extfieldsofstudy: Chemistry Medicine

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What does LTP tell us about the roles of CaMKII and PKMζ in memory?

In “Criteria for identifying the molecular basis of the engram (CaMKII, PKMζ),” Lisman proposes that elucidating the mechanism of LTP maintenance is key to understanding memory storage. He suggests three criteria for a maintenance mechanism to evaluate data on CaMKII and PKMζ as memory storage molecules: necessity, occlusion, and erasure. Here we show that when the criteria are tested, the results reveal important differences between the molecules. Inhibiting PKMζ reverses established, protein synthesis-dependent late-LTP, without affecting early-LTP or baseline synaptic transmission. In contrast, blocking CaMKII has two effects: 1) inhibiting CaMKII activity blocks LTP induction but not maintenance, and 2) disrupting CaMKII interactions with NMDARs in the postsynaptic density (PSD) depresses both early-LTP and basal synaptic transmission equivalently. To identify a maintenance mechanism, we propose a fourth criterion — persistence. PKMζ increases for hours during LTP maintenance in hippocampal slices, and for over a month in specific brain regions during long-term memory storage in conditioned animals. In contrast, increased CaMKII activity lasts only minutes following LTP induction, and CaMKII translocation to the PSD in late-LTP or memory has not been reported. Lastly, do the PKMζ and CaMKII models integrate the many other signaling molecules important for LTP? Activity-dependent PKMζ synthesis is regulated by many of the signaling molecules that induce LTP, including CaMKII, providing a plausible mechanism for new gene expression in the persistent phosphorylation by PKMζ maintaining late-LTP and memory. In contrast, CaMKII autophosphorylation and translocation do not appear to require new protein synthesis. Therefore, the cumulative evidence supports a core role for PKMζ in late-LTP and long-term memory maintenance, and separate roles for CaMKII in LTP induction and for the maintenance of postsynaptic structure and synaptic transmission in a mechanism distinct from late-LTP.

Introduction
For many years the notion of a persistently active, longterm memory storage molecule seemed superfluous. Active enzymatic processes were thought to maintain only shortterm memory, not long-term memory. This idea was consistent with the view that most molecular signaling events in cells were short-lived. As shown in model systems such as Aplysia californica, experiences that produced shortterm memory induced strong synaptic stimulation that increased the amounts of short-lived second messengers within neurons to transiently activate protein kinases [1]. The transient activation of these kinases briefly enhanced synaptic strength to modify the neuronal circuits underlying behavior for a short time. If the stimulation induced by experience was strong enough, some of these signaling molecules would also transiently upregulate gene expression, leading to a brief period of new synthesis of proteins that could support synaptic growth within the circuits [2]. Once formed, these new synapses were presumed to be stable anatomical structures that would permanently alter the circuits to maintain long-term memory without the requirement for special persistently active memory molecules.
In the mid-1980's, however, three highly creative thinkers, now sadly all departed: Francis Crick, James H. Schwartz, and John Lisman, championed the notion that memory might be maintained by active mechanisms, in particular persistently active protein kinases. All three suggested the possibility that with strong stimulation the kinases that mediate short-term memory could be converted from transiently active, second messenger-dependent forms into persistently active, second messenger-independent forms. These autonomously active enzymes, "cognitive kinases" in Schwartz's phrase [3], might then sustain enhanced synaptic transmission during persistent forms of memory. Crick proposed a theoretical model involving positive feedback between molecular dimers, analogous to the mechanism for stability of the double helix of DNA [4]. Schwartz focused on the persistent action of the cAMP-dependent protein kinase (PKA), which mediated short-term memory in Aplysia, through the degradation of its autoinhibitory regulatory unit. He proposed that this persistence underlies an intermediate-phase of memory between short-and long-term memory [5].
Lisman's proposal focused on Ca 2+ /calmodulin (CaM)dependent protein kinase II (CaMKII), an abundant protein of the PSD of glutamatergic synapses, that built on the earlier biochemical work on CaMKII by Schwartz [6] and Mary Kennedy [7]. Schwartz and Kennedy had found that, once CaMKII was activated, its autophosphorylation reduced the enzyme's requirement for Ca 2+ /CaM, converting the kinase into an autonomously active form. Lisman championed the idea that the autophosphorylation of CaMKII on threonine-286 (T286) and the subsequent autonomous activity of the enzyme described by Kennedy maintained LTP, a leading putative physiological substrate of memory in vertebrates [8,9].
In the early 1990's, a third kinase, protein kinase C (PKC), was also found to have an autonomously active form. By this time, PKC had been shown not to be the product of a single gene, but a small gene family of~11 isoforms, divided into conventional, novel, and atypical classes [10]. Most of the PKCs were activated by second messengers that bound to the kinase's regulatory domain, releasing the domain's autoinhibition of the PKC catalytic domain. One of the atypical isoforms, however, termed PKMζ, was an independent PKC catalytic domain that lacked an autoinhibitory regulatory domain, rendering it an autonomously active kinase [11]. Unlike the other isoforms that were activated only briefly in LTP by transient increases in second messengers such as Ca 2+ and diacylglycerol, PKMζ persistently increased in LTP maintenance through a protein synthesis-dependent mechanism. PKMζ was formed in neurons from a dedicated dendritic PKMζ mRNA, which normally was translationally repressed and unable to synthesize protein [11][12][13][14]. Strong synaptic activity, however, derepressed the PKMζ mRNA, driving de novo synthesis of the autonomously active kinase. Over the years, as interest in LTP as a potential mechanism of memory expanded rapidly, evidence for the roles of both CaMKII and PKMζ in these processes grew [15][16][17][18].

Inclusion vs. exclusion of evidence for CaMKII and PKMζ
In his review, Lisman compares the evidence supporting CaMKII and PKMζ in LTP and memory maintenance, focusing on three criteria that he proposes to identify a maintenance mechanism: necessity, occlusion, and erasure [16]. He concludes that there are data supporting both kinases, but the evidence for CaMKII is stronger. Lisman's conclusion, however, requires excluding evidence in support of PKMζ from a large number of studies that use the peptide PKMζ-inhibitor ZIP, which mimics the pseudosubstrate inhibition of the PKCζ regulatory domain that is missing from PKMζ. These data were excluded because of ZIP's potential off-target effects [19][20][21][22], while ignoring the direct electrophysiological evidence to the contrary in the excluded LTP and memory experiments [23][24][25].
To reach his conclusion that ZIP's effects are non-specific, Lisman excluded three sets of data. First, in normal animals, ZIP's ability to reverse LTP and erase long-term memory crucially depends upon the peptide's ability to block the specific mechanism of action by which PKMζ potentiates postsynaptic AMPAR responses [26][27][28]. PKMζ causes and maintains potentiation of postsynaptic AMPAR responses by a mechanism distinct from CaMKII or other PKCs. Instead of increases in AMPAR unit conductance or exocytosis of the receptor to the plasma membrane, PKMζ maintains synaptic potentiation by decreasing postsynaptic AMPAR endocytosis [26,29]. This action of PKMζ, which causes and sustains a doubling of the number of functional AMPAR channels at postsynaptic sites [30], is mediated specifically by inhibiting GluA2 subunit-dependent endocytosis [29]. If this GluA2-dependent endocytosis is blocked by the peptide GluR23Y, ZIP has no effect on LTP or long-term memory [26][27][28]. These results demonstrate that ZIP targets PKMζ's mechanism of action and thus reverses LTP maintenance and erases memory by acting on PKMζ or on a molecule with a very similar action.
Second, ZIP's effects in mutant PKMζ-knock-out mice on LTP and memory [19,20] are not due to non-specific effects of the drug, but rather the recruitment in the mutant mice of a different target of drug action than in wild-type mice [25]. Antisense oligodeoxynucleotides that specifically block new synthesis of PKMζ prevent the formation of late-LTP and long-term memory in wildtype mice, but not in PKMζ-knock-out mice, demonstrating that the maintenance mechanisms in these two genotypes are different. In mice lacking PKMζ, another atypical PKC isoform, PKCι/λ, which like PKMζ is sensitive to ZIP, becomes persistently active in late-LTP and long-term memory to compensate for PKMζ.
Third, bath applications of ZIP block the synaptic potentiation caused by postsynaptic perfusion of PKMζ or PKCι/λ, but not the potentiation induced by phorbol esters, activators of the full-length conventional and novel PKCs, demonstrating selectivity of the agent's action within neurons [25]. Moreover, in studies that show ZIP's ability to reverse late-LTP maintenance, ZIP has no effect on basal synaptic transmission recorded in slices [31,32] or in vivo [24,33,34], or on early-LTP maintenance [32]. Likewise, in studies that show ZIP's ability to erase established long-term memory, the drug has no effect on short-term memory [24] (but see [35]), or on long-term memory that was recently reactivated [36]. Long-term memories that have recently been recalled may undergo an active reconsolidation process that is sensitive to blockers of consolidation, such as protein synthesis inhibitors [37], but is resistant to ZIP [36,38].
Once the reconsolidation period has ended, ZIP's ability to erase memory returns. This specific effect of ZIP on PKMs/atypical PKCs, late-LTP, and long-term memory maintenance, and not on full-length conventional/novel PKC isoforms or other forms of synaptic plasticity and memory, is difficult to explain by a non-specific effect of ZIP.
In addition, ZIP's inhibitory action on PKMζ can be distinguished from its reported non-specific effects by the use of a scrambled version of the peptide as control. Whereas ZIP blocks the synaptic potentiation induced by postsynaptic perfusion of PKMζ, scrambled ZIP applied at the same doses does not [39]. In contrast, the non-specific effects reported for ZIP, such as membrane instability and "neuronal silencing," were also reported for scrambled ZIP applied at the same doses as ZIP [21,22], thus documenting inappropriate use or handling of the drug in these studies. The appropriate use of scrambled ZIP as a control, which has no effects in most studies of LTP and memory [32,40,41], provides strong necessity and erasure evidence for the maintenance role of PKMζ or a molecule with very closely related properties.
Also excluded from Lisman's review were many studies that used agents other than ZIP to inhibit the action of PKMζ that result in the same reversal of LTP and memory maintenance. These agents include: 1) dominant negative-PKMζ [31,42], 2) the inhibitor chelerythrine, which selectively inhibits PKM forms at low doses [31], and 3) RNAi that suppresses the expression of PKMζ [43]. RNAi that suppresses the other atypical isoform, PKCι/λ, does not disrupt memory maintenance [43]. Thus, if the studies using ZIP, chelerythrine, dominant negative-PKMζ, and PKMζ-RNAi had not been excluded from Lisman's review, the evidence supporting PKMζ's central role in LTP and long-term memory maintenance might have been much stronger than for CaMKII.

