Dysregulation of the mTOR Pathway Mediates Impairment of Synaptic Plasticity in a Mouse Model of Alzheimer's Disease

Article (PDF Available)inPLoS ONE 5(9) · September 2010with41 Reads
DOI: 10.1371/journal.pone.0012845 · Source: PubMed
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr protein kinase that plays a pivotal role in multiple fundamental biological processes, including synaptic plasticity. We explored the relationship between the mTOR pathway and β-amyloid (Aβ)-induced synaptic dysfunction, which is considered to be critical in the pathogenesis of Alzheimer's disease (AD). We provide evidence that inhibition of mTOR signaling correlates with impairment in synaptic plasticity in hippocampal slices from an AD mouse model and in wild-type slices exposed to exogenous Aβ1-42. Importantly, by up-regulating mTOR signaling, glycogen synthase kinase 3 (GSK3) inhibitors rescued LTP in the AD mouse model, and genetic deletion of FK506-binding protein 12 (FKBP12) prevented Aβ-induced impairment in long-term potentiation (LTP). In addition, confocal microscopy demonstrated co-localization of intraneuronal Aβ42 with mTOR. These data support the notion that the mTOR pathway modulates Aβ-related synaptic dysfunction in AD.
Dysregulation of the mTOR Pathway Mediates
Impairment of Synaptic Plasticity in a Mouse Model of
Alzheimer’s Disease
Tao Ma
, Charles A. Hoeffer
, Estibaliz Capetillo-Zarate
, Fangmin Yu
, Helen Wong
, Michael T. Lin
Davide Tampellini
, Eric Klann
, Robert D. Blitzer
, Gunnar K. Gouras
1 Department of Neurology and Neuroscience, Weill Cornell Medical Col lege, New York, New York, United States of America, 2 Center for Neural Science, New York
University, New York, New York, United States of America, 3 Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New
York, United States of America, 4 Rockefeller Unive rsity, New York, New York, United States of America
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr protein kinase that plays a
pivotal role in multiple fundamental biological processes, including synaptic plasticity. We explored the relationship
between the mTOR pathway and b-amyloid (Ab)-induced synaptic dysfunction, which is considered to be critical in the
pathogenesis of Alzheimer’s disease (AD).
Methodology/Principal Findings:
We provide evidence that inhibition of mTOR signaling correlates with impairment in
synaptic plasticity in hippocampal slices from an AD mouse model and in wild-type slices exposed to exogenous Ab1-42.
Importantly, by up-regulating mTOR signaling, glycogen synthase kinase 3 (GSK3) inhibitors rescued LTP in the AD mouse
model, and genetic deletion of FK506-binding protein 12 (FKBP12) prevented Ab-induced impairment in long-term
potentiation (LTP). In addition, confocal microscopy demonstrated co-localization of intraneuronal Ab42 with mTOR.
These data support the notion that the mTOR pathway modulates Ab-related synaptic dysfunction
in AD.
Citation: Ma T, Hoeffer CA, Capetillo-Zarate E, Yu F, Wong H, et al. (2010) Dysregulation of the mTOR Pathway Mediates Impairment of Synaptic Plasticity in a
Mouse Model of Alzheimer’s Disease. PLoS ONE 5(9): e12845. doi:10.1371/journal.pone.0012845
Editor: Mel B. Feany, Brigham and Women’s Hospital, Harvard Medical School, United States of America
Received Jun e 9, 2010; Accepted August 16, 2010; Published September 20, 2010
Copyright: ß 2010 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants NS034007 (EK), GM054508 (RDB), AG027140, AG28174, and AG09464 (GKG). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gkgouras@med.cornell.edu
Increasing evidence supports the idea that in Alzheimer’s
disease (AD) functional impairment of synaptic plasticity develops
before neurodegeneration. Extensive research has shown that
soluble oligomers of b-amyloid peptide (Ab), cleaved from the
amyloid precursor protein (APP), are capable of inhibiting long-
term potentiation (LTP) and causing learning and memory deficits
[1,2]. Formation of Ab-derived plaques, a pathological hallmark of
AD, develops after accumulation of soluble Ab oligomers. These
findings have focused attention on the early, pre-plaque stage of
AD when synaptic plasticity is already impaired by Ab [3,4]. One
of the central questions is how abnormal Ab accumulation in the
brain causes synaptic dysfunction and thus cognitive deficits. The
molecular signaling mechanisms through which Ab exerts its
synapto-toxic effects remain poorly understood.
Mammalian target of rapamycin (mTOR), an evolutionarily
conserved serine/threonine protein kinase, plays an essential role
in the control of protein translation and cell growth by responding
to multiple environmental cues including growth factors, nutrient
state, and energy level, among others. Its importance in cellular
and organismal homeostasis is reflected in the association of
dysregulated mTOR signaling with common diseases such as
cancer and diabetes [5]. More recently, mTOR has also been
shown to be important for neurons. Next to its role in long-term
synaptic plasticity, emerging evidence implicates mTOR in axon
pathfinding and regeneration, dendrite arborization and spine
morphology [6]. Control of protein translation by mTOR occurs
via phosphorylation of at least two well-established downstream
targets: p70 S6 kinase (p70S6K) and a repressor protein of the cap-
binding eukaryotic initiation factor 4E (eIF4E) termed eIF4E-
binding protein (4E-BP). A major upstream regulator of mTOR is
tuberous sclerosis complex 2 (TSC2), which integrates signals from
many other signaling molecules, including Akt (PKB) and glycogen
synthase kinase 3 (GSK3) [5].
