N-Heterocyclic carbene-catalyzed aerobic oxidation of aryl alkyl alcohols to carboxylic acids

Full text of DOI:10.1016/j.tet.2015.03.072

Neuro-peptide treatment with Cerebrolysin improves the survival of Neural
Stem Cell grafts in an APP transgenic model of Alzheimer Disease
Edward Rockenstein, Paula Desplats, Kiren Ubhi, Michael Mante, Jazmin
Florio, Anthony Adame, Stefan Winter, Hemma Brandstaetter, Dieter Meier,
Eliezer Masliah
PII:
DOI:
Reference:
S1873-5061(15)00050-1
doi: 10.1016/j.scr.2015.04.008
SCR 522
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Stem Cell Research
Received date:
Revised date:
Accepted date:
23 December 2014
31 March 2015
30 April 2015
Please cite this article as: Rockenstein, Edward, Desplats, Paula, Ubhi, Kiren, Mante,
Michael, Florio, Jazmin, Adame, Anthony, Winter, Stefan, Brandstaetter, Hemma,
Meier, Dieter, Masliah, Eliezer, Neuro-peptide treatment with Cerebrolysin improves the
survival of Neural Stem Cell grafts in an APP transgenic model of Alzheimer Disease,
Stem Cell Research (2015), doi: 10.1016/j.scr.2015.04.008
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Neuro-peptide treatment with Cerebrolysin improves the survival of Neural Stem
Cell grafts in an APP transgenic model of Alzheimer Disease
Edward Rockenstein1, *, Paula Desplats1, *, Kiren Ubhi1 , Michael Mante1, Jazmin Florio1,
Anthony Adame1, Stefan Winter2, Hemma Brandstaetter2, Dieter Meier2, Eliezer
Masliah1,3
1Department of Neurosciences, University of California San Diego, La Jolla, CA, USA,
2Clinical Research & Pharmacology, EVER Neuro Pharma GmbH, Unterach, Austria,
3Department of Pathology, University of California San Diego, La Jolla, CA, USA
* - These authors contributed equally to this work
Correspondence and reprint requests should be addressed to: Dr. E. Masliah,
Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-
0624. Phone (858) 534-8992, Fax (858) 534-6232, email: emasliah@ucsd.edu
Running title: CBL effects grafted stem cells in APP mice
This work was partially supported by NIH grant AG05131 and by a grant from EVER
Pharma.
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ABSTRACT (200 words)
Neural stem cells (NSCs) have been considered as potential therapy in
Alzheimer’s disease (AD) but their use is hampered by the poor survival of grafted cells.
Supply of neurotrophic factors to the grafted cells has been proposed as a way to
augment survival of the stem cells. In this context, we investigated the utility of
Cerebrolysin (CBL), a peptidergic mixture with neurotrophic-like properties, as an
adjunct to stem cell therapy in an APP transgenic (tg) model of AD. We grafted murine
NSCs into the hippocampus of non-tg and APP tg that were treated systemically with
CBL and analyzed after 1, 3, 6 and 9 months post grafting. Compared to vehicle-treated
non-tg mice, in the vehicle-treated APP tg mice there was considerable reduction in the
survival of the grafted NSCs. Whereas, CBL treatment enhanced the survival of NSCs
in both non-tg and APP tg with the majority of the surviving NSCs remaining as
neuroblasts. The NSCs of the CBL treated mice displayed reduced numbers of
caspase-3 and TUNEL positive cells and increased brain derived neurotrophic factor
(BDNF) and furin immunoreactivity. These results suggest that CBL might protect
grafted NSCs and as such be a potential adjuvant therapy when combined with grafting.
Key words: Alzheimer’s Disease, stem cells, neurotrophic factors, synapses.
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1. INTRODUCTION (all text 53,322 characters)
The neurogenerative process in patients with Alzheimer’s Disease (AD) is
characterized by synaptic loss [1] and loss of cholinergic, glutaminergic and GABA-ergic
neurons [2-8]. In addition, more recent studies suggest that alterations in adult
neurogenesis in the hippocampus might also contribute to the neurodegenerative
process in AD [9]. There is some controversy over whether neurogenesis is increased
[10] or decreased [11, 12] in AD. Recent studies suggest that apparent increases in
neurogenesis in AD patients may be related to glial and vasculature-associated
changes [11]. Studies in a number of animal models of Familial AD have shown that
neurogenesis is reduced [9, 13-15].
The mechanisms of neurodegeneration in AD are not completely clear, however
progressive accumulation of amyloid beta (A) with the formation of oligomers appears
to trigger a complex cascade of events that includes activation of several kinases
including glycogen synthetase kinase (GSK3) and cyclin dependent kinase-5 (CDK5)
[16-18] leading to aberrant post-transcriptional modification of the microtubule binding
protein -Tau [19]. Moreover, other studies suggest that deficient transport or expression
of neurotrophic factors (NTF) (eg, nerve growth factor [NGF], brain derived neurotrophic
factor [BDNF]) and their receptors might be involved [20-22]. For this reason,
experimental therapies for AD involves reducing the A load, blocking kinases such as
GSK3 and CDK5, reducing Tau and replacement therapies with NTFs [23-26].
However, given the advanced clinical stage at which several patients with AD
present, alternative therapies are under consideration including replacement therapy
with neuronal stem cell grafts. For example, previous studies have shown that neural
stem cells (NSC) transferred into the hippocampus of AD transgenic (tg) models
improve cognition via BDNF [24, 27] and reduce Tau and reelin accumulation in aged
Ts65DN model of Down’s syndrome [28]. Moreover, transplantation of NSC derived
from induced pluripotent stem cells (iPSC) restore memory in amyloid precursor protein
(APP) tg [29] and APP/Presenilin-1 (PS1) mice [30] supporting the notion that treatment
with NSCs might be of useful in AD patients. However, a potential problem is the
reduced viability of the transplanted cells given the noxious micro-environment in the
brain of patients with AD. For example, we have recently shown that under baseline
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conditions NSC display reduced viability when transplanted into the brains of APP mice,
however when these cells are modified to express the A degrading enzyme, neprilysin,
the stem cells are more prone to survive [31]. Therefore, adjuvant therapies that
enhance survival of grafted stem cells might be important.
Among them, we have considered the potential of combining stem cells with
Cerebrolysin™ (CBL) a peptide mixture with neurotrophic-like properties that improves
cognition in patients with mild to moderate AD [32-37]. CBL has been shown to be protective in
experimental models of excitotoxicity [38] and stroke [39-41]. In addition, CBL is neurotrophic
in APP tg models of AD by promoting synaptic formation and neurogenesis [14, 42-47]. The
protective effects of CBL in AD-like models involves different mechanisms including regulation
of GSK3 and CDK5 signaling, control of APP metabolism and anti-apoptotic effects mediated
by expression of endogenous neurotrophic factors [48]. In this context, the main objective of
this study was to investigate whether CBL is capable of enhancing the survival of transplanted
NSCs. We found NSC survival progressively declined with age in APP tg mice when
compared to controls and that adjuvant therapy with CBL enhanced survival of the BrdU
tagged grafted NSC. This study supports the notion that CBL might be a potentially useful
adjuvant therapy in combination with NSCs.
2. MATERIALS AND METHODS
2.1 Preparation and CBL testing in mouse cortical neural stem cells
Briefly, as previously described, [49, 50] mouse cortical NSCs were obtained
from Millipore. The NSCs were grown on F12/DMEM basal media supplemented with
B27 and were kept in proliferative status without induction of neuronal differentiation.
