Accepted Manuscript Title: The Ca2+ Sensor S100A1 Modulates Neuroinflammation, Histopathology and Akt Activity in the PSAPP Alzheimer’s Disease Mouse Model Author: Lauriaselle Afanador Emily A. Roltsch Leigh Holcomb Kerry S. Campbell David A. Keeling Yan Zhang Danna B. Zimmer PII: DOI: Reference:
S0143-4160(14)00082-7 http://dx.doi.org/doi:10.1016/j.ceca.2014.05.002 YCECA 1566
To appear in:
Cell Calcium
Received date: Revised date: Accepted date:
7-10-2013 15-5-2014 16-5-2014
Please cite this article as: L. Afanador, E.A. Roltsch, L.H. K.S. Campbell, D.A. Keeling, Y. Zhang, D.B. Zimmer, The Ca2+ Sensor S100A1 Modulates Neuroinflammation, Histopathology and Akt Activity in the, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The Ca2+ Sensor S100A1 Modulates Neuroinflammation, Histopathology and Akt Activity in the
cr
ip t
PSAPP Alzheimer's Disease Mouse Model
us
Lauriaselle Afanador*, Emily A. Roltsch†, Leigh Holcomb,+#,Kerry S. Campbell*, David A. Keeling*, Yan Zhang*, and Danna B. Zimmer*,a
an
*Center for Biomolecular Therapeutics, Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA and +
te
d
M
Psychiatry and Behavioral Science, Texas A&M Health Science Center, College of Medicine, and Central Texas Veterans Health Care System Neuropsychiatry Research Program, Temple, TX, USA
Ac ce p
Current address: †LSU Health Science Center, School of Medicine, 1901 Perdido St., New Orleans, LA, 70112, USA; and #Genzyme, Inc., 500 Kendall Street, Cambridge, MA 02142
TITLE RUNNING HEAD: S100A1 Modulates AD Pathobiology
a
To whom correspondence should be addressed: Danna B. Zimmer, Ph.D. Center for Biomolecular Therapeutics Department of Biochemistry & Molecular Biology University of Maryland School of Medicine 9600 Gudelsky Drive Rockville, MD 20850 Voice: 240.314.6514 Email: [email protected]
Page 1
Page 1 of 39
SUMMARY The contribution of the Ca2+ sensor S100A1 to in vivo Alzheimer's disease (AD) pathobiology has not been elucidated although S100A1 regulates numerous cellular processes
ip t
linked to AD. This study uses genetic ablation to ascertain the effects of S100A1 on neuroinflammation, beta-amyloid (A) plaque deposition and Akt activity in the PSAPP AD
cr
mouse model. PSAPP/S100A1-/- mice exhibited decreases in astrocytosis (GFAP burden),
microgliosis (Iba1 burden) and plaque load/number when compared to PSAPP/S100A1+/+ mice
us
at six and twelve months of age. The presence of detectable S100A1 staining in human AD
an
specimens is consistent with a detrimental gain of S100A1 function in AD. S100A1 ablation also reduced plaque associated and increased non-plaque associated PO4-Akt and PO4-GSK3
M
staining. S100A1Akt complexes were undetectable in PC12 cells and AD brain tissue suggesting that S100A1 indirectly modulates Akt activity. In contrast, S100A1RyR (ryanodine
d
receptor) complexes were present in human/mouse AD brain and exhibited Ca2+-dependent
te
formation in neuronal cells. This is the first direct demonstration of an S100A1 target protein complex in tissue/cells and identifies the RyR as a primary S100A1 target protein in the brain.
Ac ce p
Collectively, these data suggest that S100A1 inhibition may be a novel strategy for normalizing aberrant Ca2+ signaling in AD.