Increasing evidence has pointed to a link between mTOR and
AD. First, mTOR is critical for long-lasting forms of synaptic
plasticity and long-term memory (LTM) formation [7], which is
impaired in mouse models of AD. The importance of mTOR in
synaptic plasticity is in agreement with the central role of mTOR
in controlling mRNA translation, since de novo protein synthesis is
involved in these long-lasting forms of synaptic plasticity and LTM
[8]. Second, inhibition of the mTOR pathway was shown to
modulate aging, a well-established risk factor for AD [9,10]. Third,
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autophagy, a pathway for organelle and protein turnover, has
been implicated in the neurodegeneration of AD, and the well-
characterized mTOR inhibitor, rapamycin, is known to induce
autophagy [11]. Finally, mTOR signaling has been shown to be
altered in AD models, although data appears to be conflicting.
Down-regulation of mTOR signaling was reported in neuroblas-
toma cells treated with Ab1-42 and in brains of APP/PS1 mutant
transgenic mice [12]. In contrast, mTOR signaling was shown to
be up-regulated in 7PA2 cells over-expressing mutant APP and in
brains of another AD transgenic mouse, with rapamycin treatment
reported as protective against behavioral decline [13,14]. Howev-
er, whether these signaling changes were related to alterations in
synaptic plasticity was not explored in these studies.
In the current study we asked whether the mTOR signaling
pathway is involved in the well-established Ab-induced impair-
ment of synaptic plasticity. We report that mTOR signaling is
inhibited both in cultured neurons and hippocampal slices from
AD transgenic mice and in wild-type (WT) hippocampal slices
exposed to exogenous Ab1-42, and that this mTOR dysregulation
correlated with impairment in synaptic plasticity. Importantly, up-
regulation of mTOR signaling by both pharmacological and
genetic methods prevented Ab-induced synaptic impairment,
supporting the notion that dysregulation of the mTOR pathway
is critical for the synaptic dysfunction that characterizes AD.
Inhibition of mTOR signaling correlates with impairment
of synaptic plasticity in an AD mouse model
Increasing evidence points to a critical role for mTOR in
synaptic plasticity, aging and autophagy, all of which have been
linked to AD. We therefore examined whether the mTOR
pathway is altered in the well-established Tg2576 AD transgenic
mouse model. Tg2576 mice harbor the human APP transgene
with the Swedish mutation and develop AD-like amyloidosis and
memory deficits [15]. Western blotting was performed in acute
hippocampal slices from 3–4 month-old Tg2576 mice, an age well
before the development of Ab plaques but at which time early
physiological and functional deficits have been described [16,17].
Compared to wild-type (WT) littermate slices, hippocampal slices
from Tg2576 mice showed a significant decrease in the levels of
p70S6K phosphorylated at threonine 389 (phospho-p70S6K), a
site phosphorylated by mTOR and used as a readout for mTOR
activity [18] (Figure 1A). In addition, levels of 4E-BP phosphor-
ylated at threonine 46/47 (phospho-4E-BP) were reduced in
hippocampal slices from Tg2576 compared to WT mice
(Figure 1A). There was an analogous pattern of changes in levels
of phosphorylated p70S6K and 4E-BP in Tg2576 compared to
WT neurons in culture (Figure 1B). To further confirm reduced
phosphorylation of p70S6K in Tg2576 compared to WT mice we
performed immuno-fluorescence confocal microscopy. Consistent
with the biochemical data, immuno-fluorescence of phospho-
p70S6K was also reduced in both hippocampal slices (3–4-month-
old) and primary neurons (12 DIV) of Tg2576 compared to WT
mice (Figures 1C and 1D).
Cultured Tg2576 neurons were previously reported to have early
Ab-dependent reductions in PSD-95 but not in synapsin I [19,20].
We now provide evidence that levels of PSD-95 are similarly
reduced and levels of synapsin I unchanged in hippocampal slices
from Tg2576 compared to WT mice (Figure 1A).
Since brains from advanced human AD were shown to have
isolated increases in phospho-p70S6K, particularly in tangle-
bearing neurons [21], we next examined phospho-p70S6K in
Tg2576 transgenic mouse brains with aging. Of note, the reduced
phospho-p70S6K observed in young 3–4 month-old Tg2576
compared to wild type mice was no longer evident in brain tissue
from older (21 month-old) Tg2576 mice (Figure 1E).
Activation of the mTOR pathway is tightly associated with both
LTP and long-term depression (LTD), two important forms of
synaptic plasticity [7]. Therefore, we next treated slices either with the
adenylyl cyclase activator forskolin (FSK) or the mGluR1/5 agonist
3,5-dihydroxyphenylglycine (DHPG), which are well-established to
induce chemical LTP and LTD, respectively [22,23]. Levels of
phospho-p70S6K were significantly up-regulated in both FSK- and
DHPG-treated slices from WT mice, but these responses were
markedly blunted in slices from 3–4 month-old Tg2576 mice
(Figures 2A and 2B). These data are consistent with impaired
activation of mTOR signaling in hippocampal slices of Tg2576 mice.