Cells were labeled 48 hs prior to grafting into the mouse by infection with lentiviral
constructs containing the GFP coding sequence at a MOI=50. Additional pulse labeling
with BrdU was carried out 24 h prior to grafting. For in vitro experiments, NSCs were
treated with vehicle alone or CBL (5%) and grown in 35 mm dishes and in 6 well plates
with a coverslip in the bottom for subsequent ELISA and immunocytochemical studies
respectively.
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2.2 Generation of APP Tg Mice, grafting and Cerebrolysin treatment.
For these experiments, hAPP tg mice expressing mutated (Swedish
K670M/N671L, London V717I) human(h) APP751 under the control of the mouse (m)
Thy-1 promoter (mThy1-hAPP751) (line 41) [51] were used. These mice have been
extensively characterized [49, 52-55] and we have previously shown that these mice
display loss of synaptic contacts, defects in neurogenesis, high levels of A1-42
production, early amyloid deposition and behavioral deficits [14, 43]. Genomic DNA was
extracted from tail biopsies and analyzed by PCR amplification, as described previously
[56]. Transgenic lines were maintained by crossing heterozygous tg mice with non-
transgenic (non tg) C57BL/6 x DBA/2 F1 breeders. All mice were heterozygous with
respect to the transgene.
A total of 128 mice (3 month old) were included in this study (n=64 non-tg and
n=64 APP tg mice) that were divided into groups of n=32 corresponding to the post graft
time points at which they were analyzed (1, 3, 6 and 9 months). All animals received
bilateral stereotaxical injections of the green fluorescent protein (GFP) and BrdU tagged
NSC suspension containing 5x104 cells/l. Each of the groups (4 groups x n=32 per
time point) included n=16 non-tg (n=8 vehicle and n=8 CBL) and n=16 APP tg (n=8
vehicle and n=8 CBL) mice. CBL was provided by EVER Pharma in pre-prepared
ampoules, each milliliter of CBL contains 215.2mg of the active CBL concentrate in an
aqueous solution [57]. Mass spectrometry analysis of CBL has shown that is comprised
of amino acids (80%) and small (<10Da) peptides (20%) and previous work had shown
that this small peptides mimic the effect of neurotrophic factors including Ciliary
neurotrophic factor (CNTF), fibroblast growth factor 2 (FGF2) and Insulin-like growth
factor (IGF) [58]. Mice were injected daily for the duration of the experiment with saline
alone or CBL (i.p., 5 ml/kg, CBL Batch #92382008) for a total of 1, 3, 6 or 9 months. By
the end of the experiment for each corresponding group mice were 4, 6, 9 and 12
months old.
All experiments described were approved by the animal subjects committee at
the University of California at San Diego (UCSD) and were performed according to NIH
guidelines for animal use.
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2.3 Tissue Processing
In accordance with NIH guidelines for the humane treatment of animals, mice
were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9%
saline. Brains were removed and divided sagitally. The left hemibrain was post-fixed in
phosphate-buffered 4% paraformaldehyde (pH 7.4) at 4°C for 48 hr and sectioned at 40
µm with a Vibratome 2000 (Leica, Germany), while the right hemibrain was snap frozen
and stored at -70°C.
2.4 Immunohistochemical Analysis
Briefly, as previously described, [14, 31] blind-coded 40 µm thick vibratome
sections were immunolabeled with mouse monoclonal antibodies against BrdU (1:500,
Millipore), PCNA (1:500, Millipore), GFAP (1:500, Milllipore), NeuN (1:500, Millipore),
doublecortin (DCX) (1:500, Millipore), Ki-67 (1:100, Santa Cruz), activated caspase-3
(1:1000, Cell Signal), BDNF (1:250, Abcam), NT4 (1:100, Pierce) and furin (1:100,
Pierce) followed by horse anti mouse biotinylated secondary antibody and developed
with diaminobenzidine and imaged with an Olympus OX54 digital photomicroscope. An
additional series of sections were incubated with antibodies against the dendritic marker
MAP2 (1:500, Milllipore) and A (1:500, 82E1, IBL). After overnight incubation with the
primary antibodies, sections were incubated with FITC-conjugated horse anti-mouse
IgG secondary antibody (1:75, Vector Laboratories, Burlingame, CA), transferred to
SuperFrost slides (Fisher Scientific, Tustin, CA) and mounted under glass coverslips
with anti-fading media (Vector). Moreover, double immunolabeling experiments were
performed with the following combinations of antibodies: 1) anti-BrdU and anti-GFP; 2)
anti-NeuN and anti-GFP; 3) anti-GFAP and anti-GFP; 4) anti-olig-2 and anti-GFP; 5)
anti-BDNF and anti-GFP and 6) anti-Furin and anti-GFP. The BrdU, NeuN, GFAP, olig-
2, BDNF and furin were detected with Tyramide Red (NEN) while the GFP was detected
via amplification with FITC (green channel). All sections were processed under the
same standardized conditions. The immunolabeled sections were imaged with the
laser-scanning confocal microscope (LSCM, MRC1024, BioRad, Hercules, CA) and
analyzed as previously described [43]. Briefly, for each section a total of 8 images at
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630X were analyzed. For each image a threshold was set utilizing the Image Quant
system, the threshold was set within a dynamic range that was consistent for all cases.
The levels of pixel intensity or optical density for the area where ascertained with the
measure tool of the system. An adjacent un-stained area was used as background to
correct against. Area of images analyzed was on average 1024 by 1024 pixels. This
area included on average at least 4-5 neurons that were analyzed. The average
integrated pixels of optical density for all neurons within that area was estimated and
then averaged by the 16 images captured. The average was corrected to the
background and expressed as an overall mean by mouse per group. To confirm the
specificity of primary antibodies, control experiments were performed where sections
were incubated overnight in the absence of primary antibody (deleted) or with
preimmune serum or with primary antibody alone.
2.5 Unbiased stereology
Stereological methods were used to estimate the number of BrdU
immunoreactive cells utilizing an optical fractionator unbiased sampling design [14]. A
total of 4 alternating sections separated by approximately 250 μm containing the
hippocampus were outlined using a 4× objective attached to an Olympus BX51
microscope. Anatomical nomenclature was based on Paxinos and Franklin (2001) [59].
A systematic sampling of the outlined areas was made from a random starting point
using StereoInvestigator 8.21.1 software (Micro-BrightField, Cochester, VT). Counts
were taken at predetermined intervals (x = 152, y = 152), and a counting frame (70 × 70
μm = 4900 μm2) was superimposed on the live image of the tissue sections. Sections
were analyzed using a 60X 1.4 PlanApo oil- immersion objective with a 1.4 numerical
aperture. Section thickness was determined by focusing on the top of the section,
zeroing the z-axis and focusing on the bottom of the section. Average section thickness
was 18 μm. The dissector height was set at 12 μm. This allowed for a 2-μm top guard
zone and at least a 2-μm bottom guard zone. The forbidden zones were never included
in the cell counting. Immunoreactive neurons were only counted if the first recognizable
profile came into focus within the counting frame. This method allowed for a uniform,
systematic, and random design. Differences in stereologic counts were evaluated using
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the Mann– Whitney test for median differences (GraphPad Prism 4.0c for Macintosh,
San Diego, CA). The level of statistical significance was set at p-value<0.05.
2.6 Determination of NTF protein content by ELISA
As previously described [48], levels of Furin and BDNF were determined in whole
brain homogenates by ELISA according to the supplier's protocol (Promega, Madison,
WI). The samples were sonicated in a homogenization buffer (20 mM Tris, pH 8, 137
mM NaCl, 1% Nonidet P-40, 1,7 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml
aprotinin,1 μg/ml leupeptin and 0.5 mM sodium vanadate) at a tissue concentration of
30 μl/mg. An additional control ELISA was performed on whole brain homogenates from
non tg mice that had been treated with an amino acid control for CBL.