Inhibiting CaMKII and PKMζ reveals their different roles in synaptic transmission and LTP
Importantly, regardless of the method used, when the effects of inhibiting PKMζ and CaMKII are examined, the results reveal that the two molecules play very different roles in LTP and memory. All inhibitors of PKMζ, including ZIP [24,[31][32][33][34], chelerythrine [31,44], and dominant negative mutant forms of PKM [31,45], reverse the maintenance of late-LTP and other forms of long-term synaptic plasticity without disrupting basal synaptic transmission, recorded either in brain slices or in vivo (Fig. 1a). This ability of PKMζ inhibitors to specifically reverse late-LTP maintenance but not basal transmission or early-LTP maintenance is unique in the literature. Indeed, these experiments were the first to demonstrate that a persistently active molecular mechanism maintains late-LTP and long-term memory storage, and they remain difficult to explain by models of memory storage that are sustained solely by structurally stable synapses.
In contrast to inhibiting PKMζ, inhibiting CaMKII does not specifically reverse LTP maintenance, and the effects of the inhibition depend upon the method by which the CaMKII molecule is affected (Fig. 1b). To understand the various effects of inhibiting CaMKII, it is first important to recognize that there are two models of CaMKII's persistent effects in LTP maintenancethe original autophosphorylation model proposed by Lisman in 1988 [8], and a structural model involving translocation of the kinase from cytosol to PSD, shown for a chemical form of LTP in 2004 [46] and formalized by Lisman in 2013 [47]. Whereas in his review Lisman conflates the evidence for the two models in support of a role for CaMKII in LTP and memory, the experiments testing the enzymatic and the structural functions of CaMKII yield very different results.
Most recent studies of the role of CaMKII's enzymatic activity use cell-permeable versions of a peptide, CN21, that mimics the sequence of an endogenous CaMKII inhibitor, which at low doses effectively block both the Ca 2+ /CaM-stimulated and the autonomous activity of the enzyme [48]. These peptides prevent LTP when applied during the strong afferent stimulation that triggers LTP [48][49][50] (Fig. 1b1). But these same inhibitors when applied a few minutes after the stimulation have no effect on LTP [48][49][50]. The most temporally precise measurement used optical stimulation to release the inhibitory action of the peptide and showed LTP blockade when the inhibitor was activated concurrently with strong afferent synaptic stimulation, but no effect when activated~1 min afterward [50]. These inhibitors applied at doses that effectively block Ca 2+ /CaM-stimulated and autonomous CaMKII activity also have no effect on basal synaptic transmission. Such a transient block of LTP, in which inhibitors prevent LTP when applied during stimulation but lose their efficacy when applied after LTP is established, is characteristic of a role in LTP induction and is identical to the effects of inhibiting many other signaling molecules that have been implicated in LTP induction [51]. As such, it is strong evidence for the role of CaMKII phosphotransferase activity in LTP induction, but not LTP maintenance.
In addition to its phosphotransferase activity, however, the abundance of CaMKIIα in the PSD (~80 holoenzymes associated with a typical PSD [52]) suggests that CaMKII may also have a structural role relevant to postsynaptic function. Consistent with this notion, NMDAR activation stimulates both CaMKII autophosphorylation and its translocation from cytosol to the PSD [6,53,54], and it has been suggested that the increased abundance of CaMKII in the PSD may help form physical "slots" for AMPARs [47]. Moreover, the same C21-based inhibitory peptides that block the enzyme's activity at low doses disrupt CaMKII's interaction with NMDARs in the PSD at higher doses. At these higher doses CN21 disrupts both basal AMPAR-mediated synaptic transmission and potentiated synaptic transmission during early-LTP [55][56][57] (Fig. 1b2). The effects of the peptide on potentiated and non-potentiated AMPAR responses are equivalent [55][56][57]. Thus, in contrast to PKMζ, CaMKII's putative structural role has no specificity with respect to maintaining potentiated vs. unpotentiated synapses. Lisman speculates that a chain of molecules extend from the NMDAR-CaMKII complex to delta-catenin, to AMPA-binding protein, and then to AMPARs [47]. When the structure-disrupting peptide is washed-out,

Fig. 1 PKMζ and CaMKII inhibition differ in their effects on LTP and basal synaptic transmission. A) Above, schematic representation showing
PKMζ inhibition reverses late-LTP when inhibitors are applied after late-LTP is established. After the inhibitor is eliminated, the potentiation does not return, indicating PKMζ's role in maintaining late-LTP. Below, in a separate synaptic pathway, PKMζ inhibition has no effect on basal, untetanized synaptic transmission. PKMζ inhibition has no effect on early-LTP maintenance (not shown). B) The effects of CaMKII inhibition depend on the mechanism of inhibition. B1) Inhibition of CaMKII activity blocks LTP induction (above), but has no effect on LTP maintenance (middle) or basal synaptic transmission (below). B2) Disrupting the interaction between CaMKII and NMDAR decreases both early-LTP (above) and basal synaptic transmission (below) with no specificity for potentiated vs. unpotentiated synapses. Elimination of the blockers of CaMKII-NMDAR interaction shows incomplete reversal, indicating an effect on the maintenance of AMPAR-mediated synaptic transmission the depressed synaptic response does not fully return to the baseline response (Fig. 1b2), an effect that Lisman views as crucial evidence for a mnemonic role of CaMKII, but is equally, and perhaps more parsimoniously consistent with a role for CaMKII in maintaining postsynaptic structure.
The disruption of synaptic transmission by CaMKII inhibition influences the interpretation of CaMKII memory "erasure" experiments Because PKMζ inhibitors specifically reverse late-LTP maintenance and do not impair baseline synaptic transmission or early-LTP, PKMζ, or a molecule with very similar properties, is clearly crucial for maintaining late-LTP maintenance. Thus, the selective reversal of only potentiated synapses and not unpotentiated synapses induced by PKMζ inhibitors, as shown both in slices and in vivo, established a paradigm for causally linking LTP maintenance to memory storage by "erasure" experiments [24].
In contrast, CaMKII structural blockers irreversibly depress basal synaptic transmission and early-LTP maintenance equivalently [55][56][57]. Therefore, LTP maintenance reversal experiments that rely on CaMKII structural blockade are difficult to interpret because the inhibitors confound the possible role of CaMKII in maintaining LTP and its demonstrable role in maintaining synapse structure and general synaptic transmission. Thus, CaMKII's role in LTP maintenance is unclear.
Analogously, Lisman's recent memory erasure experiments involving overexpression of a dominant negative, K42-mutated form of CaMKII do not provide strong evidence for CaMKII's role in memory maintenance [58]. In the context of endogenous CaMKII knockdown in neurons that causes a~60% decrease in basal synaptic transmission, overexpression of wild-type CaMKIIα can support basal synaptic transmission, rescuing postsynaptic AMPAR transmission to normal levels [59]. In contrast, K42-mutants of CaMKIIα cannot support basal synaptic transmission [59]. Likewise, overexpression in neurons of K42-mutated CaMKIIα acts as a dominant negative mutation by inducing a general reduction in synaptic transmission by nearly 50% [60], similar to the reduction of strength of both potentiated and unpotentiated synapses observed with applications of the CaMKII-NMDAR blocking peptide. Such a non-specific reduction of synaptic transmission is likely to disrupt the function of pre-existing functional networks of neurons that are thought to maintain long-term memories. The non-specific reduction should prevent the expression of memory that was previously stored in the functional network, potentially without affecting the capacity of the newly reconfigured network to acquire new memories. The same pattern of impaired memory and spared learning in water maze tasks has been observed after temporary inactivation or permanent lesion of 40% of hippocampus [61,62]. Thus, similar to the observations of depressed synaptic transmission in hippocampal neurons after K42-mutated CaMKIIα overexpression, the effects of K42-mutated CaMKIIα overexpression in behaving animals are most parsimoniously attributed to structural impairment of normal synaptic transmission in hippocampal circuitry rather than to a specific impairment of memory maintenance.