Next we examined alterations in synaptic plasticity in Tg2576
mice by inducing LTP with high-frequency stimulation (HFS) at
CA3-CA1 synapses of acute hippocampal slices. Of note, LTP was
inhibited in slices from 3–4-month-old Tg2576 compared to WT
mice (Figure 2C). In contrast, LTP was normal in slices from
younger (2-month-old) Tg2576 mice (Figure S1A). Consistent with
the absence of an LTP phenotype, Western blotting of 2-month-
old Tg2576 slices also demonstrated no alterations in levels of
phosphorylated p70S6K or PSD-95 at this age (Figure S1B). These
data are consistent with prior work showing the onset of
abnormalities in Tg2576 mice at 3–4 months of age [16,17].
We next confirmed previous work showing that LTP in WT
slices was rapamycin-sensitive [24] and noted that the decay of
LTP was similar to that in Tg2576 hippocampal slices (Figure 2C).
To further compare the effects of down-regulation of mTOR
signaling by rapamycin to alterations seen in hippocampal slices
from Tg2576 mice, expression of selective synaptic proteins was
examined in the presence of rapamycin. In WT hippocampal
slices treated with rapamycin, levels of PSD-95 but not synapsin I
were significantly decreased (Figure 2D), similar to the changes
seen in Tg2576 slices (Figure 1A). However, when Tg2576 slices
were treated with rapamycin, there was no further decrease in the
levels of PSD-95 (Figure 2C).
The HFS protocol used in our experiments is known to induce
long-lasting, protein synthesis- and mTOR-dependent LTP [24].
In contrast, weaker HFS, which induces protein synthesis- and
mTOR-independent early LTP, revealed no difference in LTP
between Tg2576 and WT slices (Figure S2A). In addition, paired-
pulse facilitation (PPF) experiments were performed to assess pre-
synaptic plasticity [25], and no significant difference was observed
between 3–4-month-old Tg2576 and WT mice (Figure S2B).
Taken together, the data described above indicate that impaired
up-regulation of mTOR signaling and impaired LTP both occur
in hippocampal slices from 3–4 month old Tg2576 mice, whereas
neither occurs at 2 months of age. Thus, impaired upregulation of
mTOR signaling correlates with an impairment in LTP,
supporting a link between the mTOR signaling pathway and
AD-related synaptic dysfunction.
Up-regulation of the mTOR pathway rescues Ab-related
inhibition of LTP
Because down-regulation of mTOR signaling in Tg2576 mouse
hippocampal slices correlated with impairment in synaptic
plasticity, we next examined whether increasing mTOR signaling
could protect against Ab-related impairment in LTP. The major
negative upstream regulator of mTOR is the TSC2 complex,
which integrates multiple kinase inputs, including Akt and GSK3.
It is known that GSK3 inhibits mTOR signaling by phosphory-
lating TSC2. Since TSC2 is positively regulated by GSK3,
blocking GSK3 activity releases the inhibitory effect of TSC2 on
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mTOR and thereby increases mTOR activity [26,27]. Moreover,
inhibition of GSK3 activity has been shown to strengthen
hippocampal LTP [28]. In the presence of structurally distinct
GSK3 antagonists LiCl (10 mM) or kenpaullone (5
mM), HFS was
now able to induce long-lasting LTP in slices from Tg2576 mice
(Figures 3A and 3B), in comparison to only decremental LTP in
Tg2576 slices in the absence of GSK3 antagonists (Figure 2C). To
confirm that this potentiation of LTP in Tg2576 slices is
dependent on mTOR signaling, we determined whether LiCl-
or kenpaullone-augmented LTP was blocked by rapamycin. Slices
were pretreated with rapamycin for 30 min, followed by
application of LiCl or kenpaullone in the presence of rapamycin.
Rapamycin treatment prevented enhancement of LTP by either
LiCl or kenpaullone in Tg2576 hippocampal slices (Figures 3A
and 3B). Furthermore, Western blotting demonstrated that levels
of phosphorylated p70S6K were significantly elevated by treat-
ment of Tg2576 hippocampal slices with either LiCl or
kenpaullone (Figure 3C), providing support that up-regulation of
mTOR signaling is involved in the rescued LTP in Tg2576 slices.
Inhibition of mTOR signaling and LTP by Ab is prevent ed
in FKBP12 conditional knockout mice
Numerous studies have shown that direct exogenous application
of Ab1-42 causes deficits in hippocampal synaptic plasticity,
Figure 1. mTOR signaling is impaired in an AD mouse model. (A) Western blot of acute hippocampal slices from 3–4-month-old Tg2576 mice
showed decreased levels of phospho-p70S6K (Thr389), phospho-4E-BP (Thr37/46) and PSD-95 compared to wild-type (WT) slices. n = 9. *p,0.05. (B)
Western blot on cultured primary neurons at 12 DIV showed decreased levels of phospho-p70S6K, phospho-4E-BP and PSD-95 in Tg2576 compared
WT neurons. n = 4. *p,0.05. (C and D) Immuno-fluorescence confocal microscopy for phospho-p70S6K in the CA1 region of hippocampal slices (C)
and cultured primary neurons at 12 DIV (D) showed reduced immuno-staining in Tg2576 compared to WT mice. Representative images are shown
from three experiments. Scale bar, 75
mm. (E) In contrast to 3 and 9 month-old mice, reduced phospho-p70S6K was no longer evident of brains from
aged (21–month-old) Tg2576 compared to WT mice. n = 8 for 3-month-old; n = 4 for 9-month-old; n = 4 for 21–month-old. *p,0.05.