2.7 Statistical Analysis
Statistical analysis was performed with the Prism Graph path software package
(La Jolla, CA). All results are expressed as mean plus minus SEM. Differences among
multiple groups was assessed by one-way ANOVA with post-hoc Dunnett’s when
comparing to the control group, comparisons among the treatment groups was
performed with the Tukey posthoc test. Comparisons between 2 groups were assessed
using the two-tailed unpaired Student's t-test.
3. RESULTS
3.1 The reduced survival of grafted NSCs in the brains of APP tg mice is improved
by Cerebrolysin treatment
To investigate the survival of grafted cells in APP tg mice, cortical derived mouse
NSCs labeled with BrdU and GFP were injected into the hippocampus and mice were
analyzed 1, 3, 6 and 9m post transplantation. Immunocytochemical analysis with an
antibody against BrdU showed that the transplanted cells distributed mostly around the
CA1 region, the dentate gyrus and the sub-granular zone of the hippocampus (Fig. 1A-
C). After 1 month, in the vehicle-treated non-tg mice an average of 13,100 per unit area
were found, which was equivalent to approximately to 13.5% of the grafted cells (Fig. 1A,
D). After 3 months there was a decline to 5.8% that was stabilized after 6 and 9 months
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post transplant (6.5%) (Fig.1B, D). When compared to the non-tg vehicle control group,
in the APP tg mice there was a significant reduction in the survival of the grafted cells 1
m post-graft (5100 cells or 5.4%) (Fig.1B, D). At the later time points only few grafted
cells (less than the equivalent to 1%) were found in the vehicle APP tg group (Fig. 1C,
D). In the non-tg mice, the numbers of BrdU positive NSCs was similar in the vehicle and
CBL treated mice, however after 3, 6 and 9 months survival was higher (14%) in the CBL
treated mice compared to vehicle (5.8%) (Fig.1D). After 1 month in the CBL treated APP
tg mice the numbers of BrdU positive NSCs (18,900 or 24%) was above the non-tg
groups and the vehicle treated APP mice (Fig. 1A, D). After 3, 6 and 9 months of the
transplant the numbers of surviving NSCs was comparable to the non-tg CBL group
(12.1%) and above the vehicle-treated APP tg (less than 1%) (Fig. 1B-D). To further
corroborate that the BrdU detected cells were the grafted cells, double labeling
experiments were performed with antibodies against BrdU and GFP. By confocal
microscopy it was observed that the great majority of the GFP positive cells also
displayed BrdU reactivity (Fig. 1E, F). These results support the notion that grafted NSC
display poor survival in the brains of APP tg mice and that adjuvant treatment with CBL
reverts this effect.
3.2 Effects of Cerebrolysin and NSC grafting on AD-like neuropathology and
endogenous neurogenesis in APP tg mice.
To confirm the presence of AD-like neuropathology in the brains of APP tg mice and
to evaluate the impact of the combined effects of NSC grafting and CBL treatment
sections were immunolabeled with antibodies against A and GFAP. As expected
compared to the non-tg group the vehicle-treated APP tg mice displayed extensive A
deposition in the neocortex and hippocampus that increased over time (Fig. 2A-C upper
panels, D). In contrast, the CBL treated APP tg mice displayed a reduced amyloid
deposition that was more prominent 6 and 9 months post-graft and CBL treatment (Fig.
2C upper panels, D). With the antibody against GFAP, in both groups of APP tg mice
there was astrogliosis more prominent in the hippocampus than in the neocortex (Fig.
2A-C lower panels). Besides the expected astrogliosis along the injection site no
prominent glial reaction was observed as result of the placement of the NSC graft (Fig.
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2A-C lower panels). Next we analyzed the effects on neurodegeneration, as we have
previously shown that APP tg mice display age related synapto-dendritic loss in the
dentate gyrus of the hippocampus [44]. For this purpose sections were immunolabeled
with an antibody against MAP2 and analyzed by confocal microscopy (Fig. 3A). Analysis
at 1 month after grafting showed that non-tg and APP tg mice displayed comparable
levels of MAP2 immunoreactivity in the molecular layer of the dentate gyrus, although
compared to the vehicle, the CBL treated group had slightly higher levels of MAP2 (Fig.
3A, B). However, 3, 6 and 9m post-grafting the vehicle-treated APP tg mice displayed a
significant reduction in the % area of the neuropil covered by MAP2 immunoreactive
dendrites (Fig. 3A, B). Compared to vehicle group, grafted APP tg mice displayed levels
of MAP2 comparable to the non-tg mice (Fig. 3B).
We have previously shown that in addition to the synapto-dendritic damage, APP tg
mice display reduced neurogenesis in the hippocampus [14]. To evaluate the combined
effects of NSC grafting and CBL treatment on endogenous neurogenesis, sections were
immunostained with an antibody against double-cortin DCX (Fig. 3C). Analysis at 1
month after grafting showed that compared to vehicle treated non-tg, the APP tg mice
displayed a 47% reduction in the number of DCX positive neuroblasts in the subgranular
zone of the hippocampus (Fig. 3C, D), in contrast grafted APP tg mice treated with CBL
displayed DCX levels comparable to the un-treated non-tg mice (Fig. 3D). After 3, 6 and
9m post-grafting the vehicle treated APP tg mice displayed further reduction in the
numbers of DCX immunoreactive cells (Fig. 3C, D). A similar trend was observed 3
months after grafting in the non-tg mice and in the CBL treated APP tg (Fig. 3C, D).
However, at 6 and 9 months numbers of DCX immunoreactive cells leveled (Fig. 3C).
Taken together, these studies suggest that the combined effects of enhanced survival of
NSC with CBL reduces neurodegeneration in the hippocampus of APP tg mice by
ameliorating the degeneration of granular cells and endogenous neuroblasts in the
hippocampal dentate gyrus.
3.3 Cerebrolysin treatment promotes survival of grafted NSC with no evidence of
tumoral proliferation.
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Given that a subpopulation of grafted NSCs distributes in the dentate gyrus and that
there is reduced neurodegeneration and increased endogenous neurogenesis in the
granular zone, it is possible that CBL might promote differentiation and integration of
grafted NSCs into the hippocampus. To investigate this possibility sections were double
labeled with antibodies against a mature neuronal marker (NeuN, red) and GFP (to
enhance the visualization of the tagged grafted NSCs). Consistent with the BrdU
immunocytochemistry studies (Fig. 1A-C), GFP positive cells were identified in the sub-
granular zone of the dentate gyrus and in between granular cells (Fig. 4A). At 1 m post
grafting, compared to vehicle treated non-tg mice, the vehicle treated APP tg mice
displayed a 82% reduction in the numbers of surviving GFP positive NSCs (Fig. 4A, B).
In contrast, CBL treatment resulted in an increased number of GFP positive NSCs in the
dentate gyrus of non-tg and APP tg mice above the baseline of the controls (Fig. 4A, B).
At 3, 6 and 9 months post-grafting there was a mild decline in GFP positive cells in the
vehicle treated non-tg mice (Fig. 4A), while in the vehicle treated APP tg mice the
numbers remained very low (Fig. 4A, B). Whereas the CBL treated non-tg and APP tg
mice continued to show the presence of GFP positive NSCs above baseline controls
(Fig. 4A, B). In all four experimental groups, closer analysis of the immunolabeled
sections showed that none of the GFP positive cells were NeuN positive, neither the
GFP positive NSCs displayed the presence of neuritic extensions or integration into the
dentate gyrus (Fig. 4A arrowheads). Additional double labeling analysis was performed
with antibodies against the astroglial marker GFAP (red channel) and GFP (green
channel) (Fig. 4C). About 2% of the GFP positive NSCs displayed GFAP
immunoreactivity and a glial like morphology (Fig. 4C). Compared to non-tg mice in the
APP tg mice at 3 months post grafting there was an increase in the % of cells displayed
GFP and GFAP immunostaining (Fig. 4C, D). No significant effects were detected
following CBL treatment (Fig. 4D).
