Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain

Abstract

Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer’s disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important disease-associated proteins including the tau kinases cyclin-dependent kinase 5 and glycogen kinase synthase-3. Here, we sought to investigate the likely temporal association between these changes during the development of sporadic AD using Braak staged post-mortem brain. Quantification of protein amounts in these tissues showed increased activity of calpain-1 from Braak stage III onwards in comparison to controls, extending previous findings that calpain-1 is upregulated at end-stage disease, and suggesting that activation of calcium-sensitive signalling pathways are sustained from early stages of disease development. Increases in calpain-1 activity were associated with elevated activity of the endogenous calpain inhibitor, calpastatin, itself a known calpain substrate. Activation of the tau kinases, glycogen-kinase synthase-3 and cyclin-dependent kinase 5 were also found to occur in Braak stage II-III brain, and these preceded global elevations in tau phosphorylation and the loss of post-synaptic markers. In addition, we identified transient increases in total amyloid precursor protein and pre-synaptic markers in Braak stage II-III brain, that were lost by end stage Alzheimer's disease, that may be indicative of endogenous compensatory responses to the initial stages of neurodegeneration. These findings provide insight into the molecular events that underpin the progression of Alzheimer's disease, and further highlight the rationale for investigating novel treatment strategies that are based on preventing abnormal calcium homeostasis or blocking increases in the activity of calpain or important calpain substrates.

Electronic supplementary material
The online version of this article (doi:10.1186/s40478-016-0299-2) contains supplementary material, which is available to authorized users.

Full text

R E S E A R C H Open Access
Upregulation of calpain activity precedes
tau phosphorylation and loss of synaptic
proteins in Alzheimer’s disease brain
Ksenia Kurbatskaya
1†
, Emma C. Phillips
1†
, Cara L. Croft
1
, Giacomo Dentoni
1
, Martina M. Hughes
1
,
Matthew A. Wade
1
, Safa Al-Sarraj
2
, Claire Troakes
1,2
, Michael J. O’Neill
3
, Beatriz G. Perez-Nievas
1
,
Diane P. Hanger
1
and Wendy Noble
1*
Abstract
Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in
Alzheimer’s disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine
protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of
human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated
proteolysis regulates the activity of important disease-associated proteins including the tau kinases cyclin-
dependent kinase 5 and glycogen kinase synthase-3. Here, we sought to investigate the likely temporal association
between these changes during the development of sporadic AD using Braak staged post-mortem brain.
Quantification of protein amounts in these tissues showed increased activity of calpain-1 from Braak stage III
onwards in comparison to controls, extending previous findings that calpain-1 is upregulated at end-stage disease,
and suggesting that activation of calcium-sensitive signalling pathways are sustained from early stages of disease
development. Increases in calpain-1 activity were associated with elevated activity of the endogenous calpain
inhibitor, calpastatin, itself a known calpain substrate. Activation of the tau kinases, glycogen-kinase synthase-3 and
cyclin-dependent kinase 5 were also found to occur in Braak stage II-III brain, and these preceded global elevations in
tau phosphorylation and the loss of post-synaptic markers. In addition, we identified transient increases in total amyloid
precursor protein and pre-synaptic markers in Braak stage II-III brain, that were lost by end stage Alzheimer's disease,
that may be indicative of endogenous compensatory responses to the initial stages of neurodegeneration. These
findings provide insight into the molecular events that underpin the progression of Alzheimer's disease, and further
highlight the rationale for investigating novel treatment strategies that are based on preventing abnormal calcium
homeostasis or blocking increases in the activity of calpain or important calpain substrates.
Keywords: Alzheimer’s disease, Calpain, GSK-3, Tau, Synapse, Braak stage, Postmortem brain
Introduction
Synaptic dysfunction and neurodegeneration in Alzheimer’s
disease (AD) is associated with the presence of extracellular
deposits of β-amyloid (Aβ) in neuritic plaques and intra-
neuronal neurofibrillary tangles containing abnormally
phosphorylated and aggregated tau [50]. Considerable
evidence has shown that disruptions to Ca
2+
signalling
pathways are associated with neuronal loss in AD [6]. Ele-
vated Aβburden leads to increased intracellular Ca
2+
con-
centrations [36] by several mechanisms including increased
Ca
2+
entry through native ion channels and receptors [68,
76] or amyloid pores [15], release of Ca
2+
from intracellular
stores [18, 68] and inactivation of the ionic machinery that
extrudes excess Ca
2+
from neural cells [2]. Sustained in-
creases in intracellular Ca
2+
leads to activation of many
calcium-sensitive proteins implicated in AD including cal-
cium/calmodulin-dependent protein kinase (CAMKK2;
[44]), calcineurin [46, 67, 77], and calpains [2, 71].
* Correspondence: wendy.noble@kcl.ac.uk
†
Equal contributors
1
Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical
Neuroscience Institute, King’s College London, Institute of Psychiatry,
Psychology and Neuroscience, Rm1.25, 5 Cutcombe Road, Camberwell,
London SE5 9RX, UK
Full list of author information is available at the end of the article
© 2016 Kurbatskaya et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34
DOI 10.1186/s40478-016-0299-2
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Calpains are a family of cysteine proteases closely
linked with AD. They cleave amyloid precursor protein
(APP) to regulate Aβproduction [47], several synaptic
proteins including dynamin-1 and the NMDA receptor
subunit NR2B to affect synapse health [64], and the cla-
thrin adapter protein PICALM to modulate endocytosis
[1]. Much research has also highlighted the actions of
calpain for disease-associated changes in tau. Calpain
can cleave the N-terminus of tau directly to generate
neurotoxic tau fragments [17, 32]. Calpain-mediated
proteolysis of kinases or their activators regulates the ac-
tivity of key tau kinases, such as GSK-3 [25] and cdk5
[40], both of which promote tau phosphorylation and
tau-associated neurodegeneration in vivo [12, 23, 51, 52].
Evidence from the study of postmortem brain sup-
ports an important role for aberrant calpain regula-
tion in AD. Calpain activity is increased in end-stage
AD brain [2, 32, 61], particularly in neurofibrillary
tangle-containing neurons [25], and elevated cleavage
of many calpain substrates has been demonstrated in
postmortem AD brain [2, 42, 43]. Indeed, a recent
study using end-stage AD brain demonstrated a
strong link between calpain activation, N-terminal
cleavage and activation of GSK-3 and tau phosphoryl-
ation at several disease-relevant epitopes [32].
The aim of this study was to determine the temporal
association between changes in calpain, tau kinases, tau
and synaptic proteins during the development of spor-
adic AD using brain tissue from Braak stage II to VI AD
and age-matched controls. We observed increased activ-
ity of calpain-1 from mid-stages of AD. These increases
were associated with elevated activity of the tau kinases,
GSK-3 and cdk5 in Braak stage II-III, which were in turn
observed prior to elevated tau phosphorylation and loss
of synaptic markers. These data extend previous findings
that calpain-1 and tau kinases are upregulated at end-
stage AD, and suggest that calcium-sensitive signalling
pathways are activated very early during disease develop-
ment, prior to changes in tau phosphorylation and syn-
apse loss. These findings further highlight the rationale
for investigating novel treatment strategies for AD that
are based on preventing abnormal calcium homeostasis
or blocking increases in calpain or tau kinase activities.
Materials and methods
Preparation of post mortem human brain lysates
Frozen postmortem human temporal cortex (Table 1)
from control (n=5) and pathologically confirmed
cases of sporadic AD of Braak stage II (n=5), III (3),
IV (n=4), V (n=3) and VI (n=5) were obtained
from the MRC London Neurodegenerative Diseases
Brain Bank (Additional file 1: Table S1, Additional file
2: Table S2). Frozen tissue was homogenized (0.5–
1mgmL
−1
) in ice-cold lysis buffer containing 50 mM
Tris-buffered saline (TBS, pH 7.4), 0.1 % (v/v) Triton
X-100, 10 mM sodium fluoride, 1 mM sodium ortho-
vanadate, 2 mM ethylene glycol tetraacetic acid
(EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF)
and Complete™protease inhibitor (Roche Diagnostics
Ltd., West Sussex, UK). Homogenates were centri-
fuged at 25,000
g(av)
for 20 min at 4 °C. The resulting
supernatants were collected and stored at −20 °C
until required. Protein concentrations in supernatants
were measured using a BCA protein assay kit (Pierce
Endogen, Rockford, USA). Samples were normalised
to equal protein concentration before being analyzed
by western blotting or ELISA. Pellets were resus-
pended in 4 x sample buffer containing 50 mM Tris–
HCl pH 7.2, 2 % (w/v) SDS, 10 % (v/v) glycerol,
2.5 % (v/v) β-mercaptoethanol, 12.5 mM EDTA,
0.02 % (w/v) bromophenol blue, briefly sonicated, and
heated to 95C for 5 min prior to western blotting.