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supporting a toxic role for soluble Ab oligomers [1,2]. Western
blotting of our synthetic Ab1-42 preparation indicated the presence
of monomers and oligomers, predominantly dimers and trimers
(Figure 4A), although we cannot exclude the possibility that during
the incubation period higher molecular weight oligomer formation
may have occurred. We previously reported that application of this
Ab1-42 preparation reduced levels of PSD-95 but not of synapsin I
in cultured wild type neurons, analogous to the alterations in these
proteins in Tg2576 compared to WT neurons in culture [20]. We
now determined that levels of PSD-95 but not of synapsin I were
Figure 2. mTOR signaling dysfunction correlates with synaptic plasticity impairment in an AD mouse model. (A) Treatment of slices
with forskolin (FSK, 50
mM, 60 min) induced phosphorylation of p70S6K in WT (n = 10) but not Tg2576 mice (n = 11). Values of densitometry from
drug-treated slices are normalized to their vehicle control. *p,0.05. (B) Treatment of slices with 3,5 dihydroxyphenylglycine (DHPG, 50
mM, 60 min)
induced phosphorylation of p70S6K in WT (n = 9) but not Tg2576 mice (n = 6). Values of densitometry from drug-treated slices are normalized to their
vehicle control. *p,0.05. (C) High frequency stimulation (HFS) induced normal LTP in slices from 3–4-month-old WT mice (open circles), but only
decremental LTP in Tg2576 mice (triangles). Pretreatment of slices with rapamycin (Rapa, 1
mM, 30 min) blocked LTP induced in WT mice (dark gray
circles). n = 4 for WT and Tg; n = 6 for Rapa treated WT. Rapamycin was present throughout the recording. Scale bar, 1 mV/20 ms. The inset traces
show superimposed sample EPSPs recorded during the baseline period (black) and 60 min after HFS (red). (D) Treatment of slices with Rapa (1
120 min) reduced levels of PSD-95 in WT but not Tg2576 mice. n = 5. *p,0.05.
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also reduced in WT hippocampal slices by treatment with this
preparation of synthetic Ab1-42 (Figure 4A), paralleling the results
seen in Tg2576 compared to WT hippocampal slices (Figure 1). Of
note, treatment of WT hippocampal slices with this exogenous Ab1-
42 also caused a marked decrease in levels of phosphorylated
p70S6K and 4E-BP (Figure 4A). Similar reductions in phosphor-
ylated p70S6K and 4E-BP were seen in WT primary neurons
treated with Ab1-42 (data not shown).
To further examine mTOR signaling in Ab-related synaptic
dysfunction, we next utilized a mouse model in which the gene
encoding FK506-binding protein 12 (FKBP12) is conditionally
deleted in hippocampus and forebrain [29]. FKBP12 represses
mTOR activity, and removal of FKBP12 was demonstrated to
enhance mTOR signaling as well as LTP and memory [29]. For
this set of experiments, we confirmed that treatment with
exogenous synthetic Ab1-42 caused inhibition of LTP in
hippocampal slices of WT mice (Figure 4B). Consistent with prior
data [29], slices from FKBP12 conditional KO (cKO) mice
showed enhanced LTP compared to WT slices. Of note, and in
contrast to slices of WT mice, when FKBP12 cKO slices were
treated with Ab1-42, LTP was now successfully elicited by HFS
(Figure 4B), indicating that up-regulation of mTOR signaling from
knocking out FKBP12 prevented Ab-related impairment in
synaptic plasticity. Supporting these electrophysiology data,
Western blotting demonstrated that the decrease in levels of
phosphorylated p70S6K seen in WT slices with Ab1-42 treatment
was prevented in slices from FKBP12 cKO mice (Figure 4C). PPF
experiments confirmed that pre-synaptic function was not
significantly altered in slices from FKBP12 cKO mice [29] or in
slices from WT mice treated with Ab1-42 (Figure 4D). Taken
together, up-regulation of mTOR signaling via FKBP12 cKO
prevented Ab-related LTP impairment.
Cellular co-localization of intraneuronal Ab and
components of the mTOR pathway
The mechanism(s) by which Ab affects mTOR signaling in
neurons from Tg2576 mice remain unclear. To investigate whether
there might be a spatial relation between Ab42 and mTOR, we
performed confocal microscopy in cultured Tg2576 neurons.
Previous studies have reported that both mTOR [30] and Ab42
[31,32] localize particularly to endosomes. Consistent with this,
there was punctate co-localization of Ab42 in neurites with both
mTOR (Figure 5A) and p70S6K (Figure 5B). These findings place
mTOR signaling components in the right place to be regulated by
intraneuronal Ab. To test whether this interaction might be a
requirement for Ab-induced mTOR impairment, we studied the
effect of extracellular Ab1-42 on mTOR in hippocampal slices from
APP knockout mice [33]. We previously showed that synaptic
toxicity of extracellular Ab1-42 is blocked in APP knockout
neurons, suggesting a requirement of intracellular APP processing
and Ab generation [19]. Remarkably, in hippocampal slices
prepared from APP knockout mice, Ab1-42 treatment failed to
significantly alter levels of phosphorylated p70S6K or levels of PSD-
95 (Figure 5C), supporting a role for APP processing and
intraneuronal Ab in down-regulation of mTOR signaling.