These findings support the notion that CBL might act by enhancing the survival (or
proliferation) of grafted NSCs rather than by promoting differentiation. Based on the
initial studies showing that starting at 3 months post grafting CBL supports the viability of
NSC (Fig. 1B) and to further evaluate the effects of CBL on NSC survival and
proliferation sections of mice at the 9 month post-graft were immunolabeled with
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antibodies against activated caspase-3 and PCNA and stained with a modified version of
the TUNEL technique (Fig. 5A). Compared to vehicle treated non-tg mice, the CBL
treated mice showed reduced active capase-3 and TUNEL positive NSCs (Fig. 5B). In
contrast, vehicle treated APP tg mice displayed higher numbers of active capase-3 and
TUNEL positive NSCs (Fig. 5B, C), this effect was reversed by CBL treatment (Fig. 5B,
C). Occasional PCNA immunoreactivity was detected in the grafted NSCs; no significant
differences were detected among the four groups (Fig. 5D). Likewise, analysis with an
antibody against the proliferation marker-Ki-67, at low power no evidence of tumoral
proliferation was noted (Fig. 6A), at higher magnification occasional Ki-67 positive cells
were noted in the hippocampus (Fig. 6B arrows) ,treatment with CBL did not reveal an
increase in proliferation of NPCs in the grafted area at any of the time points including 9
months post transplantation (Fig. 6B and C). Together these results support the
possibility that CBL might enhance the survival of grafted NSCs.
3.4 Adjuvant therapy with Cerebrolysin results in increase BDNF and furin levels
in grafted NSCs
Since it has been previously shown that CBL increases the conversion of pro-NGF to
mature NGF via furin, [48] and that NSCs can protect adjacent cells via neurotrophic
factor production [23] ,we analyzed the levels of BDNF, NT4 and furin by
immunocytochemistry (Fig. 5E). This study showed that both in non-tg and tg mice levels
of BDNF and furin immunostaining were increased in the NSCs of CBL treated mice (Fig.
5E, F, H). No differences were detected for NT4 (Fig. 5E, G). To further confirm that the
grafted NSCs were producing BDNF and furin double labeling with GFP was performed
(Fig. 5I, K). These studies showed that in the saline treated animals an average of 4%
(Fig. 5I, J) and 10% (Fig. 5K, L) of the GFP positive NSCs displayed BDNF or furin
immunoreactivity respectively. Treatment with CBL resulted in an increase in the
proportion of GFP positive cells displaying BDNF Fig. 5I, J) or furin immunolabeling (Fig.
5K, L). Further analysis of CBL effects on NSCs was performed in vitro at 0, 4 and 8 DIV.
This study showed that CBL increased BrdU levels (Fig. 7A, B) and decreased TUNEL
positive cells at 4 and 8 DIV (Fig. 7C, D). At 4 and 8 DIV the % of MAP2 positive cells
increased but no differences were detected between the vehicle and CBL groups (Fig.
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7E, F). Moreover, analysis of the levels of BDNF (Fig. 7G) and furin (Fig. 7H) by ELISA
with cell lysates at 8 DIV showed increased a 20-30% increase following CBL treatment
(Fig. 7G, H). In conclusion, CBL might enhance the survival of grafted NSC via furin
mediated BDNF processing.
4. DISCUSSION
The present study showed that CBL treatment enhanced the survival of grafted
NSCs in APP tg mice. In contrast, in the vehicle-treated group there was decreased
survival of transplanted neuroblasts that was worse in the APP tg mice. This is
consistent with studies showing that the toxic microenvironment in the brain of APP tg
mice might compromise the survival of both endogenous and transplanted NSCs. For
example, a recent study showed that oligomerized Aβ42 promoted the expression of
senescence-associated biomarkers p16 and senescence-associated β-galactosidase
(SA-β-gal) in adult mouse hippocampal NSCs, and reduced the proliferation and
differentiation of these cells in vitro and in APP/PS1 tg mice [60]. Moreover, we have
observed that extracellular A triggers apoptosis of transplanted NSCs and that
genetically modified NSCs expressing neprilysin reduces amyloid load in APP tg mice
and enhances the survival of NSCs and adjacent host neurons [31], supporting the view
of beneficial effects of combined therapy with stem cells.
Previous studies have shown that transfer of rodent NSC into the hippocampus
of APP/PS1 tg mice reduces behavioral deficits without changing amyloid  burden [30]
and transplantation of NSCs derived from human iPSC reduce memory deficits in APP
mice [29]. More recent studies have utilized mesenchymal stem cells and shown CNS
trafficking [61] and neurogenesis [62] in APP tg mice. Moreover, other studies have
shown that NSC grafting reduced hippocampal Tau and reelin accumulation in aged
Ts65Dn Down syndrome mice [28] and increased synaptic density in APP tg mice [23,
63]. Together, these studies support the notion that transfer of stem cells is beneficial by
promoting neurogenesis and synaptogenesis of endogenous neuronal circuitries. In
agreement with these studies, we found that combined NSC and CBL treatment was
associated with endogenous neurogenesis and reduced degeneration of the
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hippocampal dentate gyrus in the APP tg mice.
However, it should be noted that while in previous studies the beneficial effects of
stem cell monotherapy was noticed a few weeks post-transplant in our study the
combined use of NSC with CBL continued to be helpful after a long period of time. As
recently noted, poor viability of transplanted cells could be an important problem
associated with stem cell-based therapy for PD [64] and other neurodegenerative
disorders. For example, a recent study showed that combining a collagen hydrogel
incorporating an integrin-binding protein complex as a carrier for neural stem cells
improve the viability after transplantation into the striatum [64]. Similarly, it has been
shown that co-transplantation of GDNF-overexpressing NSCs and fetal neurons
ameliorates the motor deficits in a rat model of PD [65]. Along these lines, for the
present study we combined the NSC graft with adjuvant therapy with CBL in an effort to
enhance the survival of the transplanted cells. The mechanisms through which CBL
might enhance the survival of the grafted cells are not completely clear. One possibility
is that CBL might reduce the accumulation of toxic A species that can potentially
damage the grafted NSC. In agreement with this we showed lower levels of Aload
following 6 and 9 months of CBL treatment. Moreover previous studies have shown that
CBL reduces A production by reducing maturation of APP mediated by CDK5 and
GSK3 activation [66]. In addition, we showed that CBL increased furin levels, which in
turn resulted in increased processing of pro-BDNF into BDNF in the grafted cells. This is
consistent with previous studies showing that CBL might increase levels of NGF
perhaps involving the processing of pro-NGF to its mature form [48]. The observed
increase in furin levels in CBL-treated APP tg mice is consistent with this hypothesis.
Like pro-NGF [67, 68] pro-BNDF is the precursor protein of BDNF, which is
abundant in the hippocampus [69-72]. Pro-BDNF undergoes cleavage by a family of
mammalian processing enzymes, including furin [73-75]. To date there are nine known
convertases [74, 75] with furin being the most documented. Moreover, the furin
recognition site has been reported to be essential for the proper processing of pro-
BDNF to the mature form of BDNF [76]. Previous studies showed that NSCs are
capable of producing BDNF that in turn can protect neighboring host cells [23, 63]. The
neuropathological analysis showed that CBL reduced TUNEL and activated caspase-3
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in the grafted NSCs, but had no effect of PCNA or Ki-67 at the later time points,
supporting the notion of a trophic factor mediated anti-apoptotic effect. However, it is
possible that other mechanisms might be at play, including a limited proliferative effect
soon after transplantation.