Keywords: astrocytosis, microgliosis, plaque load, neurodegeneration, GSK3, ryanodine receptor, S100B, PC12 cells
Page 2
Page 2 of 39
1. INTRODUCTION Disruption of calcium (Ca2+) homeostasis is a common event in many diseases [1, 2]. Physiological and pathological Ca2+ signals are transduced into biological responses by
ip t
members of the calmodulin/troponin/S100 superfamily of Ca2+ sensor proteins. Upon binding
cr
Ca2+, Ca2+sensor proteins undergo a large conformational change ("Ca2+ switch") that exposes a hydrophobic cleft required for interaction with their target proteins and subsequent exertion of
us
their biological effects [3]. S100s are found exclusively in vertebrates, and are distinguished from other Ca2+ sensors by their 3D structure and a highly conserved amino terminal Ca2+
an
binding loop consisting of 14 rather than the typical 12 amino acids [4, 5]. The human genome encodes 21 S100 proteins, four (S100B, S100G, S100P and S100Z) are singletons dispersed
M
throughout the genome and the remaining seventeen (S100A1-S100A16) are located within the epidermal differentiation complex (EDC) on human chromosome 1[4]. The term S100 was used
d
in the early literature to denote a mixture of the two founding family members, S100A1 and
Ac ce p
ammonium sulfate [6].
te
S100B, and refers to the solubility of these approximately 10,000 Da proteins in 100% saturated
Although S100 family members exhibit a high degree of structural similarity, they are not functionally interchangeable. With the exception of S100G, which is monomeric, S100 proteins are typically symmetric dimers [3]. Nonetheless, individual family members exhibit unique affinities for divalent metal ions (Ca2+, Zn2+, Cu2+), oligomerization properties, post-translational modifications and spatial/temporal expression patterns. S100s bind to and regulate the activity of a large number of target proteins, some of which are regulated by a single family member and others by multiple family members. Individual family members also exhibit unique binding orientations for target proteins that are due in part to differences in surface charge density [7, 8]. S100s can also compete with other Ca2+ sensors such as calmodulin for the same binding site on target proteins [9, 10]. S100s are also found in the extracellular space where they interact
Page 3
Page 3 of 39
with a variety of cell surface receptors including the Receptor for Advanced Glycation Endproducts (RAGE), G-protein coupled receptors, toll-like receptor-4, scavenger receptors, FGFreceptor 1, dopamine 2 receptor, ALCAM and bioactive spingolipids [11-14] . S100 proteins
ip t
lack a signal peptide for secretion via the conventional Golgi-mediated pathway, and there is debate as to whether extracellular S100s are actively secreted or passively released from
cr
live/necrotic cells. The diversity among S100s when coupled with their partial functional
redundancy and the unique complement of family members expressed in each cell, allows
us
individual cells to generate unique and adaptive responses to equivalent changes in intracellular Ca2+ levels.
an
Dysregulation of Ca2+ homeostasis and neuroinflammation precede the appearance of
M
toxic A peptide oligomers/plaques and hyperphosphorylated tau/neurofibrillary tangles in familial and sporadic Alzheimer's disease (AD) [15-24]. Studies using clinical specimens and animal models have implicated five S100 family members (S100A6, S100A7, S100A9, S100A12
te
d
and S100B) in aberrant Ca2+ signaling in AD [25-32]. S100B is expressed primarily in astrocytes/microglia and has been postulated to be a part of a detrimental autocatalytic cytokine
Ac ce p
cycle that drives the progression of AD [26, 27, 33]. Furthermore, inhibition/genetic ablation of S100B reduces amyloidogenesis, microgliosis, astrocytosis and tau phosphorylation in an AD mouse model [31]. S100A9 and S100A12 are inflammation-associated proteins and constitutively expressed in neutrophils as well as plaque-associated macrophages [28]. S100A9 ablation reverses memory deficits and plaque pathology in an AD mouse model [32]. S100A6 is upregulated in astrocytes that surround plaques in human AD autopsy specimens and AD mouse models [29]. S100A7, originally characterized as a marker for psoriasis [34], is present in the cerebrospinal fluid of AD patients and beneficially promotes non-amyloidogenic processing of the amyloid precursor protein (APP) [30]. While S100 family members have similar phenotypic effects in AD, their net effect (beneficial versus detrimental) and molecular mechanisms of action differ.