Figure 3. Up-regulation of the mTOR pathway via GSK3
inhibition rescues LTP impairment in Tg2576 mice. (A) Com-
pared to the LTP induced in hippocampal slices of WT mice (filled
circles, n = 4), high frequency stimulation (HFS) induced normal LTP in
slices from Tg2576 mice treated with LiCl (10 mM, open circles, n = 5),
which was inhibited by 30 min of rapamycin (Rapa; 1
mM) pretreatment
(gray triangles, n = 4). LiCl or rapamycin was present throughout the
recording. (B) Similarly, treatment of slices from Tg2576 mice with
kenpaullone (Ken, 5
mM, dark gray circles) prevented the impaired HFS
induced LTP seen in untreated Tg2576 slices; the protection in LTP
induction in Tg2576 slices by kenpaullone was inhibited by 30 min of
rapamycin (1
mM) pretreatment (open circles). n = 3. Kenpaullone or
rapamycin was present throughout the recording. (C) The decreased
levels of phospho-p70S6K in hippocampal slices from Tg2576
compared to WT (control) mice was prevented by treatment with
either Ken (5
mM) or LiCl (10 mM) for 60 min. n = 8. *p,0.05.
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Impaired neuroplasticity due to abnormal accumulation of Ab
has been proposed as a key factor in the cognitive decline with
AD pathogenesis, even preceding pl aque formation or neurode-
generation [1,2]. Develop ing a mechanistic understanding of the
ability of soluble Ab to interfere with synaptic plasticity, and in
particular the stable, translati on-dependent forms of plasticity
that are thought to mediate LTM, could yield i mportant insights
into the pathophysiology of AD. mTOR-mediated protein
synthesis is important for long-lasting forms of synaptic plasticity
and memory [34]. In the present study, we demonstrat e th at
mTOR signaling in the hippocampus is impaired in a transgenic
mouse model of AD, an effect that is mimicked by treating
hippocampi of normal mice with soluble Ab1-42. Importantly,
pharmacolog ical or genetic up-regulat ion of mTOR signaling
resto red LTP in slices from Tg2576 mice and prevented A b-
induc ed impairment in LTP. These findings provide novel
insights into the mechanis m of Ab-related synaptic dysfunction
underlying memory impairment in AD.
In agreement with the data with FKBP12 cKO mice, GSK3
inhibitors also rescued the Ab-related LTP failure. Originally
identified as a regulator of glycogen metabolism, GSK3 is a
multifunctional serine/threonine kinase that has many potential
substrates. It has been reported that GSK3 can inhibit mTOR
through phosphorylation of TSC2 [27]. Furthermore, GSK3 is
highly expressed in hippocampus and is implicated in synaptic
plasticity [35,36]. In fact, GSK3 dysfunction has been proposed as
an important underlying molecular mechanism for AD pathogen-
esis and accordingly has stimulated interest in the development of
therapeutically useful GSK3 antagonists [35,37–39]. In our study,
the rescue of LTP by GSK3 inhibitors was sensitive to rapamycin,
consistent with GSK3 acting to regulate mTOR signaling.
Inhibition of mTOR signaling was reported in the brains of
another AD transgenic mouse model [12]. On the other hand,
mTOR signaling was reported to be up-regulated in postmortem
human AD brains, particularly in tangle bearing neurons [21]. We
confirmed isolated phospho-p70S6K increases in AD vulnerable
neurons of human AD brain, which was particularly prominent in
tangle-bearing neurons (Supplementary Figure S3). Of note, we
Figure 4. Inhibition of mTOR signaling and LTP by extracellular Ab1-42 is prevented in FKBP12 cKO mice. (A) Treatment of slices from
WT mice with exogenous Ab1-42 (100 nM, 60 min) reduced levels of phospho-p70S6K, phospho-4E-BP, and PSD-95 compared to untreated WT slices
(controls). n = 4. *p,0.05. Representative Western blot from three experiments after direct loading of the Ab1-42 preparation (far right of the panel)
showed mostly Ab1-42 monomers, and also significant amounts of dimers and trimers. (B) LTP induced by HFS in WT mice (controls; red, n = 8) was
blocked by treatment with Ab1-42 (100 nM, blue, n = 7). In contrast, LTP was sustained in the presence of Ab1-42 (100 nM, green) in FKBP cKO mice
(n = 8). Ab1-42 was present throughout the recording. (C) Inhibitory effects of Ab1-42 on phospho-p70S6K were blunted in FKBP12 cKO mice. n = 5.
*p,0.05. Slices were treated with Ab1-42 for 60 min. (D) Slices from both FKBP12 cKO and WT mice treated with Ab1-42 (60 min) exhibited normal
PPF compared to the WT control. The percent facilitation, determined by the ratio of the second fEPSP to the first fEPSP (interpulse interval = 50 ms),
together with representative fEPSP traces are shown. n = 5 for WT; n = 6 for WT treated with Ab; n = 8 for FKBP12 cKO. Scale bar, 0.5 mV/25 ms.