Overexpression of CaMKII mutants and PKMζ
As another criterion, Lisman asserts that overexpression of maintenance molecules predicts saturation of synaptic transmission and loss of memory, but this is not necessarily the case. First, in contrast to early-LTP, which shows no further enhancement with repeated tetanization (i.e., saturation), late-LTP, the putative storage mechanism of long-term memory, does not saturate with repeated tetanization [63]. Indeed, saturation of LTP in awake rats fails to impair acquisition and recall of water maze memory, if the rats first learn the water maze task, explicitly demonstrating Lisman's assertion is not necessarily the case [64].
In this light, the precise nature of the overexpressed kinase and the method of overexpression must be carefully considered when interpreting overexpression experiments. In contrast to postsynaptic perfusion of PKMζ that is sufficient to mimic and occlude LTP [25,30,31], viral overexpression of PKMζ in neurons enhances only a subset of synapses [65,66], and most of the overexpressed PKMζ appears to be excluded from synaptic sites [67]. Additional NMDAR stimulation of the synapses allows overexpressed PKMζ to translocate to the PSD [67], in a process possibly related to PKMζ's role as a "plasticityrelated protein" (PRP) [32] during synaptic tagging [68]. This "capture" of PRPs by activated ("tagged") synapses may be the mechanism by which viral overexpression of PKMζ enhances long-term memory, which has been observed in several tasks [42,69,70].
Lisman's recent occlusion experiments used overexpression of this mutant T286D/T305A/T306A version of CaMKIIα, engineered to simulate persistent activation of the kinase that is also immune to deactivation [58]. This overexpression caused amnesia [58], but what it means is unclear. This is because, although overexpression of T286D/T305A/T306A mutated CaMKIIα may simulate persistent activation of the kinase, inhibition of the kinase's activity does not impair the maintenance of either LTP, as discussed above, or long-term memory [48,50], making it unlikely that CaMKII activation actually persists in LTP and long-term memory.
Indeed, the overexpression within neurons of CaMKIIα containing the T305A/T306A mutations without the T286D mutation causes synaptic potentiation that is rapidly reversed by acute applications of NMDAR antagonists [59]. These findings suggest that once T286 of CaMKII undergoes autophosphorylation within neurons, the persistence of this autophosphorylation is not maintained without continual Ca 2+ /CaM stimulation [59]. Because neither LTP maintenance nor long-term memory storage is reversed by acute applications of NMDAR antagonists [74], the persistent synaptic potentiation by CaMKII overexpression and the physiological processes of LTP and memory appear to be mechanistically distinct.

Are the actions of PKMζ and CaMKII in LTP and memory persistent?
If occlusion experiments are difficult to interpret, and the effects of CaMKII structural inhibitors on LTP and memory are confounded by their general disruption of synaptic transmission, how might one further evaluate PKMζ and CaMKII as memory maintenance molecules? An additional criterion for a maintenance mechanism might be helpfulpersistence.
Persistence is the hallmark of PKMζ's action during LTP maintenancethe increase in the autonomously active kinase lasts for hours in hippocampal slices [12,25]. Recently, persistent increases in PKMζ have been examined in various forms of long-term memory. In the hippocampus, these experience-induced increases last for at least a week following trace conditioning [43] and for at least a month following spatial conditioning [75]. In neocortex, the persistence of PKMζ increases can vary in different layers and regions, lasting for at least 40 days in the output layer 5 of motor cortex during the long-term storage of skilled motor memory [38].
In contrast to the persistent increase of PKMζ in LTP, the increase in the autonomous activity of CaMKII in LTP is transient, lasting at most a few minutes after the afferent synaptic tetanization that induces LTP, as measured by either biochemical assays of endogenous CaMKII [76] or by changes in the signal of a fluorescent version of CaMKII [77]. This brief duration of CaMKII activation after stimulation is in line with the~1 min time-window of the efficacy of CaMKII inhibitors, as discussed above [48,50].
In contrast to the very short-lived increase of autonomous CaMKII activity, CaMKII translocation to the PSD has been reported to last~1 h following chemically induced LTP [46]. This time course is consistent with a role in early-LTP, and thus, further work is required to examine whether the translocation persists into late-LTP maintenance or occurs during long-term memory storage in vivo.
Do the PKMζ and CaMKII models integrate the actions of other molecules implicated in late-LTP and long-term memory?
Lastly, one might also consider whether the models for the persistent action of PKMζ and CaMKII are integrated with the many other signaling molecules implicated in LTP. Do the models help to explain and even predict experimental data on the molecular mechanisms of all phases of LTP: induction, expression, and maintenance?
LTP induction is triggered by Ca 2+ influx through activated NMDARs, which then stimulates multiple signal transduction pathways. The persistent increase of PKMζ protein through the upregulation of PKMζ mRNA translation in LTP requires many of these transient, early signaling events, including NMDAR, CaMKII, ERK, PKA, and mTOR activation, BDNF signaling, and actin filament formation [11,[78][79][80]. In contrast, CaMKII autophosphorylation requires only increased intracellular Ca 2+ through the activated NMDAR to bind to CaM [8]. CaMKII translocation from cytosol to PSD is also triggered by NMDAR activation [46], but it is not known whether other downstream signaling mechanisms including new protein synthesis are required. Furthermore, it is unclear precisely how CaMKII-NMDAR structural associations lead to increased AMPAR-mediated synaptic transmission.
The mechanism of expression by which PKMζ enhances postsynaptic AMPAR responses during late-LTP is through stabilizing GluA2-containing AMPARs and decreasing GluA2-mediated endocytosis [26,29,66]. These findings led to the discovery that agents that destabilize postsynaptic GluA2 can also disrupt established late-LTP and long-term memory [29,81], and, conversely, that inhibitors of AMPAR endocytosis not only block the amnestic effects of ZIP [26], but also, when applied by themselves, prolong LTP and long-term memory maintenance [82][83][84]. Further work will be required to see if the models of CaMKII persistence have equivalent explanatory and predictive powers.
Finally, as discussed above, late-LTP maintenance is reversed by all known PKMζ inhibitors, and early-LTP and basal synaptic transmission are not affected by these agents. This specific action on late-LTP maintenance and not early-LTP or basal synaptic transmission is not explained by the CaMKII autophosphorylation or structural models. Conversely, however, the ability of CaMKII activity inhibitors to suppress LTP induction can be explained by observations that CaMKII activity is required to induce new PKMζ synthesis [78]. Likewise, CaMKII's structural role may be important in maintaining the postsynaptic neurotransmission that PKMζ modulates in late-LTP.

Conclusions
When the criteria of necessity, occlusion, erasure, and persistence are examined in detail, the cumulative evidence strongly supports the persistent action of PKMζ as a core molecular mechanism of late-LTP and long-term memory maintenance. CaMKII appears to have two roles: an enzymatic role that is essential for the induction of LTP, and a structural role involving interaction with the NMDAR that maintains synaptic transmission regardless of the state of potentiation. Further work will be required to evaluate whether this structural role of CaMKII is also: 1) one of several transient, post-translational mechanisms upregulated in early-LTP, 2) an expression mechanism of late-LTP and long-term memory, downstream of maintenance by PKMζ, or 3) as Lisman proposed, a maintenance mechanism of a form of synaptic plasticity, independent of PKMζ. A fundamental difference between the molecules is that CaMKII structural inhibitors generally disrupt AMPAR-mediated synaptic transmission, including basal transmission, whereas PKMζ inhibitors specifically disrupt only potentiated synaptic transmission during late-LTP. Thus, one scenario that remains to be fully investigated is that CaMKII maintains the synaptic plasticity involving the initial "AMPAfication" of NMDAR-only, "silent" synapses that occurs during development, and PKMζ maintains further potentiation of only a few of these NMDAR/AMPAR-containing synapses to sparsely encode and store information acquired during learning and experience. With John gone, we hope someone picks up his mantle to explore these and other possibilities.


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MIST1 regulates SNAI1 and acts through the PTEN/AKT signaling axis to promote anoikisresistance in human melanoma cells

Cutaneous malignant melanoma (CMM) is one of the most dangerous types of skin cancer. The prognosis of CMM patients with ulcers, regional lymph node metastasis or organ metastasis is poor. In this process, resistance to anoikis is a critical step in tumor cell metastasis. Tumor cells survive in the vascular and lymphatic system through the escape of anoikis to finally form clones in the distal tissue. The present study revealed that muscle intestine and stomach expression 1 (MIST1), a secreting cell-restricted transcription factor, was overexpressed in melanoma cells. At the same time, the expression of SNAI1 was also high. High expression of MIST1 and SNAI1 all contributed to melanoma cells bypassing anoikis. By changing the expression of MIST1, SNAI1 was indicated to be a downstream gene of MIST1. Chromatin immunoprecipitation and luciferase reporter gene technology revealed that MIST1 promoted the expression of SNAI1 by directly binding to its promoter region. Furthermore, inhibition of the phosphorylation/activity of Akt by LY294002 and knockdown of phosphatase and tensin homologue (PTEN) with simultaneous upregulation or knockdown of MIST1 revealed that SNAI1 improved the phosphorylation of Akt by inhibiting the expression of PTEN. These results suggested that MIST1 hijacked the PTEN/AKT signaling pathway through directly regulating SNAI1 and affected the anoikis resistance capacity of melanoma cells.