Page 4
Page 4 of 39
Many of the remaining S100 family members regulate cellular processes and in vitro target proteins that contribute to AD, but their direct involvement in AD has not been investigated. S100A1 is expressed primarily in neurons and regulates APP expression, tau
ip t
phosphorylation, neuronal cell sensitivity to A, dendrite/synapse formation, neuronal survival and Ca2+ homeostasis in neuronal cell lines [25, 35-37]. In addition, multiple in vitro S100A1-
cr
target proteins (ryanodine receptor (RyR), the microtubule-associated tau protein and the RAGE-receptor) exhibit altered function in AD [9, 10, 37, 38]. This study uses a genetic
us
approach to investigate S100A1's contribution to AD pathobiology in the PSAPP mouse model.
an
Although no AD mouse model exhibits all aspects of the human disease, the bigenic PSAPP (APPK670NM671L/PS-1M146L) mouse model mimics many facets of the human disease including A
M
plaque deposition, dystrophic neurites, glial activation, and memory deficits [39-43]. PSAPP/S100A1 knockout mice exhibited reductions in cortical and hippocampal inflammation
d
(astrocytosis and microgliosis) that were accompanied by decreases in plaque load/number.
te
Intracellular and plaque-associated S100A1 staining was observed in murine PSAPP and human AD specimens, and these observations are consistent with a detrimental gain of S100A1
Ac ce p
signaling in AD. Plaque associated PO4-Akt and PO4-GSK3 staining was also reduced while non-plaque associated staining was increased. Nonetheless, S100A1Akt complexes were not detectable in PC12 cells or AD brain tissue even though Akt isoforms contain an S100-target protein binding motif, suggesting that S100A1 indirectly modulates Akt activity. S100A1RyR complexes were detectable in AD brain tissue and exhibited Ca2+-dependent formation in neuronal cells. This is the first demonstration of an S100A1 target protein complex in tissue/intact cells and identifies the RyR as an S100A1 target protein in AD/neuronal cells. Collectively, these results suggest that pharmacological strategies which selectively block S100A1 action in the CNS may normalize aberrant RyR function and delay the progression of AD.
Page 5
Page 5 of 39
2. METHODS 2.1 PSAPP X S100A1 knockout mice. PSAPP/S100A1-/- and PSAPP/S100A1+/+ mice were
ip t
generated by crossing PSAPP males [39-42] with S100A1-/- knockout females [9] and subsequent intercrossing of the resulting PSAPP/S100A1+/- offspring. Genotyping was
cr
performed as previously described [9, 31]. To control for variability in genetic background, all experiments used PSAPP/S100A1+/+ and PSAPP/S100A1-/- littermates (B6.129SJL ) from F1
us
PSAPP/S100A1+/- crosses. All experiments involving animals were approved by the Institutional
an
Animal Care and Use Committee and comply with the NIH Guide for the Care and Use of Laboratory Animals.
M
2.2 Sample acquisition/processing. Brains were removed from anesthetized animals and processed as previously described [31]. Briefly, specimens were rinsed in phosphate buffered
d
saline (PBS), fixed in 4% (wt/vol) paraformaldehyde in PBS for 30 minutes and sliced into 2 mm
te
sagittal sections. After a second 30 minute fixation in 4% (wt/vol) paraformaldehyde in PBS, slices were permeabilized (2mM MgCl2, 0.01% (wt/vol) sodium deoxycholate, 0.02% (vol/vol)
Ac ce p
Nonidet P-40 in 100mM sodium phosphate buffer pH 7.5), post-fixed in 10% buffered formalin for 16 hours and embedded in paraffin. Five micron sagittal sections were mounted on glass slides for subsequent staining. Sections from human AD and control brain were obtained from the Golden Brain Bank (Coatesville, PA). 2.3 Light microscopy. GFAP (astrocytosis), Iba1 (microgliosis), Congo-red (plaque load) and S100A1 staining was performed as previously described [31]. Primary antibodies included a rabbit GFAP antibody (1-1000 dilution, Dako, Carpinteria, CA), mouse Iba1 antibody (1-300 dilution, Santa Cruz Biotechnology) and mouse S100A1 antibody (1-50 dilution of SH-A1, Santa Cruz Biotechnology). For quantification, digital images were converted to gray scale, and positive pixels, plaque size, plaque number and total plaque load quantified using Image J software (NIH Image, Bethesda, MD). Plaque load and immunoreactivity were defined as the %
