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j.neuroscience.2017.10.021

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Accepted Manuscript
Research Paper
Postnatal proteasome inhibition promotes amyloid-β aggregation in hippocampal neurons and impairs spatial learning in adult mice
Aditya Sunkaria, Aarti Yadav, Supriya Bhardwaj, Rajat Sandhir
PII:
DOI:
Reference:
S0306-4522(17)30749-2
https://doi.org/10.1016/j.neuroscience.2017.10.021
NSC 18086
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Neuroscience
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Accepted Date:
4 May 2017
16 October 2017
Please cite this article as: A. Sunkaria, A. Yadav, S. Bhardwaj, R. Sandhir, Postnatal proteasome inhibition promotes
amyloid-β aggregation in hippocampal neurons and impairs spatial learning in adult mice, Neuroscience (2017),
doi: https://doi.org/10.1016/j.neuroscience.2017.10.021
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Postnatal proteasome inhibition promotes amyloid-β aggregation in hippocampal
neurons and impairs spatial learning in adult mice
Aditya Sunkaria1#, Aarti Yadav1, Supriya Bhardwaj2, Rajat Sandhir1
1
Department of Biochemistry, Panjab University, Chandigarh, INDIA
2
Department of Dermatology, Postgraduate Institute of Medical Education and Research,
Chandigarh, INDIA
Running Title: MG132 impair spatial learning
Key words: APP-CTF; Bdnf; MG132; Proteasome; Spatial learning
#
Author to whom all correspondence be addressed.
Dr. Aditya Sunkaria
Department of Biochemistry
Panjab University
Chandigarh-160014
(India)
Email - adityasunkaria@gmail.com
1
Abstract
Ubiquitin-proteasome system (UPS) has emerged as major molecular mechanism which
modulates synaptic plasticity. However, very little is known about what happen if this system
fails during postnatal brain development. In the present study, MG132 was administered
intracerebroventricularly in BALB/c mice pups at postnatal day one (P1), a very crucial
period for synaptogenesis. Both 20S proteasome and calpain activities were found to be
reduced in the mid brain of MG132 administered pups after 24 h. Mice (P40) which received
MG132 on P1 were subjected to Morris water maze (MWM) training. Analysis showed
spatial learning and memory of MG132 mice was significantly impaired when compared to
age matched controls. Hematoxylin and eosin as well as cresyl violet staining revealed
substantial loss of neural connections, distorted architecture and increased pyknosis in
hippocampal CA1 and CA3 regions of MG132 mice. Immunohistochemical analysis of
MG132 mice showed increased accumulation of intracellular amyloid-β in hippocampal
neurons when compared to control. Moreover, double immunostaining revealed increased
expression of amyloid precursor protein C-terminal fragments (APP-CTFβ) without affecting
β-secretase expression in MG132 mice. Real-Time PCR analyses showed significant increase
in hippocampal expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptor subunit glutamate A1 (GluA1), but no change in the brain-derived
neurotrophic factor (Bdnf) expression in MG132 mice. Western blot analyses showed
decreased levels of pThr286-CaMKIIα:CaMKIIα and pSer133-CREB:CREB ratio but
increased pro:mature BDNF ratio in hippocampus of MG132 mice. Taken together, postnatal
proteasome inhibition could lead to accumulation of intracellular amyloid-β protein
aggregates, which mediate hippocampus dependent spatial memory impairments in adult
mice.
Key words: APP-CTF; Bdnf; MG132; Proteasome; Spatial learning
2
Introduction
UPS is involved in many biological processes, including aspects of neuronal development
ranging from axon morphogenesis and synapse refinement (Mabb and Ehlers, 2010).
Postnatal brain development has been considered as crucial period which comprises
formation and refinement of synaptic connections. Previous studies have shown that ubiquitin
and UPS proteins are abundant in newly formed presynaptic terminals (Speese et al., 2003,
Segref and Hoppe, 2009), and mice incapable of degrading the ubiquitinated proteins had
defective synapse formation (Wilson et al., 2002, Chen et al., 2009, Chen et al., 2011).
Although, exact period of synapse development is not completely understood but initial
postnatal weeks are crucial for the synaptogenesis in pyramidal neurons (Yuste and
Bonhoeffer, 2004). Precise synapses formation during development and their modulation in
adulthood are important for normal nervous system functioning. Therefore, early postnatal
inhibition of proteasome could affect the synaptic modulation and impart damaging effects on
learning during later stages of life.
Ever increasing evidence has shown that Aβ peptides have detrimental effects on synaptic
function. However, not much has been studied about the relationship between Aβ
aggregation, synaptic and neuron loss during early stages of brain development. It has been
shown that early intraneuronal accumulation of Aβ peptides is one of the key events leading
to synaptic and neuronal dysfunction (Gouras et al., 2000). In addition, it has also shown that
amyloid-β could inhibit proteasome, both in vitro as well as in vivo (Gregori et al., 1995,
Tseng et al., 2008). In contrast, Wegiel et al. have shown presence of intracellular Aβ
throughout the life span in control subjects as well as Down syndrome patients, and suggest
that this rather a normal neuronal metabolism than a pathological alteration (Wegiel et al.,
2007). Immunogold electron microscopy has revealed that Aβ42 can be found in neuronal
multivesicular bodies (MVBs), where it has shown to be associated with synaptic pathology
(Takahashi et al., 2002). However, exact mechanism of intraneuronal amyloid accumulation
is not completely understood.