Introduction
Cutaneous malignant melanoma (CMM) is a type of skin tumor. This malignant type of neoplasm evolves from melanoma cells at the base of the epidermis. Its incidence rate accounts for 1-3% of all malignant tumor types and 5-10% of malignant skin tumors, and has demonstrated a clear upward trend in recent years (1). It is currently thought that the pathogenesis of MM is primarily associated with solar irradiation of the skin. Ultraviolet rays in the sunlight burn the skin to induce DNA mutations. Mutations in P16 or cyclin-dependent kinase inhibitor 2A located on the short arm of chromosome 9 have been identified as the major cause of genetic pre-disposition to MM (2). Surgery, radiotherapy and chemotherapy are the major treatments of MM; however, the efficacy is low. The discovery of melanoma-associated mutations in genes in recent years and in-depth study may provide approaches for targeted therapies for a new era of MM treatment.
Normal cells gather together and adhere to the extracellular matrix (ECM). They survive through mutual substances and signal conduction. Once they lose contact with the ECM, the cells undergo programmed death, also known as apoptosis. This form of cell death was first named anoikis in 1994 (3). Anoikis is indispensable to maintain the stability of the body tissue. Its main role is to prevent abnormal cell growth or adhesion of cells to the abnormal ECM. Resistance to anoikis is a hallmark of tumor metastasis, allowing tumor cells to spread through the circulatory system to other distant organs. Tumor cells are resistant to apoptosis through autocrine and paracrine mechanisms after losing cell-to-ECM and cell-to-cell contact, and regain the ability to diffuse, metastasize and invade (4)(5)(6). Studies have demonstrated that integrins as inhibitors of anoikis have a profound effect on cell survival (7,8). The phosphoinositide 3-kinase (PI3K)/Akt and mitogen-associated protein kinase kinase/extracellular signal-regulated kinase pathways are involved in the critical step of resistance to anoikis (4).
Muscle intestine and stomach expression 1 (MIST1), also referred to as basic helix-loop-helix (bHLH) family, member a15, is a transcriptional factor. The bHLH motif, which is consistent throughout this family, contains ~60 amino acids and consists of a basic region and a HLH motif that binds to DNA (9). It has been reported that MIST1 has an important role in maintaining normal cell numbers and promoting terminal differentiation of acinar cells in pancreatic exocrine glands. These effects indicate that MIST1 is a key anti-cancer factor in pancreatic tumorigenesis (10).
The transcription factor SNAI1 is involved not only in the regulation of primordial germ cell migration, gastrulation and neural crest migration during normal embryonic development, but also has an important role in the occurrence, recurrence, invasion and metastasis of organ tumors and organ fibrosis (11)(12)(13). SNAI1 inhibits the expression of E-cadherin and has a crucial role in epithelial-to-mesenchymal transition (EMT) (14,15). Interfering with SNAI1 expression may increase the death rate of cancer cells (16). In addition, SNAI1 inhibits the expression of phosphatase and tensin homologue (PTEN) through binding to the promoter of PTEN on chromosome 10. This leads to resistance to gamma-ray-induced apoptosis (17).
PI3K/AKT is an important signaling pathway that facilitates the immortalization of cells and promotes the development of normal vascular and tumor angiogenesis (18)(19)(20)(21). PTEN is a lipid phosphatase that dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate and blocks the PI3K/AKT pathway. The effect of PTEN on p27 appears to be mediated via inhibition of the PI3K/Akt pathway. After the expression of PTEN is inhibited, cell cycle progression is accelerated through phosphorylation of Akt and glycogen synthase kinase-3 (22). It is reported that SNAI1 directly represses PTEN and leads to activation of the PI3K/Akt pathway, and then leads to inactivation and phosphorylation of the pro-apoptotic protein Bcl-2-associated death promoter. This contributes to anoikis resistance (23).
The present study initially explored the association between MIST1 and SNAI1 in human melanoma cells. It was found that MIST1 and SNAI1 contributed to the detachment of cells from the cell matrix by altering the expression of certain proteins. Their upregulation was beneficial for survival after suspension culture. The expression of SNAI1 changed with the alteration of MIST1. It was demonstrated that the PTEN/AKT signaling pathway also participated in their function; SNAI1 was found to inhibit the expression of PTEN and activate Akt. This helped cells to bypass anoikis. MIST1 was identified bind to the promoter of SNAI1 and activate the expression of SNAIL1 as a transcription factor, as indicated using chromatin immunoprecipitation (ChIP) and luciferase reporter gene technology.
Adhesion assay. Six-well plates were coated with 10 g/ml fibronectin (cat. no. F2006; Sigma Aldrich; Merck KGaA, Darmstadt, Germany). Then they were blocked at room temperature with 0.2% BSA for 2 h. A total of 1x10 5 cells were placed in each well and incubated in the 37˚C cell incubator for 30 min. Medium was discarded and excess cells were rinsed using PBS. Adhered cells were stained with crystal violet at room temperature for 10 min and photographed under a light microscope.
Cell death detection ELISA. A total of 5x10 4 cells were placed in normal and low-attachment surface 24-well plates. A Cell Death Detection ELISA PLUS (cat. no. 11774425001; Roche Diagnostics, Basel, Switzerland) was used to detect apoptosis level according to the manufacturer's instructions.

RNA isolation and polymerase chain reaction (PCR) analysis.
TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) was used for extracting total RNA from cells. ReverAid First Strand cDNA Synthesis kit (cat. no. K1622; Fermentas; Thermo Fisher Scientific, Inc.) was applied to reverse-transcribe mRNA with a polyA tail into complementary (c)DNA. PCR Master Mix (cat. no. K0172; Invitrogen; Thermo Fisher Scientific, Inc.) was used for PCR. Cycling conditions for the PCR reaction were as follows: Initial denaturation step of 3 min at 94˚C, followed by 31 cycles consisting of 30 sec at 94˚C, 30 sec at 55˚C and 30 sec at 72˚C, with a final extension period of 7 min at 72˚C.
ChIP assay. Direct combination and transcriptional activation of SNAI1 by MIST1 was predicted by the Encyclopedia of DNA Elements in the UCSC database (http://genome.ucsc.edu/ENCODE/). The JASPAR database (http://jaspar.genereg.net/) was used to predict the MIST1 binding motif. Cells were fixed with 1% formaldehyde and terminated with 2.5 mM glycine. The scraped cells were sonicated for lysis in PBS with sodium thiosulfate. The lysates were divided into three aliquots, one of which was a positive control and received no treatment and one of which was a negative control, which was incubated with immunoglobulin G (cat. no. I5006) and protein G PLUS-Agarose (cat no. P7700) 9both from Sigma-Aldrich; Merck KGaA). One third of the cell lysate was used as the test group, and was incubated with antibody against MIST1 (1:100) and Protein G PLUS-Agarose. After removal of RNA and protein, DNA was extracted with phenol-chloroform, respectively. Next, the degree of enrichment was detected using real-time quantitative PCR.
Luciferase reporter gene technology. At 24 h prior to transfection, 1x10 5 A375 and MV3 cells were seeded into 24-well plates. pGL3 luciferase reporters vector containing target fragments (~800 ng) were complexed with 1 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and then added to the cells. These target fragments included -1,199 to -753, Statistical analysis. Statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation for continuous data. One-way analysis of variance was performed for comparison analysis. Dunnett's test was used for pairwise comparisons of multiple treatment groups. All experimental groups were compared with the control groups. P<0.05 was considered to indicate a statistically significant difference.

Results
Expression of MIST1 positively correlates with SNAI1 in normal and melanoma cells. MIST1 is the first transcription factor identified as a protein specifically expressed in serous exocrine cells (24). MIST1 was identified to have an important role in pancreatic cancer. SNAI1 is involved in EMT and is closely associated with tumor metastasis, recurrence and prognosis as a classic zinc finger protein. However, the interaction between these two proteins and their function in human melanoma cells has remained to be fully elucidated. The present study found that the expression of MIST1 and SNAI1 was upregulated in the human melanoma cell lines A375 and MV3 at the RNA and protein level ( Fig. 1A and B). This indicated that MIST1 and SNAI1 are likely to have a role in the development and progression of melanoma. Furthermore, the expression of MIST1 and SNAI1 in normal or melanoma cells was substantially increased after culture in a 24-well plate with a low attachment surface for 24 h (Fig. 1C and D). Leaving the matrix stimulated the cells to express MIST1 and SNAI1. These results indicated that MIST1 and SNAI1 may help cells to bypass anoikis after loss of anchorage to their physical environment.
MIST1 and SNAI1 disrupt cell-matrix adhesion and promote anchorage independence. Previous studies have reported that SNAI1 has a pro-apoptotic function and helps cells to bypass anoikis (23). However, the association of MIST1 with anoikis has remained elusive.
To overexpress MIST1 and SNAI1, a 293T cell lentiviral packaging system was used, with which HUVEC and NHEM were transfected to endogenously express MIST1 and SNAI1 (Fig. 2). First, overexpression of MIST1 was confirmed by western blot analysis ( Fig. 2A). Subsequent cell behavioral experiments revealed that MIST1 expression resulted in a decreased adherence of normal human HUVEC and NHEM to fibronectin (Fig. 2B). As anoikis-sensitive cells, HUVEC and NHEM were used to assess the influence of MIST1 on anoikis. The ratio of dead cells in HUVEC and NHEM overexpressing MIST1 was detected after floating for 24 h by using a BD Cell Death Detection ELISA kit. After overexpression of MIST1, resistance to anoikis was obviously improved (Fig. 2C). Furthermore, HUVEC and NHEM were transfected with SNAI1 and subjected to the above experiments, revealing a similar effect of SNAI1 to that of MIST1 (Fig. 2D-F). These results indicated that MIST1 and SNAI1 promoted anchorage independence in normal human cells.  Following loss of anchorage, MIST1 and SNAI1 protected cells from anoikis.

Knockdown of MIST1 and SNAI1 increases adhesion and induces anoikis in melanoma cells.
Conversely, to determine the role of MIST1 and SNAI1 in human melanoma cells, their expression was altered in tumor cell lines. shRNAs were utilized to reduce the expression of MIST1 and SNAI1 in the A375 and MV3 human melanoma cell lines (Fig. 3). In order to avoid off-target effects, two target sequences were used to reduce expression of MIST1 and SNAI1, respectively, namely vector-shMIST1-1, vector-shMIST1-2, vector-shSNAI1-1 and vector-shSNAI1-2. The knockdown of MIST1 was detected by immunoblot (Fig. 3A). A fibronectin adhesion assay was also performed to confirm the influence of MIST1 on the attachment to the ECM. Knockdown of MIST1 increased the binding of A375 and MV3 human melanoma cell lines to fibronectin (Fig. 3B). After suspension culture for 24 h, the apoptotic rate of A375 and MV3 cells was significantly increased following MIST1 knockdown (Fig. 3C). Furthermore, knockdown of SNAI1 was revealed to have a similar effect to that of MIST1 (Fig. 3D-F). It was apparent that knockdown of endogenous MIST1 and SNAI1 inhibited anchorage independence of human melanoma cells and increased their sensitivity to anoikis. The results of the gain-and loss-of-function studies suggested that MIST1 and SNAI1 help human melanoma cells to part from the ECM and bypass anoikis, which may contribute to the formation of distant metastases.