Page 6
Page 6 of 39
area, i.e. the area of positive pixels/total pixels X 100. The data were expressed as the mean + SEM and a multicomparison ANOVA with Tukey’s HSD post hoc test or an independent samples t-test (JMP by SAS, Cary, NC) was used to determine the significance (p3) were captured with a 20x objective on a Zeiss Axio
Ac ce p
Observer.D1 microscope equipped with Zen 2012 software (Carl Zeiss Group, Germany) using uniform hardware and software settings. 2.5 Cell Culture and immunocytochemistry. PC12 cells expressing normal (PC12) and reduced levels of S100A1 (PC12S100A1-/-) were grown as previously described [36]. To activate Akt signaling, cells were serum-starved for 16 hours prior to short-term (20 min) treatment with Nerve Growth Factor (NGF) (Millipore, Temecula, CA). Total Akt (1-50 dilution of #9272, Cell Signaling Technology), PO4-Akt(Ser 473) (1-300 dilution of #4051 Cell Signaling Technology), PO4-Akt(Thr 308) (1-25 dilution of #4056, Cell Signaling Technology) and PO4-GSK3(Ser 9) (1100 dilution of #9336, Cell Signaling Technology) staining was visualized with a donkey antirabbit Alexa 488 (Invitrogen, Carlsbad, CA). Digital images from independent experiments (n=3-
Page 7
Page 7 of 39
6) were captured with a 63X oil objective and Zeiss Axio Observer.D1 microscope equipped with Zen 2012 software (Carl Zeiss Group, Germany). 2.6 Cell-based ELISAs. PO4-Akt(Ser473) and PO4-GSK3(Ser9) levels were quantified using
ip t
commercially available cell-based ELISA kit, FACE™ kits (Active Motif, Carlsbad, CA) as instructed by the manufacturer in triplicate. After normalization to the number of cells/well
cr
(crystal violet staining), the data were expressed as the mean ratio of NGF-treated
cells/untreated cells + the SEM (n= 4-5) and a t-test (GraphPad Prism, La Jolla, CA) was used
us
to determine the significance (p
S0143-4160(14)00082-7 http://dx.doi.org/doi:10.1016/j.ceca.2014.05.002 YCECA 1566
To appear in:
Cell Calcium
Received date: Revised date: Accepted date:
7-10-2013 15-5-2014 16-5-2014
Please cite this article as: L. Afanador, E.A. Roltsch, L.H. K.S. Campbell, D.A. Keeling, Y. Zhang, D.B. Zimmer, The Ca2+ Sensor S100A1 Modulates Neuroinflammation, Histopathology and Akt Activity in the, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The Ca2+ Sensor S100A1 Modulates Neuroinflammation, Histopathology and Akt Activity in the
cr
ip t
PSAPP Alzheimer's Disease Mouse Model
us
Lauriaselle Afanador*, Emily A. Roltsch†, Leigh Holcomb,+#,Kerry S. Campbell*, David A. Keeling*, Yan Zhang*, and Danna B. Zimmer*,a
an
*Center for Biomolecular Therapeutics, Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA and +
te
d
M
Psychiatry and Behavioral Science, Texas A&M Health Science Center, College of Medicine, and Central Texas Veterans Health Care System Neuropsychiatry Research Program, Temple, TX, USA
Ac ce p
Current address: †LSU Health Science Center, School of Medicine, 1901 Perdido St., New Orleans, LA, 70112, USA; and #Genzyme, Inc., 500 Kendall Street, Cambridge, MA 02142
TITLE RUNNING HEAD: S100A1 Modulates AD Pathobiology
a
To whom correspondence should be addressed: Danna B. Zimmer, Ph.D. Center for Biomolecular Therapeutics Department of Biochemistry & Molecular Biology University of Maryland School of Medicine 9600 Gudelsky Drive Rockville, MD 20850 Voice: 240.314.6514 Email: [email protected]
Page 1
Page 1 of 39
SUMMARY The contribution of the Ca2+ sensor S100A1 to in vivo Alzheimer's disease (AD) pathobiology has not been elucidated although S100A1 regulates numerous cellular processes
ip t
linked to AD. This study uses genetic ablation to ascertain the effects of S100A1 on neuroinflammation, beta-amyloid (A) plaque deposition and Akt activity in the PSAPP AD
cr
mouse model. PSAPP/S100A1-/- mice exhibited decreases in astrocytosis (GFAP burden),