Brain α-spectrin (αII-spectrin or α-fodrin) constitutes ~ 2% of the total protein content and
has been localized between presynaptic membrane and synaptic vesicles of neurons. The αIIspectrin subunit plays important role in synaptic transmission and mediates adhesion of
synaptic vesicles in nerve endings (Zhang et al., 2013). Calcium/calmodulin-dependent
protein kinase II (CaMKII) is an abundant synaptic protein, mostly present in postsynaptic
3
density (PSD). Experimental evidence has shown the relevance of AMPA-type glutamate
receptors in synaptic plasticity and learning (Kessels and Malinow, 2009, Glanzman, 2010).
The phosphorylation of GluA1 by CaMKII and AMPARs trafficking towards PSD from
cytoplasm are the most extensively studied mechanisms of AMPA-dependent plasticity
(Collingridge et al., 2004, Kristensen et al., 2011, Henley et al., 2011). It has been shown that
degradation of AMPARs is an essential event during the formation of synaptic plasticity
(Schwarz et al., 2010, Fu et al., 2011). The role of CaMKII in regulation of glutamatergic
synapse formation has been observed from study of long-term potentiation (LTP), an activity
dependent strengthening of synapses. LTP is triggered by Ca2+ entry into the postsynaptic
cell, at many excitatory synapses. Studies have shown that CaMKII detects this elevated Ca2+
levels and instigate a sequence of events that potentiates synaptic transmission. After LTP
induction, CaMKII autophosphorylates (Thr-286) in dendritic spines and acts as scaffold to
recruit proteasomes to the PSD (Bingol et al., 2010). This suggests that proteasome is a
connecting link between CaMKII and AMPARs and play a vital role during formation of
synaptic plasticity.
The most extensively studied genes modulated by experience-dependent neuronal activity in
adult brain are Creb and Bdnf. Creb responds to increase in calcium at synapse by modulating
the expression of genes, leading to synthesis of new proteins (Deisseroth et al., 1996). One of
its effector sites is the calcium-response element-3/CREB recognition element (CaRE3/CRE) regulatory region that controls the transcription of Bdnf promoter IV. The newly
expressed Bdnf mRNA is translated into Bdnf protein, which induces long-term changes in
synaptic plasticity (Zheng et al., 2011). Both Creb and Bdnf have been extensively implicated
in nervous system development, memory formation and in human cognitive disorders (Autry
and Monteggia, 2012). It has been shown that increased pro:mature Bdnf ratio could mediate
defective processing of amyloid precursor protein which promote Aβ aggregation (Matrone et
al., 2008). Bdnf pro-domain is known to possess a similar sequence polymorphism
(Val66Met) which has been linked to cognitive decline in AD (Lim et al., 2014). However,
until now very little is known about how these factors would work together when proteasome
functioning is impaired during postnatal brain development. In the present study, we
examined effect of postnatal proteasome inhibition on hippocampus based spatial memory
formation during adulthood. The molecular, behavioural and histological data suggested that
inhibiting proteasome activity during postnatal brain development could impair spatial
learning during later stages of life.
4
Experimental Procedure
Chemicals
All primary antibodies [BDNF N-20 (sc-546); β-Actin AC-15 (sc-69879); CREB-1 C-21 (sc186); p-CREB-1 Ser 133 (sc-101663); p-CaMKIIα Thr 286 (sc-12886); spectrin αII (sc48382)] were procured from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA). APP-CTF
(NBP1-76910) and BACE-1 (MAB931) were purchased from Novus Biologicals (USA). βAmyloid (D54D2) XP® Rabbit mAb (8243S) was procured from Cell Signaling Technology
Inc. (USA) Secondary antibodies [Goat anti-rabbit IgG-HRP (sc-2004) and rabbit anti-mouse
IgG-HRP (sc-358917)] were purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA,
USA). Proteasome Activity Assay Kit (K245) was purchased from BioVision, Inc. (Milpitas,
CA, USA). Fura-2, AM (F1201) was procured from ThermoFisher Scientific (MA, USA)
Trizol reagent (Life Technologies, Carlsbad, CA, USA), SYBR® Green JumpStart™ Taq
ReadyMix™ (S4438), MG-132 (C2211). All other chemicals were of analytical grade and
procured from Sigma Chemical Co. (St. Louis, MO, USA).