MIST1 confers anoikis resistance through regulating SNAI1.
As the abovementioned results indicated that MIST1 and SNAI1 had a role in the development and progression of human melanoma cells, the association between them was then assessed. To define the correlation between MIST1 and SNAI1, these two genes were ectopically overexpressed in HUVEC and NHEM cells, which do not highly express them. The results demonstrated that only MIST1 overexpression caused an increase in the expression of SNAI1, but not vice versa (Fig. 4A). After enhancement of the expression of MIST1 or SNAI1, the resistance to anoikis was obviously increased (Fig. 4B). Conversely, knockdown of MIST1 decreased the expression of SNAI1 in the human melanoma cell lines A375 and MV3, which express the two genes at relatively high levels, while knockdown of SNAI1 did not affect the levels of MIST1 (Fig. 4C). After knockdown of MIST1 or SNAI1, the resistance to anoikis was obviously decreased (Fig. 4D). As alteration of MIST1 caused a change of SNAIL1 but not vice versa, it was indicated that MIST1 is an upstream gene of SNAIL1. Furthermore, the results indicated that MIST1 promotes anchorage independence of cells through regulating SNAI1.

MIST1 hijacks the PTEN/AKT signaling pathway via SNAI1.
To investigate the molecular mechanisms via which MIST1 and SNAI1, which are highly expressed in the A375 and MV3 human melanoma cell lines, reduce their sensitivity to anoikis, the activation of Akt kinase, the downstream substrate of PI3K, was examined. Dysregulation of Akt is commonly found in tumor cells and has an important role in resistance to anoikis and promoting cell survival (25). AKT phosphorylation at the Ser473 residue was obviously increased following MIST1 overexpression in the normal human HUVEC cell line. In addition, it was previously reported that SNAI1 directly repressed PTEN, which then led to PI3K/Akt pathway activation, thus contributing to anoikis resistance (23). Consistent with these previous findings, the present study discovered downregulation of PTEN by MIST1 and SNAI1 (Fig. 5A). As a competitive inhibitor of PI3K, LY294002 is generally used to suppress p-AKT (Ser473), and was applied in the present study at 10 µM to reduce p-AKT (Ser473). After inhibition of AKT phosphorylation (Ser473), the MIST1-induced reduction in sensitivity to anoikis was restored ( Fig. 5A and B).
The expression of PTEN was increased in the A375 and MV3 human melanoma cell lines after knockdown of MIST-1. Correspondingly, the levels of p-AKT (Ser473) were obviously decreased (Fig. 5C). shRNA targeting PTEN was used to reduce its expression, which abrogated the decrease in the levels of p-AKT (Ser473) caused by MIST1 knockdown (Fig. 5C). In addition, the MIST1 knockdown-associated increases in anoikis were inhibited by simultaneous PTEN knockdown.
The results of the gain-and loss-of-function study suggested that the PTEN/Akt signaling pathway is involved in the mechanism by which MIST1 and SNAI1 mediat anoikis resistance in human melanoma cells.
MIST1 promotes SNAI1 transcription by directly binding to its promoter region. The aforementioned results confirmed that MIST1 stimulates the expression of SNAI1, but the specific mechanism required further exploration. The MIST1 binding motif predicted by the JASPAR database (http://jaspar.genereg.net/) exists in a binding sequence predicted by the ENCODE database (https://genome.ucsc.edu/ENCODE/) (Fig. 6A). ChIP was used to confirm that MIST1 directly regulated SNAI1. For this experiment, seven regions close to the transcription start site were selected (Fig. 6B). Protein G PLUS-Agarose binding with MIST1 antibody was used to pull down DNA-binding fragments. A ChIP assay using HUVEC and NHEM overexpressing MIST1, the amplification intensity of the R5 fragment from the SNAI1 promoter was significantly higher than that of the other DNA segments, as confirmed by PCR ( Fig. 6C and D). A375 and MV3, which highly express MIST1, were transfected with luciferase reporter vectors. Vectors containing the -1,199 to -753 bp fragment demonstrated enhanced luciferase reporter signaling (Fig. 6E). Taken together, these results strongly indicated that MIST1 regulates the expression of SNAI1 through binding to its promoter to ultimately confer resistance to anoikis in human melanoma cells.

Discussion
Melanoma is a melanocytic malignancy derived from the pigmented areas of the skin, mucous membrane, eye and central nervous system, with a high degree of malignancy and a poor prognosis. According to statistics, ~160,000 patients are newly diagnosed with melanoma worldwide and ~48,000 succumb to melanoma each year (26). The 5-year survival rate of patients with early, advanced and late-stage melanoma, respectively, was 91.2, 61.7 and 15.2%. In addition, the recurrence rate in melanoma patients in remission is 9 times that of other cancer types (26). Current treatments used for melanoma are surgery, radiation therapy, chemotherapy and immunotherapy. The clinical application of the cytotoxic T-lymphocyte-associated protein 4 antibody ipilimumab and the BRAF inhibitor vemurafenib, which were approved for the treatment of advanced melanoma and BRAF mutant melanoma in 2011, improved the treatment of melanoma to a certain degree, a requirement for more in-depth study of the mechanisms underlying the development and metastasis of melanoma remains, as more effective targets to combat melanoma require to be developed.
The present study focused on the key step in the metastasis of melanoma, namely resistance to anoikis. Anoikis helps tumor cells to survive during migration from the original matrix into the blood and lymph nodes. Studies have indicated that ECM as an inhibitor of anoikis and integrins has a profound effect on cell survival. The most important factors in integrin-regulated signal transduction pathways include focal adhesion kinase, integrin-linked protein kinase, tyrosine kinase, PI3K, extracellular signal-regulated kinase and connexin Shc (27). The present study elucidated the upstream signaling of the PTEN/Akt signaling pathway with regard to anoikis.
MIST1 exists in numerous tissue types, but regarding the cell type, mainly in serous exocrine cells. MIST1 is the first transcription factor identified to be a protein specifically expressed by serous exocrine cells and has a key role in the formation and maintenance of serous secretory exocytosis. As a well-known transcription factor, SNAI1 has a critical role in EMT of tumor cells. However, the association between MIST1 and SNAI1 and their role in tumor progression has remained to be elucidated.
In the present study, MIST1 was found to be upregulated in the A375 and MV3 human melanoma cell lines. Ectopic overexpression of MIST1 increased the resistance of normal cells to anoikis. Conversely, knockdown of MIST1 expression reduced the resistance of melanoma cells to anoikis. These suggested that MIST1 has a critical role in the resistance of melanoma cells to anoikis. Gain-and loss-of-function experiments and ChIP confirmed that SNAI1 was the downstream gene of MIST1. Furthermore, a luciferase reporter assay located the MIST1 recognition sequence at -1,199 to -753 bp of the SNAI1 gene promoter. It was further demonstrated that MIST1 promotes the evasion of anoikis by melanoma cells through the PTEN/Akt pathway.
To the best of our knowledge, the present study was the first to report that MIST1 binds directly to the promoter region of SNAI1 and regulates its expression. It was previously demonstrated that SNAI1 inhibits the phosphorylation of Akt by inhibiting the expression of PTEN (23). The present study provided an update regarding the upstream signaling of the PTEN/Akt pathways. This provided a novel target for the clinical treatment of melanoma. However, the specific mechanism underlying the upregulation of MIST1 in melanoma has remained elusive, and its study may provide an approach for the clinical treatment of melanoma. In addition, the role of MIST1 and SNAI1 in anoikis resistance requires further verification in a wide range of tumor types.


Electroacupuncture ameliorate learning and memory by improving N-acetylaspartate and glutamate metabolism in APP/PS1 mice

Objective To explore the precise mechanism of electroacupuncture (EA) to delay cognitive impairment in Alzheimer disease. Methods N-Acetylaspartate (NAA), glutamate (Glu) and myoinositol (mI) metabolism were measured by magnetic resonance spectroscopy, learning and memory of APP/PS1 mouse was evaluated by the Morris water maze test and the step-down avoidance test, neuron survival number and neuronal structure in the hippocampus were observed by Nissl staining, and BDNF and phosphorylated TrkB detected by Western blot. Results EA at DU20 acupuncture significantly improve learning and memory in behavioral tests, up-regulate NAA, Glu and mI metabolism, increase the surviving neurons in hippocampus, and promote the expression of BDNF and TrkB in the APP/PS1 transgenic mice. Conclusion These findings suggested that EA is a potential therapeutic for ameliorate cognitive dysfunction, and it might be due to EA could improve NAA and Glu metabolism by upregulation of BDNF in APP/PS1 mice.