In summary, the results of the present study suggest that CBL is capable of
protecting the grafted NSC in an APP tg mouse model and as such be a potential
adjuvant therapy when combined with cell based therapy.
DISCLOSURE
Authors Stefan Winter, Hemma Brandstaetter and Dieter Meier are employed by EVER
Neuro Pharma who provided the CBL used for this study.
FIGURE LEGENDS
Figure 1 –Immunocytochemical analysis of BrdU labeled grafted NSCs in CBL-
treated hAPP tg mice. Cortical NSCs labed with BrdU were transplanted into the
hippocampus of non-tg and APP tg mice (3 m/o) treated with vehicle or CBL and
immunocytochemical analysis was performed after with vibratome sections 1, 3, 6 and 9
m post graft. (A, B, C) Upper panel representative images at low power (20X) 1, 3 and 9
months post graft respectively. Lower panel is higher magnification (400X) of the area in
the upper panel marked by an open square. Arrows indicate BrdU labeled grafted NSCs
distributed along the dentate granular cell layer (DGL). (D) Computer aided image
analysis of the numbers of BrdU positive cells in the hippocampus 1, 3, 6 and 9 m post
graft. (E, F) Double labeling and confocal microscopy studies for BrdU (red) and GFP
(green) in the hippocampus of grafted animals showing high levels of co-localization
between the two markers (Bar=10 um). All results are presented as mean  SEM, each
of the four groups includes n=32 per time point (n=16 non-tg [n=8 vehicle and n=8 CBL]
and n=16 APP tg [n=8 vehicle and n=8 CBL] mice). * = p<0.05 between vehicle-treated
non-tg and APP tg by one-way ANOVA and post Dunnet’s. ** = p<0.05 between
vehicle-treated and CBL-treated groups by one-way ANOVA and post hoc Tukey-
Kramer. Bar= 250 m for low power panel and 50m for high power panel.
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Figure 2 – Neuropathological analysis of AD-like pathology in NSC grafted and
CBL-treated APP tg mice. Vibratome sections from non-tg and APP tg mice treated
with vehicle or CBL and grafted with NSCs were immunolabeled with antibodies against
A (FITC, imaged with the laser scanning confocal microscope) and GFAP (DAB,
imaged by digital video microscopy). (A, B, C) Upper panel representative images at low
power (20X) at 1, 3 and 9 months post graft respectively. Middle panel is higher
magnification (400X) of the hippocampus dentate gyrus. Arrows indicate site of the
injection and tract. Lower panel representative images at low power (20X) of the
neocortex and hippocampus with an antibody against GFAP. (D) Computer aided image
analysis of the A amyloid load in the hippocampus 1, 3, 6 and 9 m post graft. All
results are presented as mean  SEM, each of the four groups includes n=32 per time
point (n=16 non-tg [n=8 vehicle and n=8 CBL] and n=16 APP tg [n=8 vehicle and n=8
CBL] mice). ** = p<0.05 between vehicle-treated and CBL-treated APP tg mice by one-
way ANOVA and post hoc Tukey-Kramer. Bar= 250 m for low power panel and 50m
for high power panel.
Figure 3 – Immunohistochemical analysis of levels of MAP2 and endogenous
DCX immunoreactivity in the hippocampus of NSC grafted and CBL-treated APP
tg mice. Vibratome sections from non-tg and APP tg mice treated with vehicle or CBL
and grafted with NSCs were immunolabeled with an antibody against MAP2 (FITC,
imaged with the laser scanning confocal microscope) and doublecortin (DCX) and
imaged with the digital video microscope. (A) Representative images at 1, 3 and 9
months post graft respectively of the dendritic arbor (inner molecular layer) of the
dentate granular cells. (B) Computer aided image analysis of the % area of the neuropil
occupied by MAP2 dendrites at 1, 3, 6 and 9 m post graft. (C) Representative images
at 1, 3 and 9 months post graft respectively of the sub-granular zone and dentate gyrus.
(D) Computer aided stereological analysis of the estimated DCX+ cell counts at 1, 3, 6
and 9 m post graft. All results are presented as mean  SEM, each of the four groups
includes n=32 per time point (n=16 non-tg [n=8 vehicle and n=8 CBL] and n=16 APP tg
[n=8 vehicle and n=8 CBL] mice). * = p<0.05 between vehicle-treated non-tg mice and
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vehicle treated APP tg mice by one-way ANOVA and post hoc Dunnet’s. ** = p<0.05
between vehicle-treated and CBL-treated APP tg mice by one-way ANOVA and post
hoc Tukey-Kramer. Bar= 50 um.
Figure 4 – Laser scanning confocal microscopic analysis of neuronal and glial
maturation of the grafted NSCs in the hippocampus of CBL-treated APP tg mice.
Vibratome sections from non-tg and APP tg mice treated with vehicle or CBL and
grafted with NSCs (GFP tagged) were double-immunolabeled with antibodies against
NeuN (red) or GFAP (red) and GFP (green) and imaged with the laser scanning
confocal microscope. (A) Representative images at 1, 3 and 9 months post graft
respectively of the hippocampal dentate gyrus. Images illustrate granular cells (red) and
the grafted NSCs (green, arrowheads) that migrated along the sub-granular zone. (B)
Computer aided image analysis of the estimated GFP+ cells at 1, 3, 6 and 9 m post
graft. (C) Representative images at 1, 3 and 9 months post graft respectively of the
hippocampal dentate gyrus. Images illustrate astroglial cells in the dentate gyrus (red)
and the grafted NSCs (green) along the sub-granular zone. (D) Computer aided image
analysis of the % of co-localization between GFP+ and GFAP cells at 1, 3, 6 and 9 m
post graft. (E) Representative images at 1, 3 and 9 months post graft respectively of the
hippocampal dentate gyrus. Images illustrate oligodendrocytes (olig-2) in the dentate
gyrus (red) and the grafted NSCs (green) along the sub-granular zone. (F) Computer
aided image analysis of the % of co-localization between GFP+ and olig-2 cells at 1, 3,
6 and 9 m post graft. All results are presented as mean  SEM, each of the four groups
includes n=32 per time point (n=16 non-tg [n=8 vehicle and n=8 CBL] and n=16 APP tg
[n=8 vehicle and n=8 CBL] mice). * = p<0.05 between vehicle-treated non-tg mice and
vehicle treated APP tg mice by one-way ANOVA and post hoc Dunnet’s. Bar= 25 m.
Figure 5 – Immunocytochemical and double labeling analysis of markers of cell
survival and neurotrophic factors in the grafted NSCs in CBL-treated APP tg
mice. Vibratome sections from non-tg and APP tg mice treated with vehicle or CBL and
grafted with NSCs were immunolabeled with antibodies against markers of cell death
and survival (act casp3, TUNEL and PCNA) and neurotrophic factors (BDNF, NT4 and
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furin) and imaged with the digital video microscope. (A) Representative images of
vehicle and CBL treated APP tg mice at 9 months post graft respectively of the sub-
granular zone and dentate gyrus. (B, C, D) Computer aided image analysis of the
estimated active caspase 3, TUNEL and PCNA positive cells at 9 months post
graft,respectively. (E) Representative images of vehicle and CBL treated APP tg mice at
9 months post graft respectively of the sub-granular zone and dentate gyrus. (F, G, H)
Computer aided image analysis of the estimated BDNF, NT4 and Furin positive cells at
9 months post graft respectively. (I) Vibratome sections from non-tg and APP tg mice
treated with vehicle or CBL and grafted with NSCs (GFP tagged) were double-
immunolabeled with antibodies against BDNF or furin (red) and GFP (green) and
imaged with the laser scanning confocal microscope. Representative split images for
BDNF (red) and GFP (green) at 9 months post graft of NSC graft in the hippocampal
dentate gyrus of vehicle and CBL treated APP tg mice. Images illustrate BDNF co-
localization with the GFP tag in the NSCs (arrowhead). (J) Computer aided image
analysis of the % of GFP cells that co-localized with BDNF at 9 m post graft. (K)
Representative split images for furin (red) and GFP (green) at 9 months post graft of
NSCs in the hippocampal dentate gyrus of vehicle and CBL treated APP tg mice.