microgliosis (Iba1 burden) and plaque load/number when compared to PSAPP/S100A1+/+ mice
us
at six and twelve months of age. The presence of detectable S100A1 staining in human AD
an
specimens is consistent with a detrimental gain of S100A1 function in AD. S100A1 ablation also reduced plaque associated and increased non-plaque associated PO4-Akt and PO4-GSK3
M
staining. S100A1Akt complexes were undetectable in PC12 cells and AD brain tissue suggesting that S100A1 indirectly modulates Akt activity. In contrast, S100A1RyR (ryanodine
d
receptor) complexes were present in human/mouse AD brain and exhibited Ca2+-dependent
te
formation in neuronal cells. This is the first direct demonstration of an S100A1 target protein complex in tissue/cells and identifies the RyR as a primary S100A1 target protein in the brain.
Ac ce p
Collectively, these data suggest that S100A1 inhibition may be a novel strategy for normalizing aberrant Ca2+ signaling in AD.
Keywords: astrocytosis, microgliosis, plaque load, neurodegeneration, GSK3, ryanodine receptor, S100B, PC12 cells
Page 2
Page 2 of 39
1. INTRODUCTION Disruption of calcium (Ca2+) homeostasis is a common event in many diseases [1, 2]. Physiological and pathological Ca2+ signals are transduced into biological responses by
ip t
members of the calmodulin/troponin/S100 superfamily of Ca2+ sensor proteins. Upon binding
cr
Ca2+, Ca2+sensor proteins undergo a large conformational change ("Ca2+ switch") that exposes a hydrophobic cleft required for interaction with their target proteins and subsequent exertion of
us
their biological effects [3]. S100s are found exclusively in vertebrates, and are distinguished from other Ca2+ sensors by their 3D structure and a highly conserved amino terminal Ca2+
an
binding loop consisting of 14 rather than the typical 12 amino acids [4, 5]. The human genome encodes 21 S100 proteins, four (S100B, S100G, S100P and S100Z) are singletons dispersed
M
throughout the genome and the remaining seventeen (S100A1-S100A16) are located within the epidermal differentiation complex (EDC) on human chromosome 1[4]. The term S100 was used
d
in the early literature to denote a mixture of the two founding family members, S100A1 and
Ac ce p
ammonium sulfate [6].
te
S100B, and refers to the solubility of these approximately 10,000 Da proteins in 100% saturated
Although S100 family members exhibit a high degree of structural similarity, they are not functionally interchangeable. With the exception of S100G, which is monomeric, S100 proteins are typically symmetric dimers [3]. Nonetheless, individual family members exhibit unique affinities for divalent metal ions (Ca2+, Zn2+, Cu2+), oligomerization properties, post-translational modifications and spatial/temporal expression patterns. S100s bind to and regulate the activity of a large number of target proteins, some of which are regulated by a single family member and others by multiple family members. Individual family members also exhibit unique binding orientations for target proteins that are due in part to differences in surface charge density [7, 8]. S100s can also compete with other Ca2+ sensors such as calmodulin for the same binding site on target proteins [9, 10]. S100s are also found in the extracellular space where they interact
Page 3
Page 3 of 39
with a variety of cell surface receptors including the Receptor for Advanced Glycation Endproducts (RAGE), G-protein coupled receptors, toll-like receptor-4, scavenger receptors, FGFreceptor 1, dopamine 2 receptor, ALCAM and bioactive spingolipids [11-14] . S100 proteins
ip t
lack a signal peptide for secretion via the conventional Golgi-mediated pathway, and there is debate as to whether extracellular S100s are actively secreted or passively released from
cr
live/necrotic cells. The diversity among S100s when coupled with their partial functional
redundancy and the unique complement of family members expressed in each cell, allows
us
individual cells to generate unique and adaptive responses to equivalent changes in intracellular Ca2+ levels.