Animals
BALB/c mice were mated to obtain littermate for performing subsequent experiments. Mice
were housed and bred at the central animal house facility of the Institute. Both male and
female mice pups (P1) were randomly selected for postnatal biochemical studies. However,
only male BALB/c mice (P40) were selected for behavioural and biochemical studies. Mice
were kept under controlled temperature (25 ± 2°C) and relative humidity was maintained
around 50–60% with a 12/12 h light/dark cycle (8 a.m. to 8 p.m.). The mice fed with standard
mouse pellet diet (Ashirwad Industries; Kharar, India) and were given water ad libitum. The
protocols followed in present study were approved by the Institutional Animal Ethics
Committee of the University (PU/IAEC/2014/S32).
Intracerebroventricular injection
Mice pups (P1) were cryo-anaesthetised for two minutes and a sterilized glass micropipette
connected with 10 µl Hamilton syringe through a tubing, was used for administration of
MG132 or artificial cerebrospinal fluid (aCSF: 3 mM KCl, 140 mM NaCl, 1 mM MgCl 2, 2.5
mM CaCl2, 1.2 mM Na2HPO4; pH 7.4) as described by Glascock et al. (2011). The needle
was inserted 2.0 mm deep, 0.25 mm lateral to the sagittal suture and 0.50–0.75 mm rostral to
5
the neonatal coronary suture. Two µl of aCSF or MG132 (1 µM) was injected aseptically into
P1 pups brain.
Proteasome activity assay
Proteasome activity was measured according to the manufacturer protocol with slight
modifications. Briefly, brains were isolated from mice pups after 24 h of MG132
administration. Samples were assessed for proteasome activity by adding fluorogenic AMC
tagged peptide substrates in assay buffer and incubated for 30 min at 37°C. 125 mM sodium
borate buffer (pH 9.0) containing 7.5% ethanol was used to stop the reaction. Fluorescence
emitted by AMC was read at 360 nm (excitation) and 460 nm (emission) using multimode
plate reader (Tecan Infinite M200 PRO). Proteasome activity was normalized with protein
concentration and expressed as percentage of activity relative to DMSO control.
Intracellular free calcium levels
The levels of Intracellular free calcium (Ca2+) were measured by the method of Luo and Shi
(2005). The hippocampal cell suspension from pup brain was incubated with Fura-2 AM with
intermittent mixing. The cells were pellet down by centrifugation, and washed once with
Ca2+-free buffer. The suspension was centrifuged again and resulting pellet was diluted with
buffer and kept at 37°C for 6 min. The concentration of intracellular free Ca2+ was assessed
by measuring the fluorescence (340nm/380nm excitation and 510 nm emission). Maximum
fluorescence (Fmax) could be observed by lysis with 20% (w/v) SDS or 2% triton X-100, and
addition of 0.5M EGTA (pH 8.0) would give minimal fluorescence (Fmin).
The Grynkiewicz equation was used to calculate calcium levels:
Kd (for Ca2+ binding to Fura-2 at 37°C) = 225 nM; R = 340/380 ratio; Rmax = 340/380 ratio
when Ca2+ were saturated; Rmin = 340/380 ratio when Ca2+ were absent; Sfb = ratio of
baseline fluorescence (380 nm) under Ca2+-free and -bound conditions. (Kd for Ca2+ and
Fura-2 binding decreases with decrease in temperature).
6
Morris water maze analysis
Morris water maze (MWM) is a spatial learning test that depends on distant cues to navigate
from starting point along the wall of an open swimming area to locate a submerged escape
platform (Morris, 1984). The maze consists of a water tank with a diameter of 100 cm and a
hidden platform one cm below the surface of water which was placed in one of the imaginary
quadrants. Mice (1.5 months) were acclimatized to MWM apparatus for three consecutive
days for five min. Next, each mouse was trained twice a day for four consecutive days
(maximum trial run was five min) to find a hidden platform and allow to stand on it for 10
seconds. During each trial, mice were gently placed in the water with their nose facing the
wall of tank at maximum distance from hidden platform. The final acquisition was recorded
on 5th day under exactly same experimental conditions as were during training trials. Escape
latency (i.e., time to find hidden platform), path length (i.e. distance covered to find hidden
platform) and path efficiency (i.e. efficiency of path covered by mice from start position to
last position, a value of 1 indicate perfect efficiency. It is calculated by measuring straight
line distance between start position and last position divided by total distance travelled by
animal during the test) to reach hidden platform were recorded with AnyMaze video tracking
software (Stoelting Co. Wooddale, IL, USA). Both sham and MG132 administered mice were
sacrificed and hippocampi were removed for molecular analyses after two hours of final
acquisition (day 5), as genes regulating neuronal plasticity have shown modulation in their
expression within this time frame after spatial memory task (MWM) (Mizuno et al., 2000,
Porte et al., 2008, Zhou et al., 2013)
Histopathological staining
Mice were anaesthetized and perfused transcardially with ice-cold 4% paraformaldehyde in
0.1 M phosphate buffer. The brains were dissected out and stored in same fixative solution
for 24 h, paraffin embedded, sectioned (10 µm) and stored until further use. Paraffin
embedded brain sections were deparaffinized with absolute xylene and dehydrated in a
sequential ethanol dilution. The sections were then rehydrated with deionized water followed
by staining with Hematoxylin dye at room temperature for 20 min. The sections were again
rinsed with deionized water and fast dipped in 1% acid-ethanol solution. After rinsing with
deionized water, sections were stained with Eosin dye for 30 s at room temperature. DPX was
used to mount the sections, coverslip was applied and observed under light microscope.