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observed reductions in phospho-p70S6K only at early but not at
later ages in brains of Tg2576 mice. It is possible that up-
regulation of mTOR noted in human AD [12] and 3xAD-Tg mice
[13] develops later secondary to another process, such as tau
alterations, cell cycle re-entry and/or inflammation; for example,
the latter is known to stimulate mTOR signaling [40]. In addition,
although it is well established that acute inhibition of mTOR
impairs synaptic plasticity, it was recently reported that chronic
treatment with rapamycin improved behavior of AD transgenic
mice [13,14], although effects were mild. Acute compared to
chronic treatment may be critical in explaining the differences
among studies. Of note, there are two mTOR complexes, mTOR
complex 1 (mTORC1) and complex 2 (mTORC2). The
mTORC1 complex plays a critical role in synaptic plasticity. In
contrast, nothing is currently known about the role of mTORC2
in synaptic plasticity [7]. Prolonged but not acute treatment with
rapamycin has been reported to lead to interference with
mTORC2 [41], which has functions that are independent of
mTORC1 [42]. In light of these studies, we hypothesize that there
is a complex relationship between aging, Ab, and dysregulation of
the mTOR pathway in AD.
Of note, there are many other examples of similarly
contradictory-appearing data with respect to signaling in other
common age-related diseases. For example, a study reporting
down-regulation of mTOR signaling in huntingtin-accumulating
neurons in a mouse model of Huntington’s disease at the same
Figure 5. Cellular co-localization of intraneuronal Ab and components of the mTOR pathway in Tg2576 neurons. (A and B) Immuno-
fluorescence confocal microscopy of Ab42 with either mTOR (A) or p70S6K (B) in Tg2576 neurons at 12 DIV. Note the punctate co-localization of
intraneuronal Ab42 with mTOR and p70S6K. Scale bar, 50
mm. (C) Ab1-42 (100 nM, 60 min) treatment failed to induce inhibition of p70S6K
phosphorylation and to reduce PSD-95 levels in slices from APP KO compared to WT (control) mice. n = 9. *p,0.05.
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time showed that treating the mice with rapamycin was protective
[43]. In another age-related disease, adult onset diabetes, defective
insulin signaling through the insulin-PI3K-Akt pathway, which is
upstream of mTOR, is well-established, although inhibition of this
same pathway is known to extend lifespan in yeast and C. elegans
models [44]. Of note, similar conflicting data also exist on the
insulin-PI3K-Akt pathway in AD models. On one hand, Ab was
shown to impair insulin-PI3K-AKT signaling [2,45–47] and
insulin treatment was reported to improve cognitive function in
patients with early AD [48], while on the other hand, AD
transgenic mice with reduced insulin signaling were reported to be
protected against cognitive decline [49].
Although the precise molecular mechanism whereby Ab alters
the mTOR pathway is not known, our finding that Ab42 co-
localizes with mTOR and p70S6K within neurites of APP mutant
transgenic mice places it in the right location to dysregulate
mTOR signaling. Recent evidence supports that extracellular Ab
acts via APP processing and potentially also intraneuronal Ab42
[19], and therefore we examined whether extracellular Ab1-42
down-regulated mTOR in APP knockout neurons. Remarkably,
the effect of exogenous Ab on mTOR signaling was blocked in
APP knockout neurons, further supporting an emerging complex
link between extracellular and intracellular Ab.
Given the pivotal role of mTOR in maintaining cellular and
organismal homeostasis by controlling multiple fundamental
biological processes, perturbing the balance in mTOR signaling
might be key to better understanding the Ab-related impairment
in signaling involved in the synaptic dysfunction that characterizes
Materials and Methods
Hippocampal slice preparation and electrophysiology
All experiments were conducted according to and approved by
the Weill Cornell Medical College IACUC (Protocol# 0610-
550A). For preparation of acute hippocampal slices, 3 to 4 month-
old Tg2576 or wild type mice were first deeply anesthetized with
isoflurane and then decapitated. Brains were rapidly removed and
placed in ice-cold artificial cerebrospinal fluid (ACSF) containing
(in mM) 118 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25
NaH2PO4, 24 NaHCO3, and 15 glucose, bubbled with 95% O2/
5% CO2. The hippocampus was then quickly dissected out and
mm thick transverse slices were made on a tissue chopper at
4uC. The slices were maintained in an interface chamber (ACSF
and humidified 95% O2/5% CO2 atmosphere) at room
temperature for at least 2 hours before removal for experiments.