an
Dysregulation of Ca2+ homeostasis and neuroinflammation precede the appearance of
M
toxic A peptide oligomers/plaques and hyperphosphorylated tau/neurofibrillary tangles in familial and sporadic Alzheimer's disease (AD) [15-24]. Studies using clinical specimens and animal models have implicated five S100 family members (S100A6, S100A7, S100A9, S100A12
te
d
and S100B) in aberrant Ca2+ signaling in AD [25-32]. S100B is expressed primarily in astrocytes/microglia and has been postulated to be a part of a detrimental autocatalytic cytokine
Ac ce p
cycle that drives the progression of AD [26, 27, 33]. Furthermore, inhibition/genetic ablation of S100B reduces amyloidogenesis, microgliosis, astrocytosis and tau phosphorylation in an AD mouse model [31]. S100A9 and S100A12 are inflammation-associated proteins and constitutively expressed in neutrophils as well as plaque-associated macrophages [28]. S100A9 ablation reverses memory deficits and plaque pathology in an AD mouse model [32]. S100A6 is upregulated in astrocytes that surround plaques in human AD autopsy specimens and AD mouse models [29]. S100A7, originally characterized as a marker for psoriasis [34], is present in the cerebrospinal fluid of AD patients and beneficially promotes non-amyloidogenic processing of the amyloid precursor protein (APP) [30]. While S100 family members have similar phenotypic effects in AD, their net effect (beneficial versus detrimental) and molecular mechanisms of action differ.
Page 4
Page 4 of 39
Many of the remaining S100 family members regulate cellular processes and in vitro target proteins that contribute to AD, but their direct involvement in AD has not been investigated. S100A1 is expressed primarily in neurons and regulates APP expression, tau
ip t
phosphorylation, neuronal cell sensitivity to A, dendrite/synapse formation, neuronal survival and Ca2+ homeostasis in neuronal cell lines [25, 35-37]. In addition, multiple in vitro S100A1-
cr
target proteins (ryanodine receptor (RyR), the microtubule-associated tau protein and the RAGE-receptor) exhibit altered function in AD [9, 10, 37, 38]. This study uses a genetic
us
approach to investigate S100A1's contribution to AD pathobiology in the PSAPP mouse model.
an
Although no AD mouse model exhibits all aspects of the human disease, the bigenic PSAPP (APPK670NM671L/PS-1M146L) mouse model mimics many facets of the human disease including A
M
plaque deposition, dystrophic neurites, glial activation, and memory deficits [39-43]. PSAPP/S100A1 knockout mice exhibited reductions in cortical and hippocampal inflammation
d
(astrocytosis and microgliosis) that were accompanied by decreases in plaque load/number.
te
Intracellular and plaque-associated S100A1 staining was observed in murine PSAPP and human AD specimens, and these observations are consistent with a detrimental gain of S100A1
Ac ce p
signaling in AD. Plaque associated PO4-Akt and PO4-GSK3 staining was also reduced while non-plaque associated staining was increased. Nonetheless, S100A1Akt complexes were not detectable in PC12 cells or AD brain tissue even though Akt isoforms contain an S100-target protein binding motif, suggesting that S100A1 indirectly modulates Akt activity. S100A1RyR complexes were detectable in AD brain tissue and exhibited Ca2+-dependent formation in neuronal cells. This is the first demonstration of an S100A1 target protein complex in tissue/intact cells and identifies the RyR as an S100A1 target protein in AD/neuronal cells. Collectively, these results suggest that pharmacological strategies which selectively block S100A1 action in the CNS may normalize aberrant RyR function and delay the progression of AD.