Background
Alzheimer disease (AD), a sever neurodegenerative disease, characterized by a progressive memory and cognitive impairment and affects many people aged over 65 years old. With the world population grows, the incidence of AD and the cost of AD are growing at an everincreasing rate, and it has become a social issue [1]. It is generally agreed that the neuropathologic hallmarks of AD can be summarized as extracellular deposits of amyloid-β protein (Aβ), intracellular neurofibrillary tangles (NFTs) and neuronal loss in the brain [2,3]. The early accumulation of Aβ may lead a series of downstream events including neuronal degeneration, then evolved into volume atrophy and cognitive functional damage of the corresponding brain area gradually. The hippocampus plays an important role in cognitive function in the nervous system, and neurons in hippocampus are the main cells responsible for learning and memory [4,5]. The proliferation and differentiation of neurons have accompany with the process of learning and memory change [6][7][8]. Studies have shown that decreased neurons in CA1 and CA3 of hippocampus are related to cognitive deficits in those specific region [9][10][11][12].
Magnetic resonance spectroscopy (MRS) is an noninvasive method to detect the concentration of metabolites in vivo and it could found the abnormal changes of material metabolism at an early stage of AD [13]. N-Acetylaspartate (NAA), an acetylated aspartic acid compound, is synthesized in the mitochondria and located predominantly in neural bodies, dendrites, and axons [14]. As a marker of neuronal, a regional decrease of NAA may be an indication of neuronal malfunctioning due to diminished neuronal density, neuronal cell loss, and partially neuronal dysfunction [15]. Glutamate (Glu) is the most important excitatory neurotransmitter in the central nervous system which could regulate synaptic transmission, neuronal growth and differentiation, synaptic plasticity and learning and memory [16]. Glu is related to the number of available neuronal cells, and variations of Glu highly suggests that physiological or pathological changes in neurons. Myoinositol (mI) is generally considered to be the most valuable indicator to estimate in the early of AD. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family and it is the most neurotrophic factor in the body, which is tightly regulated by activity-dependent cellular processes and is strongly linked with mechanisms underlying learning and memory. BDNF activation of tyrosine receptor kinase (TrkB) stimulates intracellular signaling cascades implicated in plasticity. Studies have reported that recognition memory was associated with increased release of BDNF in the hippocampus and cortex [17].
Acupuncture is an impactful treatment to ameliorate cognitive impairment of a wide variety of neurological disorders such as Parkinson's disease [18] and vascular dementia [19]. Our previous researches have demonstrated that electroacupuncture (EA) at Baihui (DU20) acupoint could significantly improve the cognitive function and revers the aberrant cell death in 4 month-old APP/PS1 double transgenic mice [20], and promote the level of uptake rate of glucose in 12 month-old [21]. But its mechanism remain to be studied. The aim of the present study was to explore the mechanism of EA to improve learning and memory. In this study, learning and memory was evaluated in APP/PS1 transgenic mice by Morris water maze and step-down avoidance test, the material metabolism of the hippocampus were measured by MRS, the expression of BDNF and TrkB were detected by Western blot, and neuronal morphology were observed by Nissl staining.

Animals
A double transgenic APPSwe/PS1B6C3-Tg [B6C3-Tg (APPswe, PSEN1dE9)85Dbo/MmJNju] mouse model was used in this study. Male double transgenic APP/PS1 mice and wild-type mice were obtained from Model Animal Research Center of Nanjing University (SYXK2013-009). 48 mice (12 month-old) were maintained by crossing the male double transgenic mice with female wild-type mice. These mice were gene identification by PCR analysis of genomic DNA from tail biopsies. All mice were male, weighing (25 ± 2) g, housed at 21-25 °C with 60-70% humidity under controlled conditions (12-h light/dark cycle), free to access diet and water. Behavioral tests were conducted by researchers who were blind to grouping. All animal experiments involved ethical and humane treatment under a license from the Fujian Provincial Bureau of Science.

Experimental protocol
36 mice were randomly divided into three groups, 12 mice in each group: the APP/PS1 double-transgenic mice group (APP/PS1), the EA at Baihui (DU20) acupoint group (APP/PS1+DU20), the EA at non-acupoint group (APP/PS1+NA); and another 12 wild-type littermates were designated as the WT group. After the first time behavioral test, EA stimulation was applied at Baihui point (the intersection of the sagittal midline and the line between the two ears) with 0.5 mm needle inserted at a depth of 5 mm for 4 weeks, once a day, 30 min each time.
The stimulation parameters were set as disperse waves of 1 and 20 Hz and the intensity of the stimulation was 1 mA by EA apparatus (model G6805; Suzhou Medical Appliance Factory, Shanghai, China). In the APP/ PS1+NA group, the non-acupunct point (the area below the costal region, 2 cm superior to the posterior superior iliac spine and ~ 3 cm lateral to the spine) of the mouse's left side was selected to accept electric stimulation. The same needles, stimulation parameters and apparatus were used to treat the two groups (APP/PS1+DU20 and APP/PS1+NA). The APP/PS1 group and the WT group fed and grasped in the same conditions without any treatment.

Morris water maze test
In order to evaluate learning and memory, Morris water maze test was performed for 5 days as previously described [22]. The water maze apparatus (Chinese Academy of Sciences, Beijing, China) consists of a cylindrical tank (diameter 120 cm, height 50 cm, water depth 30 cm) and a camera (BS-602DVDPLUS 2.0 capture) which connected to a computer. The tank is divided into four quadrants and the platform (diameter 10 cm) is placed in the third quadrant, 2 cm from the surface of the water. During the orientation navigation, a mouse was placed in the water from an equidistant location to the platform in each quadrant for 4 days. When a mouse stayed in the platform more than 3 s within 90 s, the time will be recorded as the escape latency of the mouse in this quadrant. If the mouse did not find the platform in 90 s, it would be removed from water and put in the platform for 15 s to learning. The fifth day is probe trials to investigate the consolidation of memory. The platform was removed and the mouse was put in the first quadrant to swim 90 s. The times of each mouse crossed the original platform area and the swimming trajectory were recorded.

Step-down avoidance test
Step-down avoidance test was implement after Morris water maze test to measure the retention of memory, according to described methodology [23,24]. A mouse was put in a cylindrical insulation platform in a reaction box (20 × 20 × 60 cm) to adapt to the surrounding environment for 3 min. When the mouse stepped down the platform, it would receive an electric stimulated (voltage 32 V, current 0.25 A) for 2 s immediately. In the 5 min training session, the mouse would jump on the platform to avoid stimulation, and some mice might jump repeatedly. 24 h after training, the latency period (stepping down from the platform for the first time) and the number of errors (frequency of jumping off the platform) were recorded.

Magnetic resonance imaging
1.2-1.5% isoflurane and 30% oxygen mixed gas were applied to anesthetize mice, then fixing the mouse head in the center of the special surface coil and monitoring the respiratory rate in real time. The body temperature was maintained at 37 ± 2 °C during the scanning. T1WI (T1-weighted images) scanning was performed from sagittal, axial, and coronal to confirm the position. Then T2WI (T2-weighted images) scanning using the rapid acquisition with relaxation enhancement (RARE) sequence, TR = 2500 ms, TE = 36 ms, FOV = 32 mm × 32 mm, slice thickness = 0.8 mm, IMAGE size = 256 × 256, slices = 15, repeated 8 times, taking 5 min of each mouse.

Magnetic resonance spectroscopy
On the basis of the T2WI, volume of interest (VOI) of 1.5 × 1.5 × 1.5 mm 3 was selected in the left hippocampus. A single volume 1 H-MRS spectra was acquired with point-resolved echo spin spectroscopy (PRESS) technique with TR = 2500 ms, TE = 16.168 ms, NEX = 1. Water suppression, calibration and shimming were performed by automatic pre-scanning procedure of each voxel.
Quantification of the metabolite concentration was performed using Topspin 6.0 software package. Four dominant signals were analyzed (Fig. 4): N-acetylaspartate (NAA) at 2.02 ppm, glutamate (Glu) at 2.2 ppma, myoinositol (mI) at 3.56 ppm and creatine (Cr) at 3.02 ppm. The ratios of NAA/Cr, Glu/Cr and MI/Cr were calculated to express the level of the metabolites in the brain.

Nissl staining
Mice were sacrificed after MRS scan. 1% Pentobarbital Sodium (701O031, Solarbio, China) was used to anesthetize animals (50 mg/kg) by intraperitoneal injection. The brain was removed and kept in 4 °C environment immediately when the animal was perfused with 0.9% saline (1010110610200, Xilong chemical company, Shanghai, China) solution and 4% paraformaldehyde (20161007, Fuchen chemical reagents factory, Tianjin, China) via the left ventricle (n = 6). After 24 h, the brain samples were taken for paraffin embedding, and cut into 5 μm thick along coronal sections. Slices were put into the oven at 60 °C for 30-40 min, and dehydrated by gradient alcohol, then immersed in Nissl dyeing solution for 20 min. CA1 and CA3 of the hippocampal were observed by microscopy (Nikon TS100, Japan). Image-proplus was used to count the number of neurons in three randomly selected fields.

Western blotting
The remaining 6 mice in each group were decapitated and the hippocampus was lysated in modified radio immunoprecipitation assay buffer. The mixture was placed in a centrifuge at 4 °C, centrifuged at 14,000 r/min for 5 min, and the bicinchoninic acid (BCA) protein assay kit (23227, Thermo Fisher Scientific, USA) was used to determine the concentration of the extracted protein and calculate the volume of samples after absorbing the supernatant. At first, protein samples (50ug each group) were electrophoresed on SDS-polyacrylamide (dc3512-02, Biomiga, USA) according to the molecular weight of BDNF and TrkB, and then transferred onto PVDF (polyvinylidenefluoride) membranes. Next, incubated BDNF (ab10839, 1: 1000, Abacam, USA), TrkB (ab187041, 1: 500, Abacam, USA), p-TrkB (ab109684, 1: 600, Abacam, USA) and β-actin (20536-1-AP, 1: 8000, Abacam, USA) overnight at 4 °C after blocking 5% skimmed milk at room temperature for 2 h. Subsequently, membranes were incubated horseradish peroxidase labeled secondary antibody (ab97051, 1: 5000, Abcam, USA) at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence kit and examined with the Bio-Image Analysis System (Bio-Rad, Hercules, CA, USA). Measuring the optical density, the expression of BDNF protein was presented as the densitometric ratio of proteins to β-actin, and the expression of p-TrkB protein was presented as the densitometric ratio of TrkB.