For electrophysiological recording, slices were transferred to a
submersion chamber preheated to 30–32uC, where they were
superfused on a nylon mesh with ACSF. Monophasic, constant-
current stimuli (100
msec) were delivered with a bipolar stainless
steel electrode placed in the stratum radiatum of the CA3 region,
and the field excitatory postsynaptic potentials (fEPSPs) were
recorded in the stratum radiatum of the CA1 region with
electrodes filled with ACSF (Re = 2–4 MV). The fEPSPs were
monitored by delivering stimuli at 0.033 Hz, and the signal was
low-pass filtered at 3 kHz and digitized at 20 kHz. fEPSPs were
acquired, and amplitudes and maximum initial slopes measured,
using either Axobasic routine or pClamp 9 (Axon Instruments,
Foster City, CA). High Frequency Stimulation (HFS) protocol
consisting of two 1-s long 100 Hz trains, separated by 20 s,
delivered at an intensity that evoked a 1.5 mV fEPSP, was used to
induce LTP. In some experiments, ‘‘weak’’ HFS was used,
consisting of a single 1 s long 100 Hz train delivered at an intensity
that evoked a baseline fEPSP between 0.5 and 0.6 mV.
Primary neuronal cultures
Primary neuronal cultures were prepared from cortices and
hippocampi of embryonic day (E) 16 Tg2576 and wild-type
littermate mice as described [19]. One pup corresponds to one set
of cultures, and genotyping was done by PCR of the cerebellum.
Primary neurons were used at 12 days in vitro (DIV).
Drug treatments
Drugs were prepared as stock solutions and diluted to final
concentrations before use. For hippocampal slices, drug incuba-
tion was performed at 30–32uC in submersion maintenance
chambers containing ACSF saturated with bubbling 95% O2/5%
CO2. The final concentrations and sources were as follows:
forskolin (50
mM, Sigma), DHPG (50 mM, Sigma), Rapamycin
mM, Calbiochem), kenpaullone (1 mM, Calbiochem), and LiCl
(10 mM, Sigma). Ab1-42 stock (100
mM, Tocris) was prepared in
water and stored at 220uC overnight before use at a final
concentration of 100 nM.
Western blotting
Lysates were prepared from either primary neurons at 12 DIV or
hippocampal slices. Previously described protocols were followed
[19,20]. The following inhibitors were added into the lysis buffer (in
mM, unless indicated otherwise): 25 Na fluoride, 2 Na pyrophos-
phate, phosphatase inhibitor cocktail I & II (Calbiochem), protease
inhibitor cocktail (Roche). Protein concentrations were determined
by the Bradford technique (Bio-Rad Laboratories), and equal
amounts of protein from each sample were loaded on 4–12% Tris-
Glycine SDS-PAGE (Invitrogen) gels. After transfer, membranes
were blocked for at least 30 minutes at room temperature with
blocking buffer [BB; 5% non fat dry milk in TBS containing 0.1%
Tween 20 (TBS-T)], then probed overnight at 4uC using primary
antibodies for phospho(Thr389)-p70S6K (1:1000; Cell Signaling),
p70S6K (1:1000; Cell Signaling), phospho(Thr37/46)-4E-BP
(1:1000; Cell Signaling), 4E-BP (1:1000; Cell Signaling), PSD-95
(1:1000, Chemicon), Synapsin I (1:2000, Upstate), or actin (1:10000;
Sigma-Aldrich). Densitometric analysis of the bands was performed
using Scion Image Software. Data were analyzed using student t-test
with Origin (OriginLab Corp.) software, with significance placed at
p,0.05. Measurements of phospho-proteins were normalized to
corresponding total proteins. Summary data were presented as
group means with standard error bars.
Immunofluorescence confocal microscopy
Ice-cold 4% paraformaldehyde was used to fix both primary
neurons (20 min) and slices (overnight). The slices were further cut
into 30
mm sections. Protocols were followed as described [19,20].
Fixed neurons, hippocampal slice sections or postmortem human
brain sections were incubated overnight at 4uC with the following
antibodies: human specific Ab42 (1:250; Signet), phos-
pho(Thr389)-p70S6K (1:250; Cell Signaling), p70S6K (1:250;
Cell Signaling), and mTOR (1:100; Cell Signaling).
Supporting Information
Figure S1 (A) HFS induced normal LTP in slices from 2-month-
old Tg2576 mice, comparing to WT mice. n = 4. (B) Western
blotting showed no reduction of phospho-p70S6K and PSD95 in
slice from 2-month-old Tg2576 mice. n = 4.
Found at: doi:10.1371/journal.pone.0012845.s001 (2.73 MB
Figure S2 (A) Weak HFS (one train) induced similar early LTP
in slices from 3–4-month-old Tg2576 mice and WT mice that
Ab-Related mTOR Impairment
PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12845
decayed to baseline in about 80 minutes. n = 5. (B) Slices from 3–
4-month-old Tg2576 mice demonstrated normal PPF. n = 5. Scale
bar, 0.5 mV/25 ms.
Found at: doi:10.1371/journal.pone.0012845.s002 (2.56 MB
Figure S3 Increased labeling of phospho-p70SK6 in AD
vulnerable neurons of the hippocampus in a case with AD (A)
compared to a control (B). Inserts represent higher power views of
the black boxes within the lower power images. The inset in (A)
reveals tangle-like labeling of phospho-p70SK6 in CA1 pyramidal
neurons. Scale bar, 1 mm.
Found at: doi:10.1371/journal.pone.0012845.s003 (8.75 MB
Author Contributions
Conceived and designed the experiments: TM CCH ECZ MTL DT EK
RDB GKG. Performed the experiments: TM CCH ECZ FY HW DT.