Images illustrate furin co-localization with the GFP tag in the NSCs (arrowhead). (L)
Computer aided image analysis of the % of GFP cells that co-localized with furin at 9 m
post graft. All results are presented as mean  SEM, n=16 non-tg (n=8 vehicle and n=8
CBL) and n=16 APP tg (n=8 vehicle and n=8 CBL) mice. * = p<0.05 between vehicle-
treated non-tg mice and CBL treated non-tg mice by one-way ANOVA and post hoc
Dunnet’s. ** = p<0.05 between vehicle-treated and CBL-treated APP tg mice by one-
way ANOVA and post hoc Tukey-Kramer. Bar= 10 m.
Figure 6 – Immunocytochemical analysis of the proliferation marker Ki-67 in the
grafted NSCs in CBL-treated APP tg mice. Vibratome sections from non-tg and APP
tg mice treated with vehicle or CBL and grafted with NSCs were immunolabeled with an
antibody against Ki-67 and imaged with the digital video microscope. (A) Representative
low power images (20X) of vehicle and CBL treated mice at 1,3 and 9 months post graft
respectively of the neocortex and hippocampus, dotted open circle represent are of
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graft, bar= 250 m. (B) Representative higher power images (630X) of Ki-67 positive
cells (arrows) for the vehicle and CBL treated mice at 1,3 and 9 months post graft
respectively of the hippocampus of the corresponding site of the graft (dotted open
circle in A), bar= 25 m. (C) Computer-aided image analysis of the numbers of Ki-67
positive cells in the hippocampus of the non-tg and APP tg mice.
Figure 7 –In vitro effects of CBL on markers of cell survival and neurotrophic
factors in NSCs. Mouse cortical NSCs were grown in coverslips for 0, 4 and 8 days
and treated with vehicle or CBL at 5% in media with 1% FBS. (A, C, E ) Representative
images for NSCs immunostained with an antibody against BrdU, histochemistry for
TUNEL or immunostained with an antibody against MAP2 at day 8. (B, D, F) Computer
aided image number of cells displaying BrdU, TUNEL of MAP2 reactivity at 0, 4 and 8
DIV. * = p<0.05 between vehicle- and CBL NSCs by two-tailed, Student T-test. Bar= 10
m. (G, H) ELISA analysis for BDNF and furin in cell lysates at 8 DIV following vehicle
or CBL treatment. * = p<0.05 by two-tailed, Student T-test
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REFERENCES
1.
Overk CR, Masliah E: Pathogenesis of synaptic degeneration in Alzheimer's
disease and Lewy body disease. Biochemical pharmacology 2014, 88:508-
516.
2.
DeKosky ST, Scheff SW, Styren SD: Structural correlates of cognition in
dementia: quantification and assessment of synapse change.
Neurodegeneration 1996, 5:417-421.
3.
4.
Masliah E: Mechanisms of synaptic dysfunction in Alzheimer's disease.
HistolHistopathol 1995, 10:509-519.
Masliah E: Recent advances in the understanding of the role of synaptic
proteins in Alzheimer's Disease and other neurodegenerative disorders.
Journal of Alzheimer's disease : JAD 2001, 3:121-129.
5.
6.
7.
Masliah E, Crews L, Hansen L: Synaptic remodeling during aging and in
Alzheimer's disease. Journal of Alzheimer's disease : JAD 2006, 9:91-99.
Scheff S, DeKosky S, Price D: Quantitative assessment of cortical synaptic
density in Alzheimer's disease. NeurobiolAging 1990, 11:29-37.
Terry R, Masliah E, Salmon D, Butters N, DeTeresa R, Hill R, Hansen L,
Katzman R: Physical basis of cognitive alterations in Alzheimer disease:
synapse loss is the major correlate of cognitive impairment. AnnNeurol
1991, 30:572-580.
8.
9.
Trojanowski JQ, Shin RW, Schmidt ML, Lee VM: Relationship between
plaques, tangles, and dystrophic processes in Alzheimer's disease.
Neurobiol Aging 1995, 16:335-340; discussion 341-335.
Dong H, Goico B, Martin M, Csernansky CA, Bertchume A, Csernansky JG:
Modulation of hippocampal cell proliferation, memory, and amyloid plaque
deposition in APPsw (Tg2576) mutant mice by isolation stress.
Neuroscience 2004, 127:601-609.
10.
Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, Greenberg DA:
Increased hippocampal neurogenesis in Alzheimer's disease. Proceedings
of the National Academy of Sciences of the United States of America 2004,
101:343-347.
11.
12.
Boekhoorn K, Joels M, Lucassen PJ: Increased proliferation reflects glial and
vascular-associated changes, but not neurogenesis in the presenile
Alzheimer hippocampus. Neurobiology of disease 2006, 24:1-14.
Li B, Yamamori H, Tatebayashi Y, Shafit-Zagardo B, Tanimukai H, Chen S, Iqbal
K, Grundke-Iqbal I: Failure of neuronal maturation in Alzheimer disease
dentate gyrus. Journal of neuropathology and experimental neurology 2008,
67:78-84.
13.
14.
15.
Donovan MH, Yazdani U, Norris RD, Games D, German DC, Eisch AJ:
Decreased adult hippocampal neurogenesis in the PDAPP mouse model of
Alzheimer's disease. The Journal of comparative neurology 2006, 495:70-83.
Rockenstein E, Mante M, Adame A, Crews L, Moessler H, Masliah E: Effects of
Cerebrolysin on neurogenesis in an APP transgenic model of Alzheimer's
disease. Acta neuropathologica 2007, 113:265-275.
Haughey NJ, Nath A, Chan SL, Borchard AC, Rao MS, Mattson MP: Disruption
of neurogenesis by amyloid beta-peptide, and perturbed neural progenitor
- 20 -

ACCEPTED MANUSCRIPT
cell homeostasis, in models of Alzheimer's disease. Journal of
neurochemistry 2002, 83:1509-1524.
16.
17.
18.
19.
Engmann O, Giese KP: Crosstalk between Cdk5 and GSK3beta: Implications
for Alzheimer's Disease. Frontiers in molecular neuroscience 2009, 2:2.
Crews L, Masliah E: Molecular mechanisms of neurodegeneration in
Alzheimer's disease. Human molecular genetics 2010, 19:R12-20.
Shukla V, Skuntz S, Pant HC: Deregulated Cdk5 activity is involved in
inducing Alzheimer's disease. Archives of medical research 2012, 43:655-662.
Gong CX, Iqbal K: Hyperphosphorylation of microtubule-associated protein
tau: a promising therapeutic target for Alzheimer disease. Current medicinal
chemistry 2008, 15:2321-2328.
20.
Peng S, Wuu J, Mufson EJ, Fahnestock M: Precursor form of brain-derived
neurotrophic factor and mature brain-derived neurotrophic factor are
decreased in the pre-clinical stages of Alzheimer's disease. Journal of
neurochemistry 2005, 93:1412-1421.