Isolation of sarkosyl insoluble tau
Sarkosyl extractions were performed as previously de-
scribed by us [52]. Briefly, tissue was homogenized in
50 mM TBS (pH 7.4) containing 2 mM EGTA, 1 mM
sodium orthovanadate, 10 mM sodium fluoride and
1 mM PMSF at 100 mg/mL (w/v), and centrifuged at 20
000 gav for 20 min at 4 °C. Sarkosyl (10 % v/v) was
added to the resulting supernatant to give a final con-
centration of 1 % (v/v), and samples mixed for 30 min at
ambient temperature with rocking and then centrifuged
at 100,000 gav for 60 min at ambient temperature. The
supernatant was collected and the pellet washed twice
with 1 % sarkosyl, prior to solublization in 2 × SDS sam-
ple buffer. Thus, three fractions were generated, contain-
ing: (i) low speed supernatant (ii) sarkosyl-soluble and
(iii) sarkosyl-insoluble tau. Samples were subjected to
immunoblotting, standardising the amount of sarkosyl-
soluble or -insoluble tau to the amount of tau present in
low speed supernatants.
Gel electrophoresis and Western blotting
Protein was electrophoresed on 10–12 % (w/v) SDS-
polyacrylamide gels. Separated proteins were transferred
to nitrocellulose membranes (Whatman, Maidstone, UK)
and either blocked with 5 % (w/v) non-fat milk in TBS,
5 % (w/v) bovine serum albumin (BSA) in TBS or Odys-
sey® Blocking Buffer for 1 h. After blocking, membranes
were incubated overnight at 4 °C in blocking solution con-
taining appropriate dilutions of primary antibody. Blots
were washed and incubated with fluorophore-conjugated
secondary antibodies for 1 h at ambient temperature. Pro-
teins were visualized using an Odyssey® Infrared Imaging
system (Li-Cor Biosciences, Cambridge, UK) and quanti-
fied using ImageJ (NIH, Maryland, USA) or proprietary
Odyssey sa software (Li-Cor Biosciences, Cambridge, UK).
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 2 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Human postmortem brain samples were run on mul-
tiple gels, each containing a standard control to enable
comparison of samples across gels. Statistical analysis
was performed following standardization of total protein
amounts against neuron-specific enolase (NSE) or β-
actin amounts in each sample. White lines separating
lanes in immunoblot images indicate splicing together of
different blots or different regions of the same blot.
Antibodies
The following primary antibodies were used for Western
blotting: calpain-1 large active subunit (No. 28257, rabbit
IgG; Abcam plc, Cambridge, UK), calpastatin (CAST,
No. 4146, rabbit IgG; Cell Signalling Ltd, Beverly, MA,
USA); cleaved (active) caspase-3 (No. 13847, Asp175/
Ser376, rabbit IgG; Abcam plc, Cambridge, UK), spec-
trin, αchain (MAB1622, Clone AA6, mouse IgG; Merck
KGaA, Darmstadt, Germany), total tau (DAKO, A0024,
rabbit IgG; Agilent Technologies, Glostrup, Denmark),
tau dephosphorylated at Ser199/202 (Tau-1, MAB3420,
Clone PC1C6, mouse IgG; Merck KGaA, Darmstadt,
Germany), tau phosphorylated at Ser202 (CP13, mouse
IgG; P. Davies, Feinstein Institute for Medical Research,
NY, USA), tau phosphorylated at Ser396/404 (PHF1,
mouse IgG; P. Davies, Feinstein Institute for Medical Re-
search, NY, USA), glycogen synthase kinase-3α/βphos-
phorylated at Ser21/9 (pGSK3, No. 9331, rabbit IgG;
Cell Signalling Ltd, Beverly, MA, USA), total GSK3α/β
(GSK3, SA364-0100, Clone 1H8, mouse IgG; Enzo Life
Sciences Inc, Exeter, UK), cdk5 (sc-6247, Clone J-3,
mouse IgG, Santa Cruz Biotechnology, Dallas, Texas,
USA), p35 (sc-820, Clone C-19, rabbit IgG, Santa Cruz
Biotechnology, Dallas, Texas, USA), β-amyloid, 1–16
(6E10, SIG-39300, mouse IgG; Covance, California,
USA), NR2B (No. 06–600, rabbit IgG; Merck KGaA,
Darmstadt, Germany), PSD-95 (No. 2507, rabbit IgG;
Cell Signalling Ltd, Beverly, MA, USA), synapsin I
(AB1543P, rabbit IgG, Merck KGaA, Darmstadt,
Germany) and NSE (BBS/NC/VI-H14, mouse IgF;
DAKO, Glostrup, Denmark). For immunohistochemistry
antibodies against phosphorylated tau (clone [AT-8];
Autogen Bioclear UK Ltd, Wiltshire, UK) and amyloid β
(Aβ) (Chemicon, Temecula, CA, USA) were used.
Aβ1-40 and Aβ1-42 ELISA
Aβ1-40 and Aβ1-42 amounts in human brain samples
were quantified using ELISA kits from Life Technolo-
gies, Paisley, UK (Aβ40 ELISA KHB3481; Aβ42 ELISA
KHB3442) as previously described [73].
Immunohistochemistry
As part of the neuropathological diagnosis of each case,
7μm tissue sections were cut from formalin-fixed
paraffin-embedded blocks of AD or control human brain
tissue. Sections were deparaffinized and endogenous per-
oxidase activity was inhibited by incubating samples in
3 % (v/v) hydrogen peroxide for 30 min (for Aβ80 % for-
mic acid pretreatment for 1 h was used), and antigen re-
trieval was enhanced by microwaving in 10 mM sodium
citrate buffer, pH 6.0. Sections were blocked for 20 min in
10 % normal serum before incubating with tau/Aβanti-
bodies overnight at 4 °C. Sections were then incubated
with biotinylated secondary antibodies (DAKO) for
45 min. Sections were developed using the VECTASTAIN
Elite ABC kit (Vector Laboratories) and 0.5 mg/ml 3,3′-
diaminobenzidine chromogen (Sigma-Aldrich). All sam-
ples were counterstained with hematoxylin.
Statistical analysis
Statistical analysis was performed using GraphPad Prism
v6.0 (La Jolla, CA, USA). Western blot and ELISA data
was analyzed by nonparametric one-way analysis of vari-
ance followed by Tukey’s post-hoc tests. Correlation
analysis was performed using two-tailed Spearman tests
with linear regression. Differences were considered sta-
tistically significant when p<0.05. GraphPad Prism v6.0
was used for all statistical analyses.
Results
Temporal cortex was used for these analyses, in keeping
with previous studies of AD development [8]. Tau path-
ology, as assessed using immunohistochemistry, is min-
imal in the temporal cortex in the earliest stages of
disease [8]. Therefore, examination of this region
allowed us to determine changes in other proteins that
precede pathological changes in tau in AD. In all cases,
Braak stage II-VI tissues were compared with age-
matched control brain, these latter tissues showing no
evidence of neurodegeneration.
Progressive accumulation of phosphorylated tau in AD
Tau is a microtubule-binding protein that is abnormally
phosphorylated and progressively accumulates in NFTs
in AD [26]. Abnormal processing of tau is closely linked
with synaptic and neuronal dysfunction in AD [11], and
is an increasingly important target for the development
of new dementia therapies [53].
Postmortem brain lysates were immunoblotted using
antibodies against total tau (DAKO) and tau phos-
phorylated at Ser396/404 (PHF1), both of which
yielded bands of the expected size, approximately 50–
68 kDa (Fig. 1a). Bands of approximately 17 kDa were
also detected, which may correspond to the 17 kDa
calpain-cleaved tau fragments previously described by
others [17, 32]. Blots were also probed with an anti-
body against NSE, which acted as a control for gliosis
and/or loss of protein during neuron loss and post-
mortem delay. Total tau protein is reported to be
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 3 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

increased in degenerating regions of AD brain [35].
Following normalization of tau amounts to NSE, we
found an increase in total tau protein in mid-late
stage AD. Tau protein amounts were significantly in-
creased in Braak stage IV and V tissues compared to
control (p<0.05), with the lack of significance at
Braak stage II, III and VI likely to reflect the rela-
tively small sample set used in this study since clear
elevations in tau amounts can be observed in these
samples by western blotting (Fig. 1a). Quantification
of tau phosphorylated at Ser396/404, as detected by
the PHF-1 antibody, showed that tau phosphorylation
at this epitope is below the detectable range in con-
trol brain, and in most Braak stage II-V tissues, but
was significantly increased at end-stage AD (Braak
VI) when compared to control (p<0.001; Fig. 1a).
Similar findings were observed when these samples
were blotted with an antibody against tau phosphory-
lated at Ser202 (CP13, data not shown).
To determine if the increased abundance of tau in
these samples results from the accumulation of
degradation-resistant tau aggregates, insoluble tau was
isolated from postmortem brains with sarkosyl. This
protocol results in three tau fractions, a low speed
supernatant (S1), sarkosyl-soluble (S2) and sarkosyl-
insoluble tau (P2), all of which were immunoblotted
with antibodies against total tau and pSer396/404
(PHF1).Thesefindingsconfirmedanincreaseinin-
soluble tau as a proportion of total tau in Braak stage
V and VI tissues relative to earlier Braak stages and
controls(Fig.1b).Thus,theincreaseintotaltaupro-
tein observed in Fig. 1a likely reflects the accumula-
tion of this insoluble tau in tissue lysates, particularly
since no changes in total tau mRNA have been re-
ported in sporadic AD cortex [7, 28].