Analyzed the data: TM CCH ECZ DT EK RDB GKG. Contributed
reagents/materials/analysis tools: RDB GKG. Wrote the paper: TM MTL
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    • "mTORC1 is highly expressed in brain and regulates multiple signaling pathways related with protein synthesis, autophagy, cell growth and mitochondrial function [43]. Accumulating evidences suggest that mTORC1 plays a critical role in AD pathology [44, 45] . In a previous study, we demonstrated that interaction between Aβ and RAGE induces autophagic vacuoles via the Ca 2+ /calmodulin-dependent protein kinase kinase-AMPK pathway [29]. "
    [Show abstract] [Hide abstract] ABSTRACT: The receptor for advanced glycation end products (RAGE) has been found to interact with amyloid β (Aβ). Although RAGE does not have any kinase motifs in its cytosolic domain, the interaction between RAGE and Aβ triggers multiple cellular signaling involved in Alzheimer’s disease (AD). However, the mechanism of signal transduction by RAGE remains still unknown. Therefore, identifying binding proteins of RAGE may provide novel therapeutic targets for AD. In this study, we identified p38-regulated/activated protein kinase (PRAK) as a novel RAGE interacting molecule. To investigate the effect of Aβ on PRAK mediated RAGE signaling pathway, we treated SH-SY5Y cells with monomeric form of Aβ. We demonstrated that Aβ significantly increased the phosphorylation of PRAK as well as the interaction between PRAK and RAGE. We showed that knockdown of PRAK rescued mTORC1 inactivation induced by Aβ treatment and decreased the formation of Aβ-induced autophagosome. We provide evidence that PRAK plays a critical role in AD pathology as a key interactor of RAGE. Thus, our data suggest that PRAK might be a potential therapeutic target of AD involved in RAGE-mediated cell signaling induced by Aβ.
    Full-text · Article · Dec 2016
    • "However, elements of the autophagy system (autophagic vesicles and lysosomes) are involved in both production and degradation of Aβ (Boland et al. 2008 ). Moreover, mTOR is necessary for synaptic plasticity and memory formation and retention (Blundell et al. 2008, Parsons et al. 2006), and its loss results in long-term potentiation impairment in mouse models (Ma et al. 2010). The clinical applicability of autophagy modulators for AD treatment will therefore need to be checked carefully for efficacy and side effects. "
    [Show abstract] [Hide abstract] ABSTRACT: Ten years of remarkable progress in understanding the fundamental biochemistry of Alzheimer's disease have been followed by ten years of remarkable and increasing clinical insight into the natural progression of the disorder. The concept of a long, intermediary, prodromal phase between the first appearance of amyloid plaques and tangles and the manifestation of dementia is now well established. The major challenge for the next decade is to chart the many cellular processes that underlie this phase and link the biochemical alterations to the clinical manifestation of Alzheimer's disease. We discuss here how genetics, new cell culture systems, and improved animal models will fuel this work. We anticipate that the resulting novel insights will provide a basis for further drug development for this terrible disease. Expected final online publication date for the Annual Review of Neuroscience Volume 39 is July 08, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    Article · Apr 2016
    • "However while GSK-3β inhibition probably underlies much of lithium's positive effects on synaptic plasticity and memory, the cation's capacity to up-regulate mTOR may also play a role [101]. While the activation of this kinase has Janus faced properties in terms of neuroprotection , it must be remembered that in the CNS mTOR pathway play a key positive role in regulating synaptic remodeling and long term potentiation (LTP) [102][103][104] . Lithium may also exert direct neuroprotective effects via the activation of the pro-survival phosphatidylinositol 3- kinase/Akt and the extracellular signal-regulated kinase (ERK) pathways [105]. "
    [Show abstract] [Hide abstract] ABSTRACT: Alzheimer's disease is a progressive neurodegenerative illness characterized by the invariant existence of β-amyloid plagues and neurofibrillary tangles. Presently approved pharmaceutical approaches offer only marginal efficacy and as yet there is no effective treatment which reverses or arrests the disease. Thus far drugs targeting any single aspect of disease pathology have proved to be a failure or at best provided very slight clinical benefit. The consistent failure of drugs targeting aspects of the Aβ cascade has questioned the causal role of this pathway. There is a growing appreciation that the pathogenesis of the illness is multifactorial with Amyloid Beta, Phosphorylated Tau (ptau), inflammation, mitochondrial dysfunction, calcium dyshomeostasis, heavy metal imbalances, and GSK-3 interact in a highly complex manner to provoke a self-sustaining spiraling cascade of pathology driving disease progression. In the light of such complex pathology the failure of drugs aimed a targeting single molecules is not surprising as such approaches are usually ineffective against other complex diseases with a multifactorial pathogenesis. Combination therapies or multi target drugs might be more effective in controlling such illnesses. The putative neuroprotective effects of Lithium are achieved via the positive modulation of numerous homeostatic mechanisms regulating autophagy oxidative stress inflammation, and mitochondrial dysfunction likely achieved by inhibiting GSK-3 and inositol-145 triphosphate. Data regarding efficacy in human trials and animal models of AD are mixed, but recent data using "microdose" lithium in mild cognitive impairment is encouraging, hence lithium could be a putative multi target treatment in these patients. However, additional well designed long-term trials are needed to confirm its efficacy and safety, given that long term use is necessary to achieve reasonable therapeutic benefit.
    Article · Feb 2016
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