Page 5
Page 5 of 39
2. METHODS 2.1 PSAPP X S100A1 knockout mice. PSAPP/S100A1-/- and PSAPP/S100A1+/+ mice were
ip t
generated by crossing PSAPP males [39-42] with S100A1-/- knockout females [9] and subsequent intercrossing of the resulting PSAPP/S100A1+/- offspring. Genotyping was
cr
performed as previously described [9, 31]. To control for variability in genetic background, all experiments used PSAPP/S100A1+/+ and PSAPP/S100A1-/- littermates (B6.129SJL ) from F1
us
PSAPP/S100A1+/- crosses. All experiments involving animals were approved by the Institutional
an
Animal Care and Use Committee and comply with the NIH Guide for the Care and Use of Laboratory Animals.
M
2.2 Sample acquisition/processing. Brains were removed from anesthetized animals and processed as previously described [31]. Briefly, specimens were rinsed in phosphate buffered
d
saline (PBS), fixed in 4% (wt/vol) paraformaldehyde in PBS for 30 minutes and sliced into 2 mm
te
sagittal sections. After a second 30 minute fixation in 4% (wt/vol) paraformaldehyde in PBS, slices were permeabilized (2mM MgCl2, 0.01% (wt/vol) sodium deoxycholate, 0.02% (vol/vol)
Ac ce p
Nonidet P-40 in 100mM sodium phosphate buffer pH 7.5), post-fixed in 10% buffered formalin for 16 hours and embedded in paraffin. Five micron sagittal sections were mounted on glass slides for subsequent staining. Sections from human AD and control brain were obtained from the Golden Brain Bank (Coatesville, PA). 2.3 Light microscopy. GFAP (astrocytosis), Iba1 (microgliosis), Congo-red (plaque load) and S100A1 staining was performed as previously described [31]. Primary antibodies included a rabbit GFAP antibody (1-1000 dilution, Dako, Carpinteria, CA), mouse Iba1 antibody (1-300 dilution, Santa Cruz Biotechnology) and mouse S100A1 antibody (1-50 dilution of SH-A1, Santa Cruz Biotechnology). For quantification, digital images were converted to gray scale, and positive pixels, plaque size, plaque number and total plaque load quantified using Image J software (NIH Image, Bethesda, MD). Plaque load and immunoreactivity were defined as the %
Page 6
Page 6 of 39
area, i.e. the area of positive pixels/total pixels X 100. The data were expressed as the mean + SEM and a multicomparison ANOVA with Tukey’s HSD post hoc test or an independent samples t-test (JMP by SAS, Cary, NC) was used to determine the significance (p3) were captured with a 20x objective on a Zeiss Axio
Ac ce p
Observer.D1 microscope equipped with Zen 2012 software (Carl Zeiss Group, Germany) using uniform hardware and software settings. 2.5 Cell Culture and immunocytochemistry. PC12 cells expressing normal (PC12) and reduced levels of S100A1 (PC12S100A1-/-) were grown as previously described [36]. To activate Akt signaling, cells were serum-starved for 16 hours prior to short-term (20 min) treatment with Nerve Growth Factor (NGF) (Millipore, Temecula, CA). Total Akt (1-50 dilution of #9272, Cell Signaling Technology), PO4-Akt(Ser 473) (1-300 dilution of #4051 Cell Signaling Technology), PO4-Akt(Thr 308) (1-25 dilution of #4056, Cell Signaling Technology) and PO4-GSK3(Ser 9) (1100 dilution of #9336, Cell Signaling Technology) staining was visualized with a donkey antirabbit Alexa 488 (Invitrogen, Carlsbad, CA). Digital images from independent experiments (n=3-
Page 7
Page 7 of 39
6) were captured with a 63X oil objective and Zeiss Axio Observer.D1 microscope equipped with Zen 2012 software (Carl Zeiss Group, Germany). 2.6 Cell-based ELISAs. PO4-Akt(Ser473) and PO4-GSK3(Ser9) levels were quantified using
ip t
commercially available cell-based ELISA kit, FACE™ kits (Active Motif, Carlsbad, CA) as instructed