21.
22.
Schindowski K, Belarbi K, Buee L: Neurotrophic factors in Alzheimer's
disease: role of axonal transport. Genes, brain, and behavior 2008, 7 Suppl
1:43-56.
Scott SA, Mufson EJ, Weingartner JA, Skau KA, Crutcher KA: Nerve growth
factor in Alzheimer's disease: increased levels throughout the brain
coupled with declines in nucleus basalis. The Journal of neuroscience : the
official journal of the Society for Neuroscience 1995, 15:6213-6221.
Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Muller FJ, Loring
JF, Yamasaki TR, Poon WW, Green KN, LaFerla FM: Neural stem cells
improve cognition via BDNF in a transgenic model of Alzheimer disease.
Proceedings of the National Academy of Sciences of the United States of
America 2009, 106:13594-13599.
23.
24.
25.
Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM,
Wang L, Blesch A, Kim A, Conner JM, et al: Neuroprotective effects of brain-
derived neurotrophic factor in rodent and primate models of Alzheimer's
disease. Nature medicine 2009, 15:331-337.
Tuszynski MH, Thal L, Pay M, Salmon DP, U HS, Bakay R, Patel P, Blesch A,
Vahlsing HL, Ho G, et al: A phase 1 clinical trial of nerve growth factor gene
therapy for Alzheimer disease. Nature medicine 2005, 11:551-555.
Tuszynski MH: Nerve growth factor gene delivery: animal models to clinical
trials. Developmental neurobiology 2007, 67:1204-1215.
Nagahara AH, Mateling M, Kovacs I, Wang L, Eggert S, Rockenstein E, Koo EH,
Masliah E, Tuszynski MH: Early BDNF treatment ameliorates cell loss in the
entorhinal cortex of APP transgenic mice. The Journal of neuroscience : the
official journal of the Society for Neuroscience 2013, 33:15596-15602.
Kern DS, Maclean KN, Jiang H, Synder EY, Sladek JR, Jr., Bjugstad KB: Neural
stem cells reduce hippocampal tau and reelin accumulation in aged
Ts65Dn Down syndrome mice. Cell transplantation 2011, 20:371-379.
Fujiwara N, Shimizu J, Takai K, Arimitsu N, Saito A, Kono T, Umehara T, Ueda
Y, Wakisaka S, Suzuki T, Suzuki N: Restoration of spatial memory
26.
27.
28.
29.
- 21 -

ACCEPTED MANUSCRIPT
dysfunction of human APP transgenic mice by transplantation of neuronal
precursors derived from human iPS cells. Neuroscience letters 2013.
Zhang W, Wang PJ, Sha HY, Ni J, Li MH, Gu GJ: Neural stem cell transplants
improve cognitive function without altering amyloid pathology in an
APP/PS1 double transgenic model of Alzheimer's disease. Molecular
neurobiology 2014, 50:423-437.
Blurton-Jones M, Spencer B, Michael S, Castello NA, Agazaryan AA, Davis JL,
Muller FJ, Loring JF, Masliah E, LaFerla FM: Neural stem cells genetically-
modified to express neprilysin reduce pathology in Alzheimer transgenic
models. Stem cell research & therapy 2014, 5:46.
Alvarez XA, Cacabelos R, Laredo M, Couceiro V, Sampedro C, Varela M, Corzo
L, Fernandez-Novoa L, Vargas M, Aleixandre M, et al: A 24-week, double-blind,
placebo-controlled study of three dosages of Cerebrolysin in patients with
mild to moderate Alzheimer's disease. Eur J Neurol 2006, 13:43-54.
Ruther E, Ritter R, Apecechea M, Freytag S, Gmeinbauer R, Windisch M:
Sustained improvements in patients with dementia of Alzheimer's type
(DAT) 6 months after termination of Cerebrolysin therapy. Journal of neural
transmission 2000, 107:815-829.
Ruther E, Ritter R, Apecechea M, Freitag S, Windisch M: Efficacy of
Cerebrolysin in Alzheimer's disease. In New trends in the diagnosis and
therapy of Alzheimer's disease. Edited by Jellinger K, Ladurner G, Windisch M.
Vienna: Springer-Verlag; 1994: 131-141
30.
31.
32.
33.
34.
35.
Alvarez XA, Cacabelos R, Sampedro C, Aleixandre M, Linares C, Granizo E,
Doppler E, Moessler H: Efficacy and safety of Cerebrolysin in moderate to
moderately severe Alzheimer's disease: results of a randomized, double-
blind, controlled trial investigating three dosages of Cerebrolysin. Eur J
Neurol 2011, 18:59-68.
36.
37.
38.
Plosker GL, Gauthier S: Cerebrolysin: a review of its use in dementia. Drugs
Aging 2009, 26:893-915.
Plosker GL, Gauthier S: Spotlight on cerebrolysin in dementia. CNS Drugs
2010, 24:263-266.
Veinbergs I, Mante M, Mallory M, Masliah E: Neurotrophic effects of
Cerebrolysin in animal models of excitotoxicity. J Neural Transm Suppl 2000,
59:273-280.
39.
40.
41.
Onishchenko LS, Gaikova ON, Yanishevskii SN: Changes at the focus of
experimental ischemic stroke treated with neuroprotective agents. Neurosci
Behav Physiol 2008, 38:49-54.
Ren J, Sietsma D, Qiu S, Moessler H, Finklestein SP: Cerebrolysin enhances
functional recovery following focal cerebral infarction in rats. Restor Neurol
Neurosci 2007, 25:25-31.
Zhang C, Chopp M, Cui Y, Wang L, Zhang R, Zhang L, Lu M, Szalad A, Doppler
E, Hitzl M, Zhang ZG: Cerebrolysin enhances neurogenesis in the ischemic
brain and improves functional outcome after stroke. J Neurosci Res 2010,
88:3275-3281.
42.
Rockenstein E, Adame A, Mante M, Larrea G, Crews L, Windisch M, Moessler H,
Masliah E: Amelioration of the cerebrovascular amyloidosis in a transgenic
- 22 -

ACCEPTED MANUSCRIPT
model of Alzheimer's disease with the neurotrophic compound
cerebrolysin. J Neural Transm 2005, 112:269-282.
43.
Rockenstein E, Adame A, Mante M, Moessler H, Windisch M, Masliah E: The
neuroprotective effects of Cerebrolysin in a transgenic model of
Alzheimer's disease are associated with improved behavioral performance.
J Neural Transm 2003, 110:1313-1327.
44.
45.
46.
Rockenstein E, Mallory M, Mante M, Alford M, Windisch M, Moessler H, Masliah
E: Effects of Cerebrolysin on amyloid-beta deposition in a transgenic
model of Alzheimer's disease. J Neural Transm Suppl 2002:327-336.
Blanchard J, Chohan MO, Li B, Liu F, Iqbal K, Grundke-Iqbal I: Beneficial effect
of a CNTF tetrapeptide on adult hippocampal neurogenesis, neuronal
plasticity, and spatial memory in mice. J Alzheimers Dis 2010, 21:1185-1195.
Blanchard J, Wanka L, Tung YC, Cardenas-Aguayo Mdel C, LaFerla FM, Iqbal K,
Grundke-Iqbal I: Pharmacologic reversal of neurogenic and neuroplastic
abnormalities and cognitive impairments without affecting Abeta and tau
pathologies in 3xTg-AD mice. Acta Neuropathol 2010, 120:605-621.
Chohan MO, Li B, Blanchard J, Tung YC, Heaney AT, Rabe A, Iqbal K, Grundke-
Iqbal I: Enhancement of dentate gyrus neurogenesis, dendritic and synaptic
plasticity and memory by a neurotrophic peptide. Neurobiol Aging 2011,
32:1420-1434.