Similarly, the paraffin embedded brain sections were first deparaffinised, dehydrated and
7
then rehydrated followed by a brief (5 min) cresyl violet staining at room temperature. The
sections were washed with deionized water and mounted with DPX for microscopic
examination.
Amyloid β immunohistochemistry
Paraffin-embedded brain sections were deparaffinized and dehydrated as described earlier.
Endogenous peroxidase activity was blocked by incubation with 0.25% (v/v) H2O2 and
0.001% (w/v) sodium azide in PBS for 20 min. Antigens were retrieved by boiling the
sections (10 mM Tris-1 mM EDTA buffer; pH 9.0) in water bath for 15 min. Slides were
blocked by the 1% (w/v) BSA for 15 min and incubated with anti β-Amyloid rabbit
monoclonal antibody (1:200) at room temperature for 4 h. After being washed with PBS (pH
7.6), the slides were incubated with goat anti-rabbit HRP-conjugated secondary antibody
(1:400) at room temperature for 1 h. Nuclei were counterstained with hematoxylin. The
sections were washed with deionized water and mounted with DPX. The slides were
visualized under EVOS FL Auto Imaging System (Life Technology, USA). ImageJ software
(National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/) was used to
perform off-line Image analyses.
APP-CTF and BACE-1 double immunostaining
Paraffin-embedded brain sections were processed as described earlier. Antigens were
retrieved as described earlier. Slides were blocked by the 1% (w/v) BSA for 15 min and
incubated with APP-CTF rabbit polyclonal antibody (1:200) and BACE-1 mouse monoclonal
antibody (1:200) at room temperature for 4 h. After being washed with PBS (pH 7.6), the
slides were incubated with appropriate PE and FITC-conjugated secondary antibody (1:400)
at room temperature for 1 h. Nuclei were counterstained with DAPI. The sections were
washed with deionized water, mounted and visualized under EVOS FL Auto Imaging System
(Life Technology, USA). ImageJ software (National Institutes of Health, Bethesda, MD,
USA; http://rsb.info.nih.gov/ij/) was used to perform off-line Image analyses.
RNA isolation and Real Time PCR
Hippocampi were dissected out from mice brains and total RNA was isolated using Trizol
reagent (Life Technologies, USA). cDNA was synthesized with the help of MMLV Reverse
Transcriptase kit (Life Technologies, USA) and Real Time PCR reactions were performed
with SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma, USA) in LightCycler® 96 System
8
(Roche, USA) with the primers listed in Table 1. Primer specificity was analysed by NCBI
BLAST and verified from dissociation curve, Tm and amplicon length. The PCR cycling
program was as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 1 min at
respective annealing temperatures (Table 1), and finally 72°C for 10 s. Target RNA
expression was normalized with the housekeeping control (Eukaryotic elongation factor 2,
eEF2). Relative changes in mRNA expression levels between control and MG132 samples
were calculated as described by Pfaffl (2001).
Protein isolation and western blotting
Cells were lysed and proteins were resolved by electrophoresis and the separated proteins
were transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) as
described by Towbin et al. (1992). The molecular weights of respective proteins were
estimated with the help of pre-stained protein markers (Bio-Rad, Hercules, CA, USA). The
membranes were blocked with 5% (w/v) skimmed milk in PBS-T (Phosphate-buffered
solution pH 7.6/0.005% Tween-20) and incubated for overnight with respective primary
antibodies (1:1000) diluted in skimmed milk. Next day the membranes were washed with
PBS/PBS-T and incubated with respective horseradish peroxidase-conjugated secondary
antibodies (1:2000) for 2 h. The membranes were incubated with ECL Substrate [containing
equal volumes of peroxide reagent and luminol reagent in a 1:1 ratio (Clarity™ Western ECL
Substrate, BioRad, Hercules, CA, USA)] for 1 min. The membranes were then wrapped with
a plastic sheet and exposed to X-ray film at room temperature for 30 s to detect the
chemiluminescence. Protein bands were quantified with AlphaEase FC™ software (USA) for
densitometric analyses.
Statistical Analysis
All data are expressed as mean ± standard deviation (SD). Student’s t-test was applied to
compare the statistical difference between sham and MG132 administered mice. MWM data
were analysed with one way RM ANOVA followed by Holm-Sidak posthoc test. Statistical
analysis of the data was performed in SigmaStat 3.5 software. Values with p < 0.05 were
considered as statistically significant.