Immunohistochemical studies of fixed postmortem
cortex labelled with the AT8 phospho-antibody are
shown to confirm the Braak staging of these samples;
Fig. 1 Total tau amounts are elevated throughout AD progression, whereas increased tau phosphorylation is only detectable at end-stage disease.
aRepresentative immunoblots of cortical homogenates from postmortem brain. Blots were probed with antibodies to detect total (phosphorylated
and non-phosphorylated) amounts of tau (DAKO) at 50 to 70 kDa, and tau phosphorylated at Ser396/404 (PHF-1) at 50 to 70 kDa. Blots were also
probed with an antibody against neuron-specific enolase (NSE, 45 kDa) which acted as a loading control. Bar graphs show the amounts of DAKO and
PHF-1 once standardized to NSE content in each sample. bRepresentative immunoblots of samples from sarkosyl extraction protocols showing low
speed supernatants, sarkosyl-soluble and sarkosyl-insoluble tau probed with antibodies against total tau (DAKO). Bar charts show sarkosyl-soluble and
sarkosyl-insoluble tau as a proportion of tau in low speed supernatants as a measure of total tau. cPostmortem brain sections immunostained with an
anti-tau (AT8) antibody show Braak staging of AD brain. NFTs are absent from age-matched control brain. CTRL: control (n=5), Braak II AD (n=4), Braak
III AD (n=3), Braak IV AD (n=4), Braak V AD (n=3), Braak VI AD (n=5). Data is mean ± SEM. *p<0.05, **p<0.01
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 4 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

these show progressive appearance of characteristic
tangle-like structures in Braak IV-VI tissues (Fig. 1c).
Total APP amounts are increased in Braak stage II-III brain
Amyloid precursor protein (APP) is a type 1 transmem-
brane glycoprotein that, in AD, is pathologically cleaved to
give rise to Aβpeptides of varying length [13]. We
assessed amounts of APP holoprotein in postmortem cor-
tex by probing blots with an antibody specific for C-
terminal APP (6E10), which yielded two main bands at
106 and 113 kDa and a faint band at 130 kDa in late-stage
AD brain, together characteristic of the three major APP
isoforms found in human brain [14, 54], Fig. 2a). When
standardized to NSE, total APP amounts were significantly
increased in Braak stage II and III (p<0.005) tissue com-
pared to control, before returning to approximately con-
trol amounts in late (Braak IV-VI) stage AD (Fig. 2b). This
finding extends previous studies which have shown no
differences in total APP holoprotein amounts between
control brain, brain from non-demented aged individuals
and those with end-stage AD [54], by suggesting that there
is an upregulation of APP when the first neurodegenera-
tive changes occur in brain (Braak stage II-III), which pos-
sibly represents a compensatory CNS response to the first
signs of damage in AD.
It would also have been of interest to examine the
abundance of APP C-terminal fragments in these tissues.
Although we have previous experience in blotting APP
fragments [73, 74], it was very difficult to detect these
small protein fragments in these tissues presumably due
to rapid postmortem degradation (data not shown).
However, we measured total amounts of Aβ1-40 and
Aβ1-42 in postmortem control and AD brain using spe-
cific Invitrogen ELISAs, as we have previously described
[73, 74]. These analyses revealed that Aβ1-40 amounts
did not significant differ between any stage of AD and
Fig. 2 Transient elevations of total APP amounts in early AD, and persistent accumulation of Aβ1-42 at end-stage disease. aRepresentative immunoblots
of cortical homogenates from postmortem brain. Blots were probed with the 6E10 antibody to detect full-length amyloid precursor protein (APP) at 110 to
130 kDa. Blots were also probed with an anti-neuron-specific enolase (NSE, 45 kDa) as a loading control. bBar graph shows APP amounts in brain following
standardization to NSE protein in the same sample. AβELISAs were used to measure Aβ1-40 and Aβ1-42 amounts in pg mg
−1
in these tissues. Bar graphs
show (c)Aβ1-40 and (d)Aβ1-42 amounts in each sample. epostmortem brain sections immunostained with an anti-Aβantibody show the progressive
development of amyloid plaque pathology in AD brain. CTRL: control (n=5), Braak II AD (n=4), Braak III AD (n=3), Braak IV AD (n=4), Braak V AD (n=3),
Braak VI AD (n=5). Data is mean ± SEM. *p<0.05, **p<0.01
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 5 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

control brain (Fig. 2c), whereas Aβ1-42 burden was sig-
nificantly increased in the later stages of AD, showing
significant increases at Braak stages IV (p<0.05) and V
(p<0.001) when compared to controls (Fig. 2d). Ele-
vated Aβ1-42 amounts have previously been demon-
strated in cortical regions of sporadic AD brain [63].
Representative labelling of fixed sections with an anti-
body that detects Aβis shown to confirm the presence
of diffuse amyloid plaques in Braak II-III, and the ap-
pearance of dense core senile plaques in Braak stages
IV-VI sections, none of which were found in control tis-
sue (Fig. 2e).
The data presented here shows that marked increases
in APP amounts are found transiently in Braak stage II-
III stage AD brain, a change that might reflect an, as yet
unknown, compensatory response to early stages of
damage in the nervous system. These changes in APP
preceded the elevated Aβ1-42 production and significant
plaque deposition that was found in stage IV-VI AD
brain.
Calpain-1 activity is increased in Braak stage III brain and
is sustained throughout disease progression
Calpain-1 exists as an 80 kDa pro-enzyme that under-
goes autolysis to yield 76 and 58 kDa active fragments
[4, 75]. Blots of postmortem human brain were probed
with an antibody that specifically detects the 76 kDa ac-
tive calpain-1 subunit [2], revealing a single prominent
band (Fig. 3a). The amounts of active calpain-1 were sig-
nificantly increased in Braak stage III to VI tissues when
compared to controls (Fig. 3b), indicating that elevations
in calpain activity are prolonged from early-mid stages
of AD.
Calpastatin activity is increased at Braak stage IV to V,
but this is not sustained at end-stage disease
Calpastatin (CAST) is the endogenous inhibitor of calpain
in the brain, and thus plays an important role in respond-
ing to prolonged elevation of calpain [48]. Indeed, calpain
is known to cleave CAST to generate active fragments
with calpain inhibitory activity [25, 60]. We have previ-
ously shown that calpastatin activity is suppressed in late
stage AD when compared to age-matched control brain
[2]. To examine CAST activity throughout the develop-
ment of AD, we probed blots of brain lysates with an anti-
body against CAST which detects CAST holoprotein at
110 kDa, a number of calpain-cleaved active CAST frag-
ments at 37–75 kDa (which together with CAST holopro-
tein inhibit calpain) and bands below 37 kDa representing
inactive CAST. The smaller CAST fragments are gener-
ated by caspase-1- and caspase-3-mediated cleavage of
CAST and lack inhibitory activity [25, 60] (Fig. 3a). Active
and inactive CAST were quantified separately as a propor-
tion of total CAST (holoprotein plus all fragments). We
found that levels of active CAST (holoprotein plus 37–
75 kDa fragments) were significantly increased in Braak
IV-V AD tissue (p<0.05) compared to control (Fig. 3c).
There were also differences, some significant, in the
amounts of inactive CAST relative to total CAST in all
AD tissues (Fig. 3d), likely representing the increased total
CAST apparent in AD brain that was detected by
immunoblotting.
Active caspase-3 amounts do not change throughout AD
progression
There is much evidence of crosstalk between calpains
and caspases in the brain [48, 49], and both apoptotic
and non-apoptotic activation of caspase-3 in discrete
neurons has been demonstrated in AD brain [10, 59], al-
though the pathological relevance of this is not clear
[29]. Caspase-3 exists as a 32 kDa pro-enzyme which
has limited catalytic activity, and as active fragments of
17- and 19-kDa that are generated by the action of
caspase-8 and caspase-9, respectively. Here, blots of
brain lysates were probed with an antibody against
caspase-3 that detects both the pro-caspase and active
fragments. As we found previously [2], this antibody de-
tected predominantly a 19 kDa active caspase-3 band in
postmortem brain (Fig. 3a). When the amounts of this
active caspase-3 band were standardized to NSE
amounts in the same sample, we found no significant in-
crease in active caspase-3 in any AD group when com-
pared to controls (Fig. 3e). This finding is in keeping
with previous results from our group [2] and others [30].
Cleavage of α-spectrin increases during AD progression
We next examined cleavage of the cytoskeletal protein
α-spectrin as a surrogate marker of calpain-1 and
caspase-3 activities. Blots were probed with an antibody
against α-spectrin which detects bands of 240 kDa
(holoprotein), calpain- and caspase-cleaved fragments
(145 to 150 kDa) and caspase-3-cleaved fragments (110
to 130 kDa products) (Fig. 3a). Calpain- and caspase-
cleaved α-spectrin bands were separately quantified fol-
lowing their normalization to NSE to control for any dif-
ferences in protein loading. This quantification showed a
general trend of increased levels of 145–150 kDa calpain
and caspase-cleaved α-spectrin fragments from Braak
stage II to VI, which was significantly different from
control in Braak stage III (p<0.05) and VI (p<0.001)
brain (Fig. 3f). No differences were found in the
amounts of caspase-cleaved α-spectrin fragments be-
tween any AD tissue and control. This suggests that the
increased amounts of 145–150 kDa α-spectrin bands de-
tected in AD are due to the action of calpain and not
caspases, which is in keeping with our analysis of these
protease activities (Fig. 3a–e).
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 6 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Fig. 3 (See legend on next page.)