Statistical analysis
Date were analyzed using the SPSS20.0 software package, expressed as mean ± SEM. In the Morris water maze test, escape latency statistically difference between groups using repeated measurements of variance analysis, and others were compared by one-way analysis of variance, when P < 0.05 presents the statistically difference was significant.

EA could ameliorate the learning and memory impairment in APP/PS1 transgenic mice
In order to confirm the efficacy of EA at DU20 acupuncture, the behavioral tests were performed twice. In Morris water maze test, swimming paths of different groups were shown in Figs. 1a, 2a. Compared with the WT group, the APP/PS1 group demonstrated that increased of escape latency (Fig. 1b), and reduced of the number of crossing the platform (Fig. 1c) at the baseline (P < 0.01). No difference was observed in the APP/PS1 group, the APP/PS1+DU20 group and the APP/PS1+NA group (P > 0.05). However, EA at DU20 acupuncture significantly reduced the escape latency compared with the APP/PS1 group (P < 0.05) as shown in Fig. 2b; and increased the number of mice crossing the platform in the probe trials (P < 0.05) as shown in Fig. 2c. Furthermore, there was a difference between the APP/PS1+EA group and the APP/PS1+NA group in the Morris water maze test (P < 0.05). These findings suggested that the learning and memory function was improved by EA at DU20 acupuncture in APP/PS1 double transgenic mice.
In step-down avoidance test, compared with the WT group, both of the latency and the number of errors were Fig. 1 The baseline of different groups in Morris water maze task. a Representative swimming paths from different groups. b The average latency to discover the hidden platform. c Platform location crosses. *P < 0.01 versus WT markedly higher in the APP/PS1 group (Fig. 3a, b) before EA treatment (P < 0.01). No difference was observed in the APP/PS1 group, the APP/PS1+DU20 group and the APP/PS1+NA group (P > 0.05). Through 4 weeks EA treatment (Fig. 3c, d), latency was longer and the number of errors was less in the APP/PS1+EA groups than these in the APP/PS1 group (P < 0.01); moreover, latency of the APP/PS1+NA group was longer than the APP/ PS1+DU20 group (P < 0.05). The results of step-down avoidance test were demonstrated that the damaged learning and memory was improved by EA at DU20 acupuncture in APP/PS1 double transgenic mice.
The level of NAA and Glu were increased by EA at DU20 acupuncture in APP/PS1 transgenic mice As shown in Fig. 4a, c, the ratios of NAA/Cr, Glu/Cr and mI/Cr in the left hippocampus were selected as VOI by 1 H-MRS. At the baseline (Fig. 4b), compared with the APP/PS1 group, the contents of NAA/Cr and Glu/Cr were less (P < 0.01), while the content of mI/Cr was higher in the WT group (P < 0.01). These indicated that 12 months old APP/PS1 double transgenic mice had material metabolism changes in the brain. And the level of NAA/Cr, Glu/Cr, mI/Cr were no difference in the APP/PS1 group, the APP/PS1 + DU20 group and the APP/PS1+NA group (P > 0.05). After 4 weeks intervention (Fig. 4d), 1 H-MRS scan had been shown that NAA/Cr and Glu/Cr in the APP/PS1+EA group were higher than those in the APP/PS1 group (P < 0.05), and there was no difference of mI/Cr in the APP/PS1 group, APP/PS1+EA group and APP/PS1+NA group (P > 0.05). The results of 1 H-MRS indicated that the material metabolism including NAA, Glu, and mI could be regulated by EA at DU20 acupuncture in the APP/ PS1 transgenic mice, and these metabolites which were associated with the pathological of AD. The average latency to discover the hidden platform. c Platform location crosses. # P < 0.05 versus APP/PS1 group; ## P < 0.01 versus APP/PS1 group; ▲ P < 0.05 versus APP/PS1+NA group The integrity of neurons in CA1 and CA3 were protected by EA at DU20 acupuncture in APP/PS1 transgenic mice Nissl staining was conducted to observe the neuronal morphology and quantity in CA1 and CA3 of hippocampus. As shown in Fig. 5a, the neurons were significantly shrunken and weakly stained in the APP/PS1 group and APP/PS1+NA group, which indicated that neurons were diffusely deteriorated and a great number of Nissl bodies had been lost. While the neuronal cells destruction in the APP/PS1+DU20 group were significantly decrease.
Compared with the WT group (Fig. 5b), the number of surviving neurons of CA1 and CA3 of the APP/ PS1 group was significantly decreased (P < 0.01). However, EA could increase the surviving neurons (P < 0.05), and there was no difference of neuronal expression between the APP/PS1+DU20 group and APP/PS1+NA group in CA1(P > 0.05); but the population of surviving neurons was increased in CA3 of the APP/PS1+NA group (P < 0.05) compared to the APP/PS1+DU20 group. It is manifested that the number of surviving neurons was increased and their structural integrity in CA1 and CA3 regions were protected by EA in APP/PS1 transgenic mice.

The expression of BDNF and TrkB were up-regulated by EA in APP/PS1 transgenic mice
According to the Western Blotting (Fig. 6a, b), the expression of BDNF and TrkB were reduced in APP/PS1 mice compared with the WT mice (P < 0.01); however EA could up-regulate the level of BDNF and TrkB (P < 0.01), and there was a significantly difference between the APP/PS1+DU20 group and the APP/PS1+NA group (P < 0.05). The results of Western Blot shown that EA

Discussion
As the most frequently used, Baihui (DU20) acupuncture, meeting point on the Governing vessel with the six yang channels, some researches suggested that EA at DU20 acupuncture could ameliorate memory-related performance in many behavioral tests as well as modulate cholinergic neurons and improve cerebral blood flow [25][26][27]. In this present study, Morris water maze test and step-down avoidance test had been shown that the learning and memory were significantly injured in APP/ PS1 transgenic mice. We investigated that treatment with EA at DU20 acupuncture could ameliorate behavioral impairment. However, the improvement was not observed in no-acupunct group. The results suggested that EA at DU20 acupuncture had specificity of improve the learning and memory impairment in APP/PS1 transgenic mice.
AD is an age-related disease, and its diagnosis mainly depends on clinical symptoms and neuropsychological assessments, but these can not reflect neuronal changes Fig. 4 Metabolites measured by using 1H-MRS. a The baseline of the 4 groups by using 1H-MRS spectra. b NAA/Cr, Glu/Cr, mI/Cr of the 4 groups were detected before EA treatment. c NAA/Cr, Glu/Cr, mI/Cr of the 4 groups were detected after EA treatment. d NAA/Cr, Glu/Cr, mI/Cr of the 4 groups were detected after EA treatment for 4 weeks. A: WT group, B: APP/PS1 group, C: APP/PS1+DU20 group; D: APP/PS1+NA group. The square represents the detection area. *P < 0.01 versus WT; # P < 0.05 versus APP/PS1 group; ▲ P < 0.05 versus APP/PS1+NA group in the specific region [28]. Therefore, establish a preclinical diagnosis standard before appearance clinically symptom is urgent. MRI plays an important role in the diagnosis of AD, and it could identify subtle pathophysiological changes before brain structure and behavioral abnormal. MRS is a quantitative analysis and imaging technique by specific nuclear chemical shifts [29]. The change of metabolites in the brain may alter the intracellular metabolic environment, tissue structure, and affect cognitive function. We can measure a range of cognitive-related material metabolism in the brain with MRS, which could be used to diagnosis AD and evaluate the physiological and pathological process accurately.
NAA can be used to quantify the damage of neuronal cells [15]; and Glu can reflect the number of surviving neuronal cells. In our study, the contents of NAA and Glu were lower, and mI was higher in the APP/PS1 group than those in the WT group. At the same time, behavioral tests showed that mice had learning and memory  The expression of the TrkB of the APP/PS1 transgenic mice of the 4 groups. BDNF, brain-derived neurotrophic factor; TrkB, tyrosine kinase receptor B; *P < 0.01 versus WT; ## P < 0.01 versus APP/PS1 group; ▲ P < 0.05 versus APP/PS1+NA group disorders with material metabolic changes, and both of those were the result of neuronal absence [21]. However, EA at DU20 acupuncture could increase the level of NAA and Glu, and improve behavioral test of 12 month-old APP/PS1 double transgenic mice. In the Nissl staining, the changes of neuronal cell number and structure in the APP/PS1+DU20 group reflect that EA at DU20 acupuncture has unique advantage to ameliorate the learning and memory impairment in APP/PS1 double transgenic mice. Researches had been demonstrated that NAA and NAA/ Cr were significantly decreased in the hippocampus or cortex in APP/PS1 double transgenic mice, and it was thought to be associated with deletion of neuronal cells [30]. Besides, Oberg had been shown that compared with 6-9 months old WT mice, Glu had a significantly reduced in APP/PS1 double transgenic mice [31].
The activation of the BDNF-TrkB signal pathway is necessary for consolidate hippocampus-dependent learning and memory. In our study, the absence and structural damage of neuronal cells resulted in inhibition of BDNF-TrkB in the APP/PS1 group, appearing cognitive impairment which performance as learning and memory dysfunction. The contents of NAA and Glu in the APP/ PS1+DU20 group were significantly higher than those in the model group, which promoted the repair of neuronal cell proliferation. Variety of BDNF expression can directly affect NAA concentrations in the brain of patients with degenerative disease [32]. Study had shown that reduced of BDNF mRNA could lead to similarly a low level of NAA, and it is closely related to age-dependent cognitive behavioral decline [33]. In addition, BDNF induced Glu release through activation of the TrkB/phospholipase C-γ (PLC-γ) pathway [34][35][36]. It indicates that the PLC-γ pathway which activated by the BDNF-TrkB signal is necessary to stimulate Glu release. Use exogenous drugs or other agents to increased TrkB expression, could promote the release of Glu [37]. Moreover, the mechanism of how to EA improve the contents of material metabolism in the brain and increase the expression of related proteins needs to be explored in the future.