Results
Postnatal MG132 administration resulted in decreased 20S proteasome and calpain
activities
9
MG132 is a specific inhibitor of 20S proteasome but it can also inhibit calpain activity to
some extent, therefore, first we checked the status of both enzyme activities in mice pup
brains after 24 h of MG132 administration (Fig. 1A). Proteasome activity was measured with
the help of fluorogenic AMC tagged peptide substrate. The peptidase activity was found to be
inhibited by almost 70% (t8 = 41.25, p < 0.001, n = 5) tby the addition of 1 µM MG132 (Fig.
1B). Next, we checked intracellular calcium levels which were significantly increased (0.55
fold, t8 = 30.04, p < 0.002, n = 5) after 24 h of MG132 administration (Fig. 1C). It has been
shown that calpain activation required elevated levels of intracellular calcium but even in the
presence of increased Ca2+, we observed significantly decreased calpain mediated αIIspectrin cleavage in MG132 administered mice pup brains. Densitometric analysis showed
significant reduction in levels of αII-spectrin breakdown products i.e. 150 kDa (t8 = 6.87, p <
0.001, n = 5) and 120 kDa (t8 = 2.68, p = 0.028, n = 5) proteins (Fig. 1D and 1E).
Postnatal proteasome inhibition impaired spatial memory in adult mice
Morris water maze (MWM) test was used to study the mechanisms of spatial learning in
rodents. Both sham and MG132 administered mice (P40) were allowed to swim (five min) in
MWM for three consecutive days. These mice were then trained to reach the hidden platform
for four consecutive days (two trials per day). MG132 administered mice took significantly
more time to reach the hidden platform on each training day when compared to sham.
However, average speed remains unaltered throughout the experiment (Fig. 2B). Acquisition
analysis revealed significant increase (20.68 fold, t8 = 18.04, p < 0.001, n = 5) in the escape
latency to reach hidden platform by MG132 administered mice when compared to sham (Fig.
2C). Moreover, MG132 administered mice consistently showed longer path length (1.89 fold,
t8 = 7.78, p < 0.01, n = 5) with decreased path efficiency (0.66 fold, t8 = 3.13, p = 0.014, n =
5) to reach the hidden platform when compared to sham (Fig. 2D and 2E). In addition,
MG132 administered mice have spent significantly less (0.31 fold, p < 0.01) time in
exploring the quadrant containing platform (Quad 3) when compared to sham (Fig. 2F).
Representative track plots showed that sham had more targeted approach to find the hidden
platform when compared to MG132 administered mice during acquisition analysis (Fig. 2G).
These findings indicate that spatial learning could get impaired in adult life when
proteasomes were inhibited during early postnatal period, a crucial phase for synapse
development.
Histological alterations in hippocampus after postnatal MG132 administration
10
Hematoxylin and eosin staining of MG132 mice brain sections showed distorted neuronal
architecture when compared to sham. Hippocampal CA1 and CA3 regions showed
significantly increased pyknosis in pyramidal cell layers in MG132 mice brain when
compared to sham brain (Fig. 3A). Moreover, cresyl violet staining also supported these
alterations in MG132 administered mice (Fig. 3B). These observations suggested that
impairment in postnatal proteasome functioning could severely affect the orderly
arrangement of hippocampal cells.
Immunohistochemical staining on consecutive sections has also showed increased amyloid-β
positivity and was significantly (CA1 = 0.21 fold, t4 = 22.32, p < 0.01, n = 3; CA3 = 0.40
fold, t4 = 20.68, p < 0.01, n = 3) more prevalent in hippocampal region (Fig. 3C). Moreover,
double immunostaining have shown marked increase (CA1 = 1.09 fold, t4 = 26.23, p < 0.01,
n = 3; CA3 = 1.18 fold, t4 = 25.80, p < 0.01, n = 3) in aggregation of intracellular APP-CTFs,
without affecting BACE-1 expression, in hippocampal region when compared to sham (Fig.
3D). These results suggest that postnatal proteasome inhibition could induce intracellular
accumulation of amyloid-β structures in hippocampus.
Effect of postnatal administration of MG132 on molecules involved in memory
formation
Real-Time PCR analyses revealed significant increase (0.45 fold, t8 = 4.28, p ≤ 0.029, n = 5)
in the expression of GluA1 mRNA in MG132 administered mice when compared to sham
(Fig. 4A). The translated product of GluA1 has shown to be a phosphorylation substrate of
CaMKII. It is a serine/threonine protein kinase believed to regulate synaptic plasticity as well
as neurotransmission in response to calcium signalling produced by neuronal activity.
Western blot analysis of pT286-CaMKII showed significant decrease (0.71 fold, t8 = 45.27, p
≤ 0.001, n = 5) in MG132 administered mice when compared to sham (Fig. 4B).
Activity-dependent gene transcription is a rapid process, mediated by free intracellular Ca2+
as second messenger, and is regulated by a group of immediate early genes (IEG) whose
expression does not need de novo protein synthesis (Morgan and Curran, 1988). We observed
no change in the expression of Creb in its non-phosphorylated state. However, the level of its
phosphorylated (pS133-Creb) state was found to be significantly decreased (0.51 fold, t8 =
35.94, p ≤ 0.001) in MG132 administered mice when compared to sham (Fig. 4C).