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 7 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Cdk5/p25 is elevated in Braak III brains and is sustained
to late-stage disease
Cyclin-dependent kinase 5 (cdk5) is a proline-directed
serine/threonine kinase that is somewhat controversially
implicated in AD pathogenesis [19, 55, 69]. Cdk5 is acti-
vated when it forms a complex with one of its neuronal
activators, such as p35. When cleaved by calpain, p35
yields the more stable and potent activator, p25, sus-
tained expression of which is associated with increased
tau phosphorylation and tau-associated synaptic and
neuronal loss in vivo [12, 52]. Here, blots of AD brain ly-
sates were probed with antibodies against cdk5, yielding
a band of 33 kDa, and p35 which detects both p35
(35 kDa) and p25 (25 kDa) (Fig. 4a). Quantification of
these results showed no significant changes in total cdk5
protein, p35 or p25 amounts in AD brain when com-
pared to control (Fig. 4b, c). However, when p25 was
measured as a proportion of p35, we found a significant
increase in the p25/p35 ratio in Braak stage III to V
brain (p<0.05 for all) compared to control (Fig. 4d), the
same disease stages in which calpain activity was found
to be significantly elevated. This was indicative that in-
creased calpain-mediated p25 generation and therefore
increased cdk5 activity occurs from an early stage of AD
Fig. 4 Changes in cdk5 and GSK-3 activities with AD progression. aRepresentative blots of cortical homogenates from postmortem brain. Blots
were probed with antibodies against cyclin dependent kinase 5 (cdk5) to detect holoprotein at 33 kDa, p35 to detect holoprotein at 35 kDa and
calpain-cleaved 25 kDa fragments (p25) at 25 kDa, total glycogen synthase kinase 3α/β(totGSK3) at 47 and 51 kDa, respectively and GSK3α/β
phosphorylated at Ser21/9 (pGSK3). Blots were also probed with an antibody against neuron-specific enolase (NSE, 45 kDa) as a loading control.
Bar graphs show amounts of (b) cdk5 relative to NSE, (c) p35 following normalisation to cdk5, (d) the p25/p35 ratio, (e) GSK3αand (f) GSK3βrela-
tive to NSE, and (g) phosphorylated (inactive) GSK3 normalised to total GSK-3 protein. CTRL: control (n=5), Braak II AD (n=4), Braak III AD (n=3),
Braak IV AD (n=4), Braak V AD (n=3), Braak VI AD (n=5). Data is mean ± SEM. *p<0.05, **p<0.01, ***p<0.001
(See figure on previous page.)
Fig. 3 Active calpain-1 amounts are elevated early in AD and are sustained throughout disease progression. aRepresentative blots of cortical
homogenates from postmortem brain. Blots were probed with antibodies to detect active calpain-1 at 76 kDa and active/cleaved caspase-3 at
19 kDa. An antibody against calpastatin (CAST) was used to detect CAST holoprotein at 110 kDa, active CAST at> 25 kDa and inactive CAST at <25 kDa.
An antibody against α-spectrin was used to detect holoprotein at 240 kDa, calpain- and caspase-cleaved fragments at 140 to 150 kDa and
caspase-cleaved fragments at 110 to 130 kDa. Blots were also probed with an antibody against neuron-specific enolase (NSE, 45 kDa) as a loading
control. Bar graphs show amounts of (b) active calpain-1 relative to NSE in each sample, (c) active CAST and (d) inactive CAST both as a proportion of
total CAST (e) active caspase-3 relative to NSE, (f) caspase- and calpain- cleaved 140–150 kDa α-spectrin fragments and (g) caspase-cleaved 110–125
kD α-spectrin fragments, both standardized to NSE. CTRL: control (n=5), Braak II AD (n=4), Braak III AD (n=3), Braak IV AD (n=4), Braak V AD (n=3),
Braak VI AD (n=5). Data is mean ± SEM. *p<0.05, **p<0.01, ***p<0.001
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 8 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

development and is sustained throughout disease pro-
gression. It is worth noting that p25 levels are also re-
ported to be decreased in AD [reviewed in 20], with
increased p25 linked to synaptogenesis [20]. While the
reasons for the differences between these studies and
ours are not clear, it is possible that the elevated p25
amounts that we observe may be associated with the in-
creased abundance of synaptic proteins, at least in early
Braak stages.
GSK3 expression and activity are increased in late stage
AD brain
Glycogen synthase kinase 3 (GSK3) is a proline-directed
serine/threonine kinase that plays a central role in AD
pathology [26]. GSK-3 exists as two isoforms, GSK3α
and GSK3β, which are phosphorylated at Ser21 and
Ser9, respectively, to suppress kinase activity. GSK3 can
be activated by calpain-mediated cleavage of the N-
terminal portion of the kinase which removes Ser21/9 to
allow an active kinase conformation [24]. Blots of brain
lysates were probed with an antibody against GSK3α/β,
yielding two bands at 51 and 47 kDa, which represent
GSK3αand GSK3β, respectively (Fig. 4a). When normal-
ized to NSE amounts in each sample, we found signifi-
cantly increased levels of GSK3α(p<0.001) and GSK3β
(p<0.0001) in Braak VI AD tissue compared to control
(Fig. 4e, f). Blots were also probed with an antibody spe-
cific for GSK3αand GSK3βphosphorylated at Ser21/9
(pGSK3). We detected one prominent band in these tis-
sues (Fig. 4a), which is likely to represent pSer9 on GSK-
3βsince the antibody we used exhibits preference for
this site. We therefore did not differentiate between -α
and –βisomers in our quantitative analysis. Our results
indicated that phosphorylation of GSK3 is significantly
reduced in Braak stage II AD brain when compared to
control (Fig. 4g), indicating increased GSK3 activity at
the very earliest stages of AD development. GSK-3 activ-
ity was not sustained throughout disease and was rather
variable in later disease stages.
Pre- and post-synaptic proteins are upregulated at Braak
stage II-III and are lost in late-stage AD brain
Alterations in intracellular Ca
2+
and calpain activities, as
well as the accumulation of phosphorylated tau, are linked
with disrupted synaptic function in AD [11, 78]. We there-
fore assessed changes in synaptic markers in postmortem
brain lysates. Blots were probed with an antibody against
the pre-synaptic protein synapsin I, a neuron specific
phosphoprotein localized to the cytoplasmic side of small
synaptic vesicles that plays an important role in the release
of neurotransmitters [3], which yielded two bands at ap-
proximately 70 and 74 kDa (Fig. 5a). To assess post-
synaptic changes, antibodies against the NR2B subunit of
N-methyl-D-aspartate (NMDA) receptors (170 kDa) and
postsynaptic density-95 (PSD95, 95 kDa) were used
(Fig. 5a). NR2B is a post-synaptic ionotropic glutamate re-
ceptor that conducts Ca
2+
and mediates excitotoxic cell
death in models of AD [29]. PSD-95 is an integral scaf-
folding component of the postsynapse that is also com-
monly used a marker for loss of synapses in AD models
(e.g. [16]). All synaptic protein levels were normalized
against NSE prior to statistical analysis.
Quantitative analysis revealed a similar pattern for all
markers studied in the supernatant fraction, with increased
protein levels apparent in Braak stages II-III relative to con-
trols, followed by a recovery to normal levels or loss at end-
stage disease. Synapsin-I and NR2B protein amounts were
significantly increased in Braak stage II-III tissues, but were
not different from control amounts in later stage AD (Fig. 5a).
PSD95 protein amounts were also significantly increased in
Braak III brains (p<0.05) and were reduced below control
amounts at end-stage AD (Braak stage VI) (Fig. 5a). These
results perhaps suggest an increase in synapse number or ac-
tivity during the early stages of AD, concomitant with in-
creased APP protein amounts, which is lost as disease
progresses and synapses degenerate.
In case the preparation of these samples resulted in
synaptic proteins being lost in the pellets deposited by
centrifugation, we also solubilized the respective protein
pellets and immunoblotted these samples as described
above. β-actin was used to normalize these blots so that
the influence of neuron loss could be taken into account.
In general, these blots showed a similar pattern to that
observed when probing supernatants (Fig. 5b), with the
exception of PSD-95 amounts which were much more
stable across Braak stages. It is possible that this relates
to the observation that PSD-95 is present in both cyto-
plasmic and postsynaptic membrane compartments [27];
the results here may suggest that there is tighter regula-
tion of membrane-associated PSD-95 in disease.
We also detected NR2B fragments of approximately
150 kDa (Fig. 5b) in pellet fractions. These degradation
products have previously been reported as an important
measure of synaptic integrity [5]. Their presence indi-
cates that there has been degradation of synaptic pro-
teins in the tissues analysed here. However, we observed
a direct correlation between the amounts of NR2B
degradation products and full-length protein (r=0.5017,
p=0.0339) therefore this degradation is believed not
contributed to the changes in protein amounts reported
here with respect to Braak stage.
Calpain-1 correlates with Aβ1-42 burden, tau accumulation
and tau kinase activity
Previous studies have linked elevated intracellular Ca
2+
to
Aβoverproduction, tau phosphorylation and synaptic dys-
function in AD [38, 83]. We therefore sought to determine
whether elevated calpain-1 activity in postmortem brain
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 9 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

of different AD stages correlated with other pathological
findings, including tau phosphorylation and amounts, tau
kinase activity, Aβburden, and synaptic protein expres-
sion (Fig. 6).