Conclusion
The present study revealed that electroacupuncture at "Baihui" (DU20) acupunct could increase the levels of NAA and Glu, decrease neuronal damage and up-regulate BDNF and p-TrkB in hippocampus. It might be the mechanism underlying EA improve learning and memory and it also provides the foundation for the clinical treatment and research of cognitive dysfunction in the future.


Does rapamycin slow down time?

According to Einstein’s theory, time is relative and can be made to move slower by increasing the speed of the observer. In the famous twin paradox, this slowing of time enables one identical twin to live longer than the other. Animals treated with rapamycin, an inhibitor of mTOR, also live longer [1-5]. Of course, that does not mean that rapamycin slows time in the Einsteinian sense. Instead, it figuratively slows biological time by slowing seemingly opposite processes. On the one hand, rapamycin (and other mTOR inhibitors) retards cell proliferation, while on the other hand, it retards loss of proliferative potential [6, 7]. In other words, rapamycin decelerates proliferation while preserving the potential to proliferate. In that way, rapamycin suppresses both cell growth and geroconversion (conversion to senescence). It has been calculated that rapamycin slows geroconversion by approximately 3-fold [6]. By doing so, rapamycin slows development and aging, reproduction and menopause, and hyperfunction and functional decline [8]. This is because in each case one process is a continuation of the other (Figure 1, 2). For example, aging is a continuation of developmental growth (Figure 1), and functional decline (loss of function, Figure 2) results from earlier hyperfunction [8]. The slowing of biological time entails “suppression plus preservation.” For example, by suppressing betacell function, rapamycin preserves beta-cell function in the long run [9-11]; or by suppressing reproduction, it preserves the oocytes, thereby delaying menopause [1214]. In theory, a woman who wants to have children later in life could postpone reproduction using rapamycin. This can be seen as “freezing the ovaries” until later in life. Rapamycin suppresses cellular hyperfunction and thus delays all diseases of aging, from cancer to Alzheimer’s [8]. Pathological processes such as age-related diseases are continuations (or exacerbations) of physiological processes. Geroconversion is a continuation of growth (Figure 1), hyperfunction is a continuation of tissuespecific cellular function (Figure 2), age-related hypertension and presbyopia are continuations of developmental trends (see Figure 3 in ref. 15). Therefore, aging is both hyperfunctional and quasi-programmed [8, 16, 17]. (A quasi-program is a purposeless continuation of a developmental program.) Hyperfunction eventually leads to organ damage and functional decline [8, 15]. By suppressing hyperfunction, rapamycin delays organ damage (e.g., infarction) and loss of organ function [8, 15]. In addition to their therapeutic effects, the side effects of rapalogs are also consequences of slowing down time. For example, by slowing cell proliferation, high doses of rapamycin induce reversible anemia, mucositis and skin rash. So why does rapamycin do all that? Rapamycin is produced by the bacterium Streptomyces hygroscopicus, which lives in the soil of Easter island [18]. This wonderful microbe had no intention of slowing time on its mysterious island; instead, it sought to slow down the growth of fungi, its natural enemy. But since rapamycin slows growth, it should also slow aging if aging is a continuation of growth. In fact, it does just that; rapamycin prolongs the lifespan of yeast [19]. Rapalogs (rapamycin, everolimus, temsirolimus and deforolimus) are allosteric inhibitors of mTOR complex 1, a central regulator of RNA translation and cellular growth and metabolism [20-23]. mTOR enhances translation of TOP and TOP-like mRNAs [22, 23]. Rapamycin and, especially, the pan-mTOR inhibitor Torin1 slow this translation [22, 23]. This raises the question, is slowing translation equivalent to slowing time and, if so, can biological time be measured based on the speed of translation? Editorial Essay

Does rapamycin slow down time? Mikhail V. Blagosklonny
According to Einstein's theory, time is relative and can be made to move slower by increasing the speed of the observer. In the famous twin paradox, this slowing of time enables one identical twin to live longer than the other. Animals treated with rapamycin, an inhibitor of mTOR, also live longer [1][2][3][4][5]. Of course, that does not mean that rapamycin slows time in the Einsteinian sense. Instead, it figuratively slows biological time by slowing seemingly opposite processes. On the one hand, rapamycin (and other mTOR inhibitors) retards cell proliferation, while on the other hand, it retards loss of proliferative potential [6,7]. In other words, rapamycin decelerates proliferation while preserving the potential to proliferate. In that way, rapamycin suppresses both cell growth and geroconversion (conversion to senescence). It has been calculated that rapamycin slows geroconversion by approximately 3-fold [6]. By doing so, rapamycin slows development and aging, reproduction and menopause, and hyperfunction and functional decline [8]. This is because in each case one process is a continuation of the other (Figure 1, 2). For example, aging is a continuation of developmental growth (Figure 1), and functional decline (loss of function, Figure  2) results from earlier hyperfunction [8].
The slowing of biological time entails "suppression plus preservation." For example, by suppressing betacell function, rapamycin preserves beta-cell function in the long run [9][10][11]; or by suppressing reproduction, it preserves the oocytes, thereby delaying menopause [12][13][14]. In theory, a woman who wants to have children later in life could postpone reproduction using rapamycin. This can be seen as "freezing the ovaries" until later in life. Rapamycin suppresses cellular hyperfunction and thus delays all diseases of aging, from cancer to Alzheimer's [8]. Pathological processes such as age-related diseases are continuations (or exacerbations) of physiological processes. Geroconversion is a continuation of growth ( Figure 1), hyperfunction is a continuation of tissuespecific cellular function ( Figure 2), age-related hypertension and presbyopia are continuations of developmental trends (see Figure 3 in ref. 15). Therefore, aging is both hyperfunctional and quasi-programmed [8,16,17]. (A quasi-program is a purposeless continuation of a developmental program.) Hyperfunction eventually leads to organ damage and functional decline [8,15]. By suppressing hyperfunction, rapamycin delays organ damage (e.g., infarction) and loss of organ function [8,15]. In addition to their therapeutic effects, the side effects of rapalogs are also consequences of slowing down time. For example, by slowing cell proliferation, high doses of rapamycin induce reversible anemia, mucositis and skin rash.
So why does rapamycin do all that? Rapamycin is produced by the bacterium Streptomyces hygroscopicus, which lives in the soil of Easter island [18]. This wonderful microbe had no intention of slowing time on its mysterious island; instead, it sought to slow down the growth of fungi, its natural enemy. But since rapamycin slows growth, it should also slow aging if aging is a continuation of growth. In fact, it does just that; rapamycin prolongs the lifespan of yeast [19].
Rapalogs (rapamycin, everolimus, temsirolimus and deforolimus) are allosteric inhibitors of mTOR complex 1, a central regulator of RNA translation and cellular growth and metabolism [20][21][22][23]. mTOR enhances translation of TOP and TOP-like mRNAs [22,23]. Rapamycin and, especially, the pan-mTOR inhibitor Torin1 slow this translation [22,23]. This raises the question, is slowing translation equivalent to slowing time and, if so, can biological time be measured based on the speed of translation? The answer to that question is, not entirely. Although mTOR inhibitors may in principle "slow time" by slowing rapamycin-sensitive translation from mRNA to protein, this does not completely explain the gerosuppressive effects of rapamycin or pan-mTOR inhibitors. In fact, inhibitors of S6 kinase slightly exhibit gerosuppressive activities [24], even though S6 kinase is not crucial for RNA translation [23]. So, mTOR inhibitors may affect the speed of aging by suppressing geroconversion ( Figure  1) and cellular function and hyperfunction ( Figure 2) independently of TOP mRNA translation. In addition, rapamycin also slows age-related methylation, or the epigenetic clock [25][26][27][28][29].

Editorial Essay
Another intriguing possibility is that rapamycin slows time by slowing the circadian clock. mTOR inhibition slows the circadian clock and dampens clock oscillations, whereas mTOR activation accelerates the clock and enhances clock oscillations at the level of cells, tissues and mice [30]. Conversely, circadian clock mediators affect the mTOR pathway and aging [31]. Because mTOR activity is itself part of the circadian clock, its sensitivity to rapamycin can vary widely depending on the time of the day and the phase of the clock [32]. This should be taken into account when comparing the numerous studies in mouse models. It should also be taken into account when designing rapamycin-based therapies for aging.
Rapamycin has been combined with several lifeextending drugs in the "Koschei" formula [5]. This rapamycin-based drug combination has been successfully used as an anti-aging therapy at the Alan Green clinic https://rapamycintherapy.com https://roguehealthandfitness.com/rapamycinanti-aging-medicine-an-interview-with-alan-s-green-md/?print=pdf The older we become, the faster time flies. It is initially measured in days, then in weeks, the four seasons, and finally "Winter-Summer" cycles. Of course, this is an illusion, but an annoying one. Would treatment with rapamycin enable us to notice Spring again?