11
It has been shown that Creb control a transcriptional program which includes brain derived
neurotrophic factor (Bdnf) (Tao et al., 1998). Bdnf has shown to be essential for synaptic
plasticity and spatial memory formation (Yamada et al., 2002). The phosphorylated Creb
binds to Bdnf promoter and up-regulates its expression. The expression of Bdnf mRNA was
checked and observed no statistical change in MG132 administered mice when compared to
sham (Fig. 4D). Western blot analyses showed a significant decrease in mature:pro Bdnf ratio
(0.78 fold, t8 = 4, p ≤ 0.001) in MG132 administered mice when compared to sham (Fig. 4E).
Mature Bdnf levels are found to be decreased in brains of Alzheimer’s disease (AD) patients,
which suggest a pathogenic involvement of Bdnf in AD progression.
Discussion
In the present study, we used behavioural, histological and molecular analyses to find the
effect of postnatal proteasomal inhibition on hippocampus dependent memory functions
during adult life. Previously, UPS was known to degrade biologically non-functional proteins
(Hershko and Ciechanover, 1998). However, this system can specifically control the
expression of proteins involved in neural transmission and in long-term synaptic plasticity
(Patrick, 2006, Yi and Ehlers, 2007). Until today, much of the research has focused on
proteasome dysfunction in age related neurodegenerative disorders. However, the effects of
postnatal proteasome inhibition on hippocampus based memory in adult mice have been little
studied. These results suggested that postnatal proteasome inhibition could induce
intraneuronal amyloid-β accumulation and affect spatial memory functions of hippocampus.
It has been shown that spectrin breakdown is involved in specific processes which modulate
the glutamatergic synaptic transmission or could directly act on numerous glutamate
receptors of synaptic membranes by regulating the plasticity of membrane skeleton (Lynch et
al., 2007). Impairment in these highly regulated processes has shown to interfere with
learning and memory of mice (Meary et al., 2007). In line with these reports, we observed
that postnatal MG132 administration could reduce the 20S proteasome activity as well as
calpain mediated αII-spectrin cleavage and thus could affect the modulation of synaptic
plasticity during crucial phase of brain development.
Exposure to excitatory toxins during postnatal period could induce neurobehavioural
impairments in adulthood (Levin et al., 2006, Gill et al., 2010). Previously, it has been shown
that proteasome inhibition during postnatal stage could results in profound behavioural
alterations during adult stage in mice (Romero-Granados et al., 2011). So, we wanted to
12
check whether postnatal MG132 administration had any effects on spatial memory during
adulthood. The results of MWM test revealed that postnatal proteasome inhibition could
induce significant impairment in hippocampus based spatial learning and memory functions
of adult mice. Histopathological assessment of hippocampal CA1 and CA3 regions showed
disturbed cellular architecture and increased pyknosis, which could be responsible for the
observed neurobehavioural impairments. Our findings were in line with previous report
where it has been shown that both CA1 and CA3 lesions could disrupt the accurate relocation
of a previously visited place (Lee et al., 2005). These results suggest that postnatal
proteasome inhibition could induce detrimental effects on hippocampal cells, which leads to
impaired processing of signals and responsible for poor spatial learning.
Next, we wanted to check whether postnatal proteasome inhibition could also trigger protein
aggregation in adult life. Studies have suggested that the intracellular Aβ accumulation might
be an early event in the pathogenesis of neurological disorders like AD and Down syndrome
(Gouras et al., 2000). Aβ is proteolytic product of amyloid precursor protein (APP) (Kang et
al., 1987). The APP is first cleaved by β-secretase, which produce C-terminal fragment (APPCTFβ), followed by γ-secretase mediated cleavage to release toxic Aβ (Nunan and Small,
2000). Our results have shown significant increase in intracellular accumulation of APPCTFβ and amyloid-β in hippocampal region of MG132 administered mice. Patients with mild
cognitive impairment have shown be positive for intraneuronal amyloid-β in hippocampus
and entorhinal cortex, which might explain these patients are more susceptible to develop
early AD pathology (Gouras et al., 2000). Moreover, intracellular amyloid-β aggregation has
also shown to promote extracellular amyloid plaque formation in Down syndrome (Gyure et
al., 2001). Immunogold electron microscopic analysis has revealed that Aβ42 could be found
in multivesicular bodies (MVBs) of neurons (Takahashi et al., 2002). Aβ immunotherapy has
shown to effectively clear the extracellular plaques and improve cognition in murine models
of AD (Schenk et al., 1999, Janus et al., 2000). In addition, removal of extracellular Aβ
plaques from brains of 3xTg-AD mice has also shown to promote clearance of intraneuronal
accumulation of Aβ (Oddo et al., 2004). Based on these reports, it could be possible that
extracellular Aβ might originate from intraneuronal amyloid structures and a dynamic
balance must exist between them, such that when extracellular plaques are removed,
intraneuronal structures are also diminished. Recently, it has been shown that Aβ could gain
access in neurons of ‘learning ganglia’ and leads to memory impairment at behavioural level
without any sign of neuronal death in Lymnaea (Ford et al., 2015). Aβ-induced detrimental
13
effects on synaptic plasticity have shown to occur before cell death, however, how
intraneuronal amyloid-β accumulation could mediate loss of spatial memory is not fully
understood. Therefore, next we checked the effect of postnatal proteasome inhibition on
spatial learning and memory formation.