47.
48.
Ubhi K, Rockenstein E, Vazquez-Roque R, Mante M, Inglis C, Patrick C, Adame
A, Fahnestock M, Doppler E, Novak P, et al: Cerebrolysin modulates pronerve
growth factor/nerve growth factor ratio and ameliorates the cholinergic
deficit in a transgenic model of Alzheimer's disease. Journal of neuroscience
research 2013, 91:167-177.
49.
50.
Crews L, Patrick C, Adame A, Rockenstein E, Masliah E: Modulation of
aberrant CDK5 signaling rescues impaired neurogenesis in models of
Alzheimer's disease. Cell death & disease 2011, 2:e120.
Desplats P, Spencer B, Crews L, Pathel P, Morvinski-Friedmann D, Kosberg K,
Roberts S, Patrick C, Winner B, Winkler J, Masliah E: alpha-Synuclein induces
alterations in adult neurogenesis in Parkinson disease models via p53-
mediated repression of Notch1. The Journal of biological chemistry 2012,
287:31691-31702.
51.
52.
Rockenstein E, Mallory M, Mante M, Sisk A, Masliaha E: Early formation of
mature amyloid-beta protein deposits in a mutant APP transgenic model
depends on levels of Abeta(1-42). Journal of neuroscience research 2001,
66:573-582.
Crews L, Adame A, Patrick C, Delaney A, Pham E, Rockenstein E, Hansen L,
Masliah E: Increased BMP6 levels in the brains of Alzheimer's disease
patients and APP transgenic mice are accompanied by impaired
neurogenesis. The Journal of neuroscience : the official journal of the Society
for Neuroscience 2010, 30:12252-12262.
53.
Havas D, Hutter-Paier B, Ubhi K, Rockenstein E, Crailsheim K, Masliah E,
Windisch M: A longitudinal study of behavioral deficits in an AbetaPP
transgenic mouse model of Alzheimer's disease. J Alzheimers Dis 2011,
25:231-243.
- 23 -

ACCEPTED MANUSCRIPT
54.
Rockenstein E, Ubhi K, Pham E, Michael S, Doppler E, Novak P, Inglis C, Mante
M, Adame A, Alvarez XA, et al: Beneficial effects of a neurotrophic
peptidergic mixture persist for a prolonged period following treatment
interruption in a transgenic model of Alzheimer's disease. J Neurosci Res
2011, 89:1812-1821.
55.
56.
Spencer B, Marr RA, Rockenstein E, Crews L, Adame A, Potkar R, Patrick C,
Gage FH, Verma IM, Masliah E: Long-term neprilysin gene transfer is
associated with reduced levels of intracellular Abeta and behavioral
improvement in APP transgenic mice. BMC neuroscience 2008, 9:109.
Rockenstein EM, McConlogue L, Tan H, Power M, Masliah E, Mucke L: Levels
and alternative splicing of amyloid beta protein precursor (APP) transcripts
in brains of APP transgenic mice and humans with Alzheimer's disease.
The Journal of biological chemistry 1995, 270:28257-28267.
57.
58.
EBEWENeuroPharmaGmbH: Cerebrolysin ® solution for injection: summary
of product characteristics. 2009.
Chen H, Tung YC, Li B, Iqbal K, Grundke-Iqbal I: Trophic factors counteract
elevated FGF-2-induced inhibition of adult neurogenesis. Neurobiol Aging
2007, 28:1148-1162.
59.
60.
Paxinos G, Franklin K: The Mouse Brain in Stereotaxic Coordinates. Second
edn. San Diego: Academic Press; 2001.
He N, Jin WL, Lok KH, Wang Y, Yin M, Wang ZJ: Amyloid-beta(1-42) oligomer
accelerates senescence in adult hippocampal neural stem/progenitor cells
via formylpeptide receptor 2. Cell death & disease 2013, 4:e924.
Danielyan L, Beer-Hammer S, Stolzing A, Schafer R, Siegel G, Fabian C, Kahle
P, Biedermann T, Lourhmati A, Buadze M, et al: Intranasal delivery of bone
marrow derived mesenchymal stem cells, macrophages, and microglia to
the brain in mouse models of Alzheimer's and Parkinson's disease. Cell
transplantation 2014.
61.
62.
63.
64.
Yan Y, Ma T, Gong K, Ao Q, Zhang X, Gong Y: Adipose-derived mesenchymal
stem cell transplantation promotes adult neurogenesis in the brains of
Alzheimer's disease mice. Neural regeneration research 2014, 9:798-805.
Zhang W, Wang GM, Wang PJ, Zhang Q, Sha SH: Effects of neural stem cells
on synaptic proteins and memory in a mouse model of Alzheimer's
disease. Journal of neuroscience research 2014, 92:185-194.
Nakaji-Hirabayashi T, Kato K, Iwata H: In vivo study on the survival of neural
stem cells transplanted into the rat brain with a collagen hydrogel that
incorporates laminin-derived polypeptides. Bioconjugate chemistry 2013,
24:1798-1804.
65.
66.
Deng X, Liang Y, Lu H, Yang Z, Liu R, Wang J, Song X, Long J, Li Y, Lei D, Feng
Z: Co-transplantation of GDNF-overexpressing neural stem cells and fetal
dopaminergic neurons mitigates motor symptoms in a rat model of
Parkinson's disease. PloS one 2013, 8:e80880.
Rockenstein E, Torrance M, Mante M, Adame A, Paulino A, Rose JB, Crews L,
Moessler H, Masliah E: Cerebrolysin decreases amyloid-beta production by
regulating amyloid protein precursor maturation in a transgenic model of
Alzheimer's disease. J Neurosci Res 2006, 83:1252-1261.
- 24 -

ACCEPTED MANUSCRIPT
67.
68.
69.
Fahnestock M, Michalski B, Xu B, Coughlin MD: The precursor pro-nerve
growth factor is the predominant form of nerve growth factor in brain and
is increased in Alzheimer's disease. Mol Cell Neurosci 2001, 18:210-220.
Peng S, Wuu J, Mufson EJ, Fahnestock M: Increased proNGF levels in
subjects with mild cognitive impairment and mild Alzheimer disease. J
Neuropathol Exp Neurol 2004, 63:641-649.
Lessmann V, Brigadski T: Mechanisms, locations, and kinetics of synaptic
BDNF secretion: an update. Neuroscience research 2009, 65:11-22.
Barker PA: Whither proBDNF? Nature neuroscience 2009, 12:105-106.
West AE, Pruunsild P, Timmusk T: Neurotrophins: transcription and
translation. Handbook of experimental pharmacology 2014, 220:67-100.
Bekinschtein P, Cammarota M, Medina JH: BDNF and memory processing.
Neuropharmacology 2014, 76 Pt C:677-683.
70.
71.
72.
73.
Chen Y, Zhang J, Deng M: Furin mediates brain-derived neurotrophic factor
upregulation in cultured rat astrocytes exposed to oxygen-glucose
deprivation. J Neurosci Res 2015, 93:189-194.
74.
75.
76.
Fugere M, Day R: Cutting back on pro-protein convertases: the latest
approaches to pharmacological inhibition. Trends Pharmacol Sci 2005,
26:294-301.
Seidah NG, Chretien M: Proprotein and prohormone convertases: a family of
subtilases generating diverse bioactive polypeptides. Brain Res 1999,
848:45-62.
Lim KC, Tyler CM, Lim ST, Giuliano R, Federoff HJ: Proteolytic processing of
proNGF is necessary for mature NGF regulated secretion from neurons.
Biochem Biophys Res Commun 2007, 361:599-604.
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