We found that increased calpain-1 activity significantly
correlated with increased total amounts of Aβ1-42 (p<
0.001; Fig. 6a), indicating that calpain may regulate APP
processing or be activated by Aβin AD brain, both of
Fig. 5 Pre- and post- synaptic protein amounts are altered during AD development. Representative blots of (a) supernatants and (b) pellets from
cortical homogenates of postmortem brain. Blots were probed with antibodies against synapsin I holoprotein (74 kDa), the NR2B subunit of N-methyl
D-aspartate receptor (NR2B,170 kDa) and post-synaptic density 95 protein (PSD95, 95 kDa. Blots were also probed with antibodies against neuron-specific
enolase (NSE, 45 kDa) or β-actin (42 kDa) as loading controls. Bar graphs show amounts of synapsin I, NR2B and PSD95, all normalised to their respective
loading control. CTRL: control (n=5), Braak II AD (n=4), Braak III AD (n=3), Braak IV AD (n=4), Braak V AD (n=3), Braak VI AD (n=5). Data is mean ± SEM.
*p<0.05, **p<0.01
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 10 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

which mechanisms have previously been reported in
vitro [45, 71]. Correlation of calpain-1 with calpain-
cleaved α-spectrin and p25 amounts provided further
confirmation of aberrant calpain-1 proteolytic activity in
AD brain (p<0.05 for both; Fig. 6b, c). Correlations be-
tween other proteins examined in this work did not yield
any positive results (data not shown).
In summary, our findings demonstrate that the activity
of the calcium-regulated protease, calpain, is elevated at
early Braak stages (II-III), occurring alongside activation
of the tau kinases cdk5 and GSK-3, and preceding accu-
mulation of Aβ1-42, increases in phosphorylation of tau at
disease relevant epitopes, and loss of synaptic markers at
end-stage AD (Fig. 6d). In addition, we show increases in
the amounts of APP, pre- and post-synaptic markers in
Braak stage II-III AD brain that may represent some, as
yet, unknown response of the nervous system to counter-
act the influence of early neurodegenerative changes.
Fig. 6 Calpain-1 activities in AD brain correlate with Aβ1-42 burden, cytoskeletal protein cleavage and kinase activities. Scatter plots show the
correlation between amounts of active calpain-1 and (a)Aβ1-42, (b) calpain- and caspase-cleaved α-spectrin fragments and (c) p25 in all tissue
samples. Correlation analysis was used to generate correlation co-efficients (r values) and significance. *p<0.05, **p<0.01, ***p<0.001. dQualitative
plot illustrating the stage of disease at which changes were observed in calpain-1 activity, total APP protein, Aβ1-42 amounts, active cdk5 (p25/cdk5),
active GSK-3 (reductions in pGSK-3), p-tau (tau phosphorylated at Ser396/404), pre- (synapsin-1) and post- (PSD-95) synaptic marker amounts. Relative
amounts are indicated in grey scale, with low protein amounts signified by pale shading and large amounts by dark shading
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 11 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Discussion
Here we have used postmortem brain from all Braak
stages to examine at which stage of disease development
changes occur in key neurodegenerative disease proteins.
We demonstrate that there is increased activity of
calpain-1 from Braak stage III onwards in comparison to
controls, extending previous findings that calpain-1 is
upregulated at end-stage disease. In addition, activation
of the tau kinases, GSK-3 and cdk5 were also found to
occur in Braak stage II-III tissues, and these preceded
global elevations in tau phosphorylation and the loss of
post-synaptic markers observed in late-stage AD. In
addition, we identified transient increases in total APP
and pre-synaptic markers in Braak stage II-III, that were
lost by end-stage AD, that may be indicative of endogen-
ous compensatory responses to the initial stages of neu-
rodegeneration. Our human brain data substantiate
findings from many experimental models which have
supported the hypothesis that sporadic AD arises in re-
sponse to Aβ-mediated dysregulation of calcium signal-
ling [6, 34, 37, 65, 66, 70].
Activation of calpain-1 was used as a marker for
calcium dysregulation in this study. Calpain-1 is an
intracellular cysteine protease that is activated upon
autoproteolytic cleavage of the inactive precursor at its
N-terminus in low micromolar (μM) concentrations of
calcium. Increased truncation and activation of calpain-1
has previously been reported in late stage (Braak V-VI)
AD brain [2, 25, 32, 61]. In addition, biomarker studies
have recently demonstrated increased calpain activity in
cerebrospinal fluid, and corresponding reductions of
calpain activity in serum and plasma, in AD patients
relative to non-cognitively impaired controls [39]. This
is not surprising since calpain-mediated proteolysis has
been implicated in many neurodegenerative pathways
including the processing of amyloid precursor protein to
generate Aβspecies and resulting synaptic dysfunction
[45, 72], cleavage and phosphorylation of tau by cdk5,
GSK-3 and dual specificity tyrosine-phosphorylation reg-
ulated kinase 1A DYRK1A [25, 32, 50, 71], and altered
learning and memory abilities via processing of synaptic
proteins and suppression of LTP [33, 41].
In addition, recent evidence has implicated the calpain
substrate and endogenous inhibitor, calpastatin in a
novel autodestruction pathway linked to neurodegenera-
tion [80, 81]. Rapid Wallerian degeneration of injured
axons was shown to occur following activation of calpain
alongside depletion of calpastatin inhibitory activity [31].
Induction of this calpain-calpastatin-mediated degeneration
pathway was subsequently shown to occur downstream
of nicotinamide mononucleotide adenylyltransferase 1-
mediated changes in Sarm1 and mitogen activated protein
kinase activities, and depletion of ATP [21, 32]. In addition
to playing an important role in pruning processes during
neuronal development [31], this pathway is likely to be in-
volved in a wide spectrum of neurodegenerative diseases.
Subsequent investigations will likely provide more insight
into the importance of this signalling cascade for AD.
Another area in which dysregulation of calcium and/or
calpain signalling is likely to be an important influence
is the prion-like propagation of protein aggregates, a
topic of intensive research in neurodegenerative disease
research. Both Aβand tau aggregates are reported to be
transmitted through AD brain along anatomically
connected pathways [82]. Although all of the mecha-
nisms underlying pathology spread are not completely
understood, stimulating electrical activity, or activating
calcium-dependent NMDA and AMPA receptors, was
shown to induce the release of tau from neurons in pri-
mary culture and in mouse models of disease [9, 58, 79].
Thus, it is possible that dysregulation of calcium-calpain
pathways may contribute to tau spread in neurodegener-
ative tauopathies, including AD.
There are several questions raised by experimental
models that were not addressed in this study. For example,
calpain-mediated cleavage of the NR2B subunit of
NMDARs has been shown to give rise to active NMDAR
forms that could exacerbate excitotoxicity [22, 64]. We
did not observe calpain-cleaved NR2B fragments in this
study, which could have been due to the effects of post-
mortem degradation of rapidly turned over proteins, or
the levels of these fragments being below detectable levels.
The transient increase in NR2B holoprotein that we
observe at Braak stage II-III in supernatant fractions could
imply that calpain-mediated cleavage of NR2B occurs
from mid-stage AD. Alternatively, it is possible that an
early compensatory response resulting in increased NR2B
in Braak II-III tissues is overcome as AD develops.
In addition, we observed loss of only post-synaptic pro-
teins in supernatants from late-stage AD cortical homoge-
nates, with the pre-synaptic marker synapsin 1 being
increased at Braak stage III and returning to control levels
at end-stage AD. This result is in discrepancy to previous
findings showing reductions in synapsin-1 amounts in lam-
ina 3 of the posterior cingulate cortex in Braak stage V-VI
AD brain, relative to early Braak stage and non-cognitively
impaired controls [62]. However, connections from the pos-
terior cingulate are very different to those from the tem-
poral cortex [56], and this may account for the difference in
these findings. In addition, the relatively small sample set
used in this study may have masked subtle changes in
protein amounts during AD progression. Furthermore,
the control group used in this study included some
individuals younger than average in comparison to
the experimental groups. This is believed not to have
skewed the findings since these samples did not appear to
differ significantly from older controls. However, it would
be interesting in future work to assess the contribution of
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 12 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

normal aging to the changes described here, perhaps using
resources such as that collected from the MRC-CFAS
study [19] or the Lothian Birth Cohort [57].
Conclusions
In conclusion, in this study we have used postmortem hu-
man brain to examine protein changes in different stages
of AD. We provide evidence to show that alterations in
calpain activity occurs relatively early in the disease
process, concurrent with increased Aβ1-42 production
and activation of tau kinases, and prior to increased tau
phosphorylation and loss of post-synaptic markers
(Fig. 6d). Our findings therefore suggest that aberrant
regulation of calpain is an important early step in disease
development, supporting ongoing pre-clinical and clinical
studies focused on correcting disrupted calcium channel
activation and calpain activation in Alzheimer’s disease
and related neurodegenerative conditions. Moreover, our
results suggest that there are synaptic compensatory
mechanisms during early Braak stages. Further experi-
mentation may reveal the mechanisms underlying these
events and perhaps indicate strategies to prolong this sup-
posed endogenous neuroprotective response.
Ethics approval and consent to participate
Postmortem human brain was obtained from the MRC
London Neurodegenerative Diseases Brain Bank (REC
reference: 08/MRE09/38 + 5).