Spatial learning has been considered as a potential substrate of hippocampal synaptic
plasticity (Morris et al., 1986, Martin et al., 2000). It is mediated by NMDA receptor
activation which require controlled incorporation of GluA1 subunit into AMPA receptors
(Zamanillo et al., 1999, Malinow and Malenka, 2002, Collingridge et al., 2009). GluA1 has
shown to play an important role in rapid but short-lasting potentiation (Hoffman et al., 2002,
Romberg et al., 2009). In GluA1 knockout (GluA1-/-) mice, acquisition of spatial reference
memory was found to be normal, as they could successfully discriminate between rewarded
and non-rewarded locations. However, they were unable to discriminate between these
locations during spatial working memory test (Zamanillo et al., 1999, Reisel et al., 2002,
Schmitt et al., 2003). In the present study, we observed significantly increased GluA1 mRNA
levels in MG132 administered mice when compared with sham mice. AMPARs have
emerged as important mediators of synaptic plasticity in the brain (Seeburg et al., 2001,
Malinow and Malenka, 2002). In contrast with NMDA glutamate receptors, which always
permit Ca2+ entry upon activation, AMPARs constitute an activity-dependent switch, which
controls glutamate-evoked entry of Ca2+ into neurons (Burnashev et al., 1992). It has been
observed that GluA1 subunit is highly expressed in regions that demonstrate a high density of
calcium-permeable AMPARs (Engelman et al., 1999, Hartmann et al., 2004).
It has been shown that GluA1 is phosphorylated by CaMKII and form a short-term memory
trace that help in remembering recently visited locations (Sanderson et al., 2008). Recently, it
has been shown that MWM training could phosphorylated the CaMKII followed by elevation
of GluA1 in the hippocampus (Jiang et al., 2015). However, we observed decreased
expression of pT286-CaMKII in MG132 administered mice when compared with sham mice.
Previously, it has been shown that short term memory is a labile state which requires
activation and/or posttranslational modifications of pre-existing molecules, whereas
consolidation of long term memory requires gene expression and de novo protein synthesis
for converting new information into more stable state (Davis and Squire, 1984, McGaugh,
2000). Many types of long term memory consolidation in rodents require phosphorylation at
Ser133 of Creb by cAMP- or Ca2+-dependent protein kinase (Trifilieff et al., 2006, Brightwell
et al., 2007). In addition, in vitro studies have shown that the CaMKII can also phosphorylate
14
Ser133 of Creb (Dash et al., 1991, Sheng et al., 1991). In the present study, western blot
analysis revealed a significant decrease in the levels of pSer133-Creb in MG132 administered
mice, which could be attributed to the decreased levels of CaMKII.
Several transcription factors are known to regulate Bdnf promoters. Among them, Creb could
mediate the regulation of Bdnf promoters I and III (Tabuchi et al., 2002). We observed
increased Bdnf mRNA levels in MG132 administered mice when compared to control mice.
Bdnf is produced in an activity-dependent manner and synthesized as a precursor (pro-Bdnf),
which is proteolytically processed into mature Bdnf (Lu, 2003b). The activity-dependent
secretion of Bdnf has shown to play an important role in hippocampus based memory in
human (Egan et al., 2003, Lu, 2003a). The pro-Bdnf interacts preferentially with panneurotrophin receptor p75 (p75NTR), whereas mature Bdnf selectively binds and activates the
receptor tyrosine kinase TrkB (Chao and Bothwell, 2002, Ibanez, 2002). If not processed,
pro-Bdnf has displayed antagonistic effects against its mature counterpart (Greenberg et al.,
2009, Deinhardt and Chao, 2014). These include, accelerating cell death (Teng et al., 2005),
blocking neuronal migration (Xu et al., 2011) and reducing neurite outgrowth (Sun et al.,
2012). Western blot analyses revealed significant decrease in the hippocampal mature:pro
Bdnf ratio. These results suggest that elevated pro-Bdnf levels might be responsible for
impairments in the spatial learning of MG132 administered mice. Our results are in
accordance with the findings of Chen et al. (2016), who have also shown that pro-Bdnf could
inhibit neural proliferation in the hippocampal dentate gyrus of the aged mice, and showed
poor performances in the Morris water maze test. Ever increasing evidence has suggested a
link between decreased mature Bdnf levels and AD pathogenesis. Studies have shown that
cortex and hippocampus are associated with learning and memory, both regions exhibited
extensive amyloid pathology as well as decreased Bdnf levels in AD (Connor et al., 1997,
Peng et al., 2005).
