profile and side effects make them less likely to be useful for long-term use in disease-modifying therapies."
ASG note: This is a most extraordinary research paper regarding the pathogenesis of PD and how rapamycin can be used to stop disease progression. The tragedy of rapamycin is that researchers repeat the "old wives tale" about "side-effects" based on experience with rapamycin use in transplant medicine and fail to understand that rapamycin can be used intermittently to reduce mTORC1 without the side-effects of daily use in transplant medicine.
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2017 paper from China. Zhang.
"Rapamycin protects mice against MPTP induced loss of dopaminergic neurons, which is an established model for Parkinson's disease". We demonstrate that rapamycin preserves astrocytic expression of glutamate transporters and glutamate reuptake. the protectiveeffect was also observe in astrocyte cultures, indicating that rapamycin acts directly on astrocytes.
In the MPTP model, rapamycin caused reduced expression (of ubiquitin) and reduced colocalization of glutamate transporters with ubiquitin." [Ubiquitin associated with destruction with proteases, so reduction of colocalization with ubiquitin indicates preservation structures]
"Rapamycin increased interleukin 6 expression, which was associated with reduced expression of inflammatory cytokines."
"These results demonstrate for the first time in a Parkinson's disease animal model that the neuroprotective effects of rapamycin are associated with glial [astrocytes] and anti-inflammatory effects."
The motor symptoms of Parkinson's disease (PD) are predominantly due to degeneration of dopaminergic neurons in the pars compacta of substantia nigra (SNpc), and multiple mechanisms are believed to contribute to neurodegeneration in PD, including alpha-synuclein toxicity, mitochondrial impairment, reactive oxygen species, glutamate excitotoxicity and increased inflammation.
The MPTP of PD model recapitulates the motor symptoms and preferential cell loss in the SNpc. Its features also include mitochondrial dysfunction, glutamate excitotoxicity, inflammatory response, and so on."
"a previous study revealed that RTP801 is a proapoptotic protein that is sufficient and necessary to induce neuronal death in cellular and animal models of PD, and rapamycin blockes expression of RTP801 in DA neurons and preserves phosphorylation of phosphoinositide 3-kinaseat a critical residue. [Malagelada study discussed above]
In this study, we demonstrate that rapamycin directly targets glial cells to limit two well-establishedcontributing factors to PD pathology: astrocytic activation and the inflammatory response.
1. Rapamycin is neuroprotective in the MPTP model of PD.
The mice exposed to MPTP developed the typical behavor and histopathological deficits including reduced holding time, increased climbing time, and marked reduction of tyrosine in Snpc. and cell death and alpha-synuclein expression in Snpc. Treatment with rapamycin prevented these pathological developments and cell death.
"these results verify protective effects of rapamycin in the MPTP mouse model of PD.
[the rest of results examine the specific mechanism of action]
2. "Rapamycin increases the expression and function of glutamate transporters in in vitro and in vivo models of PD."