Consent for publication
Not applicable.
Additional files
Additional file 1: Table S1. Characteristics of postmortem brain samples
(DOCX 75 kb)
Additional file 2: Table S2. Summary of postmortem brain characteristics
(DOC 28 kb)
Abbreviations
AD: Alzheimer’s disease; Aβ:β-amyloid; AMPA: α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid; APP: amyloid precursor protein; CAST: calpastatin;
CDK5: cyclin-dependent kinase 5; GSK3: glycogen synthase kinase 3;
MAPK: mitogen activated protein kinase; NFT: neurofibrillary tangle; NMDAR:
N-methyl-D-aspartate receptor; NMNAT1: nicotinamide mononucleotide
adenylyltransferase 1; NSE: neuron specific enolase; PSD95: postsynaptic density-
95; PICALM: phosphatidylinositol binding clathrin assembly protein;
SARM1: sterile alpha and TIR motif-containing protein.
Competing interests
Michael J. O’Neill is an employee of Eli Lilly.
Author contributions
KK, ECP, GD, CLC, MAW, MMH, BGP-N and CT performed the experiments and
analyzed the data. KK and WN designed the research and KK, CT, BGP-N, MO,
DPH and WN wrote and revised the paper. All authors read and approved the
final manuscript.
Acknowledgements
We are grateful to Professor Peter Davies (Feinstein Institute for Medical
Research, NY, USA) for his generous gift of tau antibodies.
Funding
This work was supported by Alzheimer’s Research UK (ARUK-ESG2014-2 to WN;
ARUK-RF2014-2 to BGP-N), Rosetrees Trust (JS15/M367 to WN), BBSRC/Eli Lilly (BB/
K501219/1 to WN) and the National Centre for the Replacement, Refinement and
Reduction of Animals in Research (NC3Rs, NC/K500343/1 to WN).
Author details
1
Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical
Neuroscience Institute, King’s College London, Institute of Psychiatry,
Psychology and Neuroscience, Rm1.25, 5 Cutcombe Road, Camberwell,
London SE5 9RX, UK.
2
King’s College London, MRC London
Neurodegenerative Diseases Brain Bank, London, UK.
3
Eli Lilly and Company,
Erl Wood Manor, , Windlesham, Surrey GU20 6PH, UK.
Received: 8 February 2016 Accepted: 15 March 2016
References
1. Ando K, Brion JP, Stygelbout V, Suain V, Authelet M, Dedecker R, Chanut A,
Lacor P, Lavaur J, Sazdovitch V, Rogaeva E, Potier MC, Duyckaerts C. Clathrin
adaptor CALM/PICALM is associated with neurofibrillary tangles and is
cleaved in Alzheimer’s brains. Acta Neuropathol. 2013;125:861–78.
doi:10.1007/s00401-013-1111-z.
2. Atherton J, Kurbatskaya K, Bondulich M, Croft CL, Garwood CJ, Chhabra R,
Wray S, Jeromin A, Hanger DP, Noble W. Calpain cleavage and inactivation
of the sodium calcium exchanger-3 occur downstream of Aβin Alzheimer’s
disease. Aging Cell. 2014;13:49–59. doi:10.1111/acel.12148.
3. Bähler M, Benfenati F, Valtorta F, Greengard P. The synapsins and the
regulation of synaptic function. Bioessays. 1990;12:259–63. doi:10.1002/
bies.950120603.
4. Baki A, Tompa P, Alexa A, Molnár O, Friedrich P (1996) Autolysis parallels
activation of mu-calpain. Biochem J 318 (Pt 3:897–901.
5. Bayés À, Collins MO, Galtrey CM, Simonnet C, Roy M, Croning MD, Gou
G, van de Lagemaat LN, Milward D, Whittle IR, Smith C, Choudhary JS,
Grant SG. Human post-mortem synapse proteome integrity screening
for proteomic studies of postsynaptic complexes. Mol Brain. 2014;7:88.
doi:10.1186/s13041-014-0088-4.
6. Bezprozvanny I, Mattson MMP. Neuronal calcium mishandling and the
pathogenesis of Alzheimer’s disease. Trends Neurosci. 2008;31:454–63.
doi:10.1016/j.tins.2008.06.005.Neuronal.
7. Boutajangout A, Boom A, Leroy K, Brion JP. Expression of tau mRNA and
soluble tau isoforms in affected and non-affected brain areas in Alzheimer’s
disease. FEBS Lett. 2004;576:183–9. doi:10.1016/j.febslet.2004.09.011.
8. Braak H, Thal DR, Ghebremedhin E, Del Tredici K. Stages of the Pathologic
Process in Alzheimer Disease. J Neuropathol Exp Neurol. 2011;70:960–9.
doi:10.1097/NEN.0b013e318232a379.
9. Bright J, Hussain S, Dang V, Wright S, Cooper B, Byun T, Ramos C, Singh A,
Parry G, Stagliano N, Griswold-Prenner I. Human secreted tau increases
amyloid-beta production. Neurobiol Aging. 2015;36:693–709. doi:10.1016/j.
neurobiolaging.2014.09.007.
10. De Calignon A, Fox LM, Pitstick R, Carlson GA, Bacskai BJ, Spires-Jones TL,
Hyman BT. Caspase activation precedes and leads to tangles. Nature. 2010;
464:1201–4. doi:10.1038/nature08890.
11. Crimins JL, Pooler A, Polydoro M, Luebke JI, Spires-Jones TL. The
intersection of amyloid beta and tau in glutamatergic synaptic dysfunction
and collapse in Alzheimer’s disease. Ageing Res Rev. 2013;12:757–63.
doi:10.1016/j.arr.2013.03.002.
12. Cruz JC, Tseng HC, Goldman JA, Shih H, Tsai LH. Aberrant Cdk5 activation
by p25 triggers pathological events leading to neurodegeneration and
neurofibrillary tangles. Neuron. 2003;40:471–83. doi:10.1016/S0896-
6273(03)00627-5.
13. Dawkins E, Small DH. Insights into the physiological function of the β-
amyloid precursor protein: Beyond Alzheimer’s disease. J Neurochem. 2014;
129:756–69. doi:10.1111/jnc.12675.
14. Delvaux E, Bentley K, Stubbs V, Sabbagh M, Coleman PD. Differential
processing of amyloid precursor protein in brain and in peripheral blood
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 13 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

leukocytes. Neurobiol Aging. 2013;34:1680–6. doi:10.1016/j.neurobiolaging.
2012.12.004.
15. Demuro A, Smith M, Parker I. Single-channel Ca2+ imaging implicates Abeta
1–42 amyloid pores in Alzheimer’s disease pathology. J Cell Biol. 2011;195:
515–24. doi:10.1083/jcb.201104133.
16. Dorostkar MM, Burgold S, Filser S, Barghorn S, Schmidt B, Anumala UR,
Hillen H, Klein C, Herms J. Immunotherapy alleviates amyloid-associated
synaptic pathology in an Alzheimer’s disease mouse model. Brain. 2014;137:
3319–26. doi:10.1093/brain/awu280.
17. Ferreira A, Bigio EH. Calpain-mediated tau cleavage: a mechanism leading
to neurodegeneration shared by multiple tauopathies. Mol Med. 2011;17:
676–85. doi:10.2119/molmed.2010.00220.
18. Ferreira IL, Ferreiro E, Schmidt J, Cardoso JM, Pereira CMF, Carvalho AL, Oliveira
CR, Rego AC. Abeta and NMDAR activation cause mitochondrial dysfunction
involving ER calcium release. Neurobiol Aging. 2015;36:680–92. doi:10.1016/j.
neurobiolaging.2014.09.006.
19. Garwood C, Faizullabhoy A, Wharton SB, Ince PG, Heath PR, Shaw PJ, Baxter
L, Gelsthorpe C, Forster G, Matthews FE, Brayne C, Simpson JE; MRC
Cognitive Function and Ageing Neuropathology Study Group. Calcium
dysregulation in relation to Alzheimer-type pathology in the ageing brain.
Neuropathol Appl Neurobiol. 2013;39:788–99. doi:10.1111/nan.12033.
20. Giese KP. Generation of the Cdk5 activator p25 is a memory mechanism
that is affected in early Alzheimer’s disease. Front Mol Neurosci. 2014;7:36.
doi:10.3389/fnmol.2014.00036.
21. Gilley J, Orsomando G, Nascimento-Ferreira I, Coleman MP. Absence of
SARM1 Rescues Development and Survival of NMNAT2-Deficient Axons. Cell
Rep. 2015;10:1974–81. doi:10.1016/j.celrep.2015.02.060.
22. Gladding CM, Sepers MD, Xu J, Zhang LYJ, Milnerwood AJ, Lombroso PJ,
Raymond LA. Calpain and STriatal-Enriched protein tyrosine Phosphatase
(STEP) activation contribute to extrasynaptic NMDA receptor localization in
a huntington’s disease mouse model. Hum Mol Genet. 2012;21:3739–52.
doi:10.1093/hmg/dds154.
23. Gómez-Sintes R, Hernández F, Bortolozzi A, Artigas F, Avila J, Zaratin P,
Gotteland JP, Lucas JJ. Neuronal apoptosis and reversible motor deficit in
dominant-negative GSK-3 conditional transgenic mice. EMBO J. 2007;26:
2743–54. doi:10.1038/sj.emboj.7601725.
24. Goñi-Oliver P, Lucas JJ, Avila J, Hernández F. N-terminal cleavage of GSK-3
by calpain: A new form of GSK-3 regulation. J Biol Chem. 2007;282:22406–
13. doi:10.1074/jbc.M702793200.
25. Grynspan F, Griffin WR, Cataldo A, Katayama S, Nixon RA. Active site-
directed antibodies identify calpain II as an early- appearing and pervasive
component of neurofibrillary pathology in Alzheimer’s disease. Brain Res.
1997;763:145–58. doi:10.1016/S0006-8993(97)00384-3.
26. Hanger DP, Anderton BH, Noble W. Tau phosphorylation: the therapeutic
challenge for neurodegenerative disease. Trends Mol Med. 2009;15:112–9.
doi:10.1016/j.molmed.2009.01.003.
27. Henstridge CM, Jackson RJ, Kim JM, Herrmann AG, Wright AK, Harris SE,
Bastin ME, Starr JM, Wardlaw J, Gillingwater TH, Smith C, McKenzie CA, Cox
SR, Deary IJ, Spires-Jones TL. Post-mortem brain analyses of the Lothian
Birth Cohort 1936: extending lifetime cognitive and brain phenotyping to
the level of the synapse. Acta Neuropathol Commun. 2015;3:53. doi:10.1186/
s40478-015-0232-0.
28. Hyman BT, Augustinack JC, Ingelsson M. Transcriptional and conformational
changes of the tau molecule in Alzheimer’s disease. Biochim Biophys Acta -
Mol Basis Dis. 2005;1739:150–7. doi:10.1016/j.bbadis.2004.06.015.
29. Ittner LM, Ke YD, Delerue F, Bi M, Gladbach A, van Eersel J, Wölfing H,
Chieng BC, Christie MJ, Napier IA, Eckert A, Staufenbiel M, Hardeman E, Götz
J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer’s
disease mouse models. Cell. 2010;142:387–97. doi:10.1016/j.cell.2010.06.036.
30. Jellinger KA, Stadelmann C. Mechanisms of cell death in neurodegenerative
disorders. J Neural Transm Suppl. 2000;59:95–114.
31. Jin N, Qian W, Yin X, Zhang L, Iqbal K, Grundke-Iqbal I, Gong CX, Liu F. CREB
regulates the expression of neuronal glucose transporter 3: A possible
mechanism related to impaired brain glucose uptake in Alzheimer’s disease.
Nucleic Acids Res. 2013;41:3240–56. doi:10.1093/nar/gks1227.
32. Jin N, Yin X, Yu D, Cao M, Gong C, Iqbal K, Gu X, Liu F. Truncation and
activation of GSK-3βby calpain I: a molecular mechanism links to tau
hyperphosphorylation in Alzheimer’s disease. Sci Rep. 2015;5:8187.
doi:10.1038/srep08187.
33. Kelly BL, Vassar R, Ferreira A. β-amyloid-induced dynamin 1 depletion in
hippocampal neurons: A potential mechanism for early cognitive decline in
Alzheimer disease. J Biol Chem. 2005;280:31746–53. doi:10.1074/jbc.
M503259200.
34. Khachaturian ZS. Calcium, membranes, aging and Alzheimer’s disease:
Introduction and overview. Ann N Y Acad Sci. 1989;568:1–4. doi:10.1111/j.
1749-6632.1989.tb12485.x.
35. Khatoon S, Grundke-Iqbal I, Iqbal K. Levels of normal and abnormally
phosphorylated tau in different cellular and regional compartments of
Alzheimer disease and control brains. FEBS Lett. 1994;351:80–4.
36. Kuchibhotla KV, Goldman ST, Lattarulo CR, Wu HY, Hyman BT, Bacskai BJ.
Abeta Plaques Lead to Aberrant Regulation of Calcium Homeostasis In Vivo
Resulting in Structural and Functional Disruption of Neuronal Networks.
Neuron. 2008;59:214–25. doi:10.1016/j.neuron.2008.06.008.
37. LaFerla FM. Calcium dyshomeostasis and intracellular signalling in
Alzheimer’s disease. Nat Rev Neurosci. 2002;3:862–72. doi:10.1038/nrn960.
38. LaFerla FM, Oddo S. Alzheimer’s disease: Abeta, tau and synaptic dysfunction.
Trends Mol Med. 2005;11:170–6. doi:10.1016/j.molmed.2005.02.009.
39. Laske C, Stellos K, Kempter I, Stransky E, Maetzler W, Fleming I,
Randriamboavonjy V. Increased cerebrospinal fluid calpain activity and
microparticle levels in Alzheimer’s disease. Alzheimers Dement. 2015;11:465–
74. doi:10.1016/j.jalz.2014.06.003.
40. Lee MS, Kwon YT, Li M, Peng J, Friedlander RM, Tsai LH. Neurotoxicity
induces cleavage of p35 to p25 by calpain. Nature. 2000;405:360–4.
doi:10.1038/35012636.
41. Li S, Jin M, Koeglsperger T, Shepardson NE, Shankar GM, Selkoe DJ. Soluble Aβ
oligomers inhibit long-term potentiation through a mechanism involving
excessive activation of extrasynaptic NR2B-containing NMDA receptors.
J Neurosci. 2011;31:6627–38. doi:10.1523/JNEUROSCI.0203-11.2011.
42. Liang B, Duan BY, Zhou XP, Gong JX, Luo ZG. Calpain activation promotes
BACE1 expression, amyloid precursor protein processing, and amyloid
plaque formation in a transgenic mouse model of Alzheimer disease. J Biol
Chem. 2010;285:27737–44. doi:10.1074/jbc.M110.117960.
43. Liu F, Iqbal-Grundke I, Iqbal K, Oda Y, Tomizawa K, Gong CX. Truncation and
activation of calcineurin A by calpain I in Alzheimer disease brain. J Biol
Chem. 2005;280:37755–62. doi:10.1074/jbc.M507475200.
44. Mairet-Coello G, Courchet J, Pieraut S, Courchet V, Maximov A, Polleux F.
The CAMKK2-AMPK Kinase Pathway Mediates the Synaptotoxic Effects of Aβ
Oligomers through Tau Phosphorylation. Neuron. 2013;78:94–108.
doi:10.1016/j.neuron.2013.02.003.
45. Mathews PM, Jiang Y, Schmidt SD, Grbovic OM, Mercken M, Nixon RA.
Calpain activity regulates the cell surface distribution of amyloid precursor
protein. Inhibition of calpains enhances endosomal generation of ??-cleaved
C-terminal APP fragments. J Biol Chem. 2002;277:36415–24. doi:10.1074/jbc.
M205208200.
46. Mohmmad Abdul H, Baig I, LeVine H, Guttmann RP, Norris CM. Proteolysis
of calcineurin is increased in human hippocampus during mild cognitive
impairment and is stimulated by oligomeric Abeta in primary cell culture.
Aging Cell. 2011;10:103–13. doi:10.1111/j.1474-9726.2010.00645.x.
47. Morales-Corraliza J, Berger JD, Mazzella MJ, Veeranna NTA, Ghiso J, Rao MV,
Staufenbiel M, Nixon RA, Mathews PM. Calpastatin modulates APP
processing in the brains of beta-amyloid depositing but not wild-type mice.
Neurobiol Aging. 2012. doi:10.1016/j.neurobiolaging.2011.11.023.
48. Murachi T. Calpain and calpastatin. Rinsho Byori. 1990;38:337–46.
doi:10.1016/0968-0004(83)90165-2.
49. Nakagawa T, Yuan J. Cross-talk between two cysteine protease families:
Activation of caspase-12 by calpain in apoptosis. J Cell Biol. 2000;150:887–
94. doi:10.1083/jcb.150.4.887.
50. Nicholson AM, Methner DNR, Ferreira A. Membrane cholesterol modulates
{beta}-amyloid-dependent tau cleavage by inducing changes in the
membrane content and localization of N-methyl-D-aspartic acid receptors.
J Biol Chem. 2011;286:976–86. doi:10.1074/jbc.M110.154138.
51. Noble W, Hanger DP, Miller CCJ, Lovestone S. The importance of tau
phosphorylation for neurodegenerative diseases. Front Neurol. 2013.
doi:10.3389/fneur.2013.00083.
52. Noble W, Olm V, Takata K, Casey E, Mary O, Meyerson J, Gaynor K,
LaFrancois J, Wang L, Kondo T, Davies P, Burns M, Veeranna, Nixon R,
Dickson D, Matsuoka Y, Ahlijanian M, Lau LF, Duff K. Cdk5 is a key factor in
tau aggregation and tangle formation in vivo. Neuron. 2003;38:555–65. doi:
10.1016/S0896-6273(03)00259-9.
53. Noble W, Pooler AM, Hanger DP. Advances in tau-based drug discovery.
Expert Opin Drug Discov. 2011;6:797–810. doi:10.1517/17460441.2011.
586690.
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 14 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

54. Nordstedt C, Gandy SE, Alafuzoff I, Caporaso GL, Iverfeldt K, Grebb JA,
Winblad B, Greengard P. Alzheimer beta/A4 amyloid precursor protein
in human brain: aging-associated increases in holoprotein and in a
proteolytic fragment. Proc Natl Acad Sci U S A. 1991;88:8910–4.
doi:10.1073/pnas.88.20.8910.
55. Patrick GN, Zukerberg L, Nikolic M, de la Monte S, Dikkes P, Tsai LH.
Conversion of p35 to p25 deregulates Cdk5 activity and promotes
neurodegeneration. Nature. 1999;402:615–22. doi:10.1038/45159.
56. Pearson JM, Heilbronner SR, Barack DL, Hayden BY, Platt ML. Posterior
cingulate cortex: Adapting behavior to a changing world. Trends Cogn Sci.
2011;15:143–51. doi:10.1016/j.tics.2011.02.002.
57. Petersen JD, Chen X, Vinade L, Dosemeci A, Lisman JE, Reese TS.
Distribution of postsynaptic density (PSD)-95 and Ca2+/calmodulin-
dependent protein kinase II at the PSD. J Neurosci. 2003;23:11270–8.
58. Pooler AM, Phillips EC, Lau DHW, Noble W, Hanger DP. Physiological release
of endogenous tau is stimulated by neuronal activity. EMBO Rep. 2013;14:
389–94. doi:10.1038/embor.2013.15.
59. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death
Differ. 1999;6:99–104. doi:10.1038/sj.cdd.4400476.
60. Rao MV, Mohan PS, Peterhoff CM, Yang D-S, Schmidt SD, Stavrides PH,
Campbell J, Chen Y, Jiang Y, Paskevich PA, Cataldo AM, Haroutunian V,
Nixon RA. Marked calpastatin (CAST) depletion in Alzheimer’sdisease
accelerates cytoskeleton disruption and neurodegeneration:
neuroprotection by CAST overexpression. J Neurosci. 2008;28:12241–54.
doi:10.1523/JNEUROSCI.4119-08.2008.
61. Saito K, Elce JS, Hamos JE, Nixon RA. Widespread activation of calcium-
activated neutral proteinase (calpain) in the brain in Alzheimer disease: a
potential molecular basis for neuronal degeneration. Proc Natl Acad Sci U S
A. 1993;90:2628–32. doi:10.1073/pnas.90.7.2628.
62. Scheff SW, Price DA, Ansari MA, Roberts KN, Schmitt FA, Ikonomovic
MD, Mufson EJ. Synaptic change in the posterior cingulate gyrus in the
progression of Alzheimer’s disease. J Alzheimers Dis. 2015;43:1073–90.
doi:10.3233/JAD-141518.
63. Shinohara M, Fujioka S, Murray ME, Wojtas A, Baker M, Rovelet-Lecrux A,
Rademakers R, Das P, Parisi JE, Graff-Radford NR, Petersen RC, Dickson DW,
Bu G. Regional distribution of synaptic markers and APP correlate with
distinct clinicopathological features in sporadic and familial Alzheimer’s
disease. Brain. 2014;137:1533–49. doi:10.1093/brain/awu046.
64. Simpkins KL, Guttmann RP, Dong Y, Chen Z, Sokol S, Neumar RW, Lynch DR.
Selective activation induced cleavage of the NR2B subunit by calpain.
J Neurosci. 2003;23:11322–31.
65. Stutzmann GE, Mattson MP. Endoplasmic reticulum Ca(2+) handling in
excitable cells in health and disease. Pharmacol Rev. 2011;63:700–27.
doi:10.1124/pr.110.003814.
66. Stutzmann GE, Smith I, Caccamo A, Oddo S, Parker I, Laferla F. Enhanced
ryanodine-mediated calcium release in mutant PS1-expressing Alzheimer’s
mouse models. Ann N Y Acad Sci. 2007;1097:265–77. doi:10.1196/annals.1379.025.
67. SunT,WuX-S,XuJ,McNeilBD,PangZP,YangW,BaiL,QadriS,
Molkentin JD, Yue DT, Wu L-G. The role of calcium/calmodulin-activated
calcineurin in rapid and slow endocytosis at central synapses. J
Neurosci. 2010;30:11838–47. doi:10.1523/JNEUROSCI.1481-10.2010.
68. Supnet C, Bezprozvanny I. The dysregulation of intracellular calcium in
Alzheimer disease. Cell Calcium. 2010;47:183–9. doi:10.1016/j.ceca.2009.
12.014.
69. Taniguchi S, Fujita Y, Hayashi S, Kakita A, Takahashi H, Murayama S, Saido
TC, Hisanaga S, Iwatsubo T, Hasegawa M. Calpain-mediated degradation of
p35 to p25 in postmortem human and rat brains. FEBS Lett. 2001;489:46–50.
doi:10.1016/S0014-5793(00)02431-5.
70. Thibault O, Gant JC, Landfield PW. Expansion of the calcium hypothesis of
brain aging and Alzheimer’s disease: Minding the store. Aging Cell. 2007;6:
307–17. doi:10.1111/j.1474-9726.2007.00295.x.
71. Town T, Zolton J, Shaffner R, Schnell B, Crescentini R, Wu Y, Zeng J,
DelleDonne A, Obregon D, Tan J, Mullan M. p35/Cdk5 pathway mediates
soluble amyloid-?? peptide-induced tau phosphorylation in vitro. J Neurosci
Res. 2002;69:362–72. doi:10.1002/jnr.10299.
72. Trinchese F, Liu S, Zhang H. Inhibition of calpains improves memory and
synaptic transmission in a mouse model of Alzheimer disease. J Clin Invest.
2008;118:2796–807. doi:10.1172/JCI34254DS1.
73. Vagnoni A, Perkinton MS, Gray EH, Francis PT, Noble W, Miller CCJ. Calsyntenin-
1 mediates axonal transport of the amyloid precursor protein and regulates aβ
production. Hum Mol Genet. 2012;21:2845–54. doi:10.1093/hmg/dds109.
74. Vagnoni A, Rodriguez L, Manser C, De Vos KJ, Miller CCJ. Phosphorylation of
kinesin light chain 1 at serine 460 modulates binding and trafficking of
calsyntenin-1. J Cell Sci. 2011;124:1032–42. doi:10.1242/jcs.075168.
75. Veeranna KT, Boland B, Odrljin T, Mohan P, Basavarajappa BS, Peterhoff C,
Cataldo A, Rudnicki A, Amin N, Li BS, Pant HC, Hungund BL, Arancio O,
Nixon RA. Calpain mediates calcium-induced activation of the erk1,2 MAPK
pathway and cytoskeletal phosphorylation in neurons: relevance to
Alzheimer’s disease. Am J Pathol. 2004;165:795–805. doi:10.1016/S0197-
4580(04)80585-2.
76. Wang Y, Mattson MP. L-type Ca2+ currents at CA1 synapses, but not CA3 or
dentate granule neuron synapses, are increased in 3xTgAD mice in an age-
dependent manner. Neurobiol Aging. 2014;35:88–95. doi:10.1016/j.
neurobiolaging.2013.07.007.
77. Wu H, Hudry E, Hashimoto T, Kuchibhotla K, Fan Z, Spires-jones T, Xie H,
Arbel-ornath M, Cynthia L, Bacskai BJ, Hyman BT. Amyloid Beta (A-beta)
induces the morphological neurodegenerative tried of spine loss, dendritic
simplification, and neuritic dystrophies through calcineurin (CaN) activation.
J Neurosci. 2010;30:2636–49. doi:10.1523/JNEUROSCI.4456-09.2010.Amyloid.
78. Wu H-Y, Lynch DR. Calpain and synaptic function. Mol Neurobiol. 2006;33:
215–36. doi:10.1385/MN:33:3:215.
79. Yamada K, Holth JK, Liao F, Stewart FR, Mahan TE, Jiang H, Cirrito JR, Patel
TK, Hochgräfe K, Mandelkow E-M, Holtzman DM. Neuronal activity regulates
extracellular tau in vivo. J Exp Med. 2014;211:387–93. doi:10.1084/jem.
20131685.
80. Yang J, Weimer RM, Kallop D, Olsen O, Wu Z, Renier N, Uryu K, Tessier-
Lavigne M. Regulation of axon degeneration after injury and in
development by the endogenous calpain inhibitor calpastatin. Neuron.
2013;80:1175–89. doi:10.1016/j.neuron.2013.08.034.
81. Yang J, Wu Z, Renier N, Simon DJ, Uryu K, Park DS, Greer PA, Tournier C,
Davis RJ, Tessier-Lavigne M. Pathological Axonal Death through a MAPK
Cascade that Triggers a Local Energy Deficit. Cell. 2015;160:161–76.
doi:10.1016/j.cell.2014.11.053.
82. Yin R-H, Tan L, Jiang T, Yu J-T. Prion-like Mechanisms in Alzheimer’s Disease.
Curr Alzheimer Res. 2014;11:755–64. doi:10.2174/156720501108140910121425.
83. Zempel H, Thies E, Mandelkow E, Mandelkow E-M. Abeta oligomers cause
localized Ca(2+) elevation, missorting of endogenous Tau into dendrites,
Tau phosphorylation, and destruction of microtubules and spines.
J Neurosci. 2010;30:11938–50. doi:10.1523/JNEUROSCI.2357-10.2010.
• We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
Submit your next manuscript to BioMed Central
and we will help you at every step:
Kurbatskaya et al. Acta Neuropathologica Communications (2016) 4:34 Page 15 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com