Taken together, our findings suggest that proteasomes play a vital role in maintaining
synaptic plasticity during early brain development. Defects in proteasome functioning during
this phase could promote aggregation of intracellular amyloid-β in the hippocampus and
results in impaired spatial learning during later stages of life.
15
Acknowledgments
This work was supported by Department of Science and Technology, New Delhi – StartUp
Research Grant for Young Scientist Scheme to Dr. Aditya Sunkaria [SB/YS/LS-172/2014];
Department of Science and Technology, New Delhi – Inspire SRF Scheme to Aarti Yadav
[DST-IF120643] Indian Council of Medical Research, New Delhi – SRF Scheme to Supriya
Bhardwaj [67/19/2013-Imm/BMS].
Conflict of interest
None of the authors have any conflicts of interest to declare.
16
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Figure Legends
Figure 1: Early molecular events were assessed in mice pup brains after 24 h of postnatal
MG132 administration. (A) Experimental time line; (B) 20S proteasome activity was
measured by fluorogenic AMC tagged peptide substrates; (C) intracellular calcium levels
were measured with Fura-2; (D) western blots of αII-spectrin and its breakdown products; (E)
densitometric analysis of spectrin breakdown product (150 kDa)/αII-spectrin (240 kDa)
normalized with β-actin; (F) densitometric analysis of spectrin breakdown product (120 kDa)/
αII-spectrin (240 kDa) normalized with β-actin. Experiments were performed in triplicate and
data were expressed as Mean ± SD (n = 5 each group); *p < 0.05.
Figure 2: Hippocampus based spatial learning and memory function was assessed by Morris
water maze test. (A) Experimental time line, quadrants and platform placement in MWM
apparatus; (B) average speed of sham and MG132 mice during test; (C) escape latency,
average time taken by sham and MG132 mice to reach hidden platform after each training
and acquisition sessions; (D) path length, average distance covered by sham and MG132
mice to reach hidden platform; (E) path efficiency of sham and MG132 mice to reach hidden
platform; (F) percentage of time spent in exploring each quadrant by sham and MG132 mice
during test; (G) representative track plots of sham and MG132 mice during each training day
and acquisition test. Each animal was tested for learning and memory function and data were
expressed as Mean ± SD [five animals per group i.e. sham (n=5) and MG132 (n=5) group.
The MWM test was performed thrice by each animal with at least 3 h of test interval.]; *p <
0.05.
Figure 3: Effect of postnatal MG132 administration on hippocampus. (A) H&E staining
shows hippocampal CA1 and CA3 regions of sham and MG132 mice. Inset show magnified
regions marked with yellow asterisks. Percentage of pyknotic cells quantified by ImageJ
software; (B) cresyl violet staining shows hippocampal CA1 and CA3 regions of sham and
MG132, mice inset show magnified regions marked with red asterisks; (C) Aβ
immunohistochemistry of consecutive sections of CA1 and CA3 regions of sham and MG132
mice. Percentage of Aβ positive neurons was quantitated with IHC toolbox plugin of ImageJ
software; (D) double immunostaining of APP-CTF and BACE-1 of CA1 and CA3 regions of
sham and MG132 mice. Image analysis was performed on three consecutive sections for each
region and data were expressed as Mean ± SD; *p < 0.05. H&E magnification = 40×; cresyl
24
violet magnification = 20×, Aβ immunohistochemistry and double immunostaining
magnification = 40×, scale bar represents 100 µm.
Figure 4: Effect of postnatal proteasome inhibition on various molecules involved in learning
and memory. (A) qPCR expression analysis of GluA1 normalized with eEF2; (B) western
blots of pT286-CaMKIIα and β-actin, densitometric analysis of pCaMKIIα normalized with
β-actin; (C) Western blots of pS133-Creb, Creb and β-actin, densitometric analysis of pS133Creb/Creb normalized with β-actin; (D) qPCR expression analysis of Bdnf normalized with
eEF2; (E) western blots of pro-Bdnf, mature Bdnf and β-actin, densitometric analysis of
mature/pro Bdnf normalized with β-actin; Experiments were performed in triplicate and data
were expressed as Mean ± SD (n = 5 each group); *p < 0.05.
25
Table 1: Sequence of primers used for real time PCR
Gene
NCBI ID
Forward Primer (5′→3′)
Reverse Primer (5′→3′)
Bdnf
AY057907.1
CTGGATGAGGACCAGAAG
CCTCCAGCAGAAAGAGTAG
58
GluA1
NM_001113325.2
CGGAAGTAAGGACAAGAC
CTACGGGATTTGTAGCAG
57
eEF2
NM_007907.2
GATAAGGGTGAGGTGTCT
GTGATGTCGGTGAGAATG
57
Tm (°C)
26
27
28
29
30
Highlights



Postnatal MG132 administration leads to decreased proteasome and calpain activities
Defected postnatal proteasome could promote Aβ accumulation in hippocampal
neurons
Postnatal proteasome inhibition impaired spatial memory in adult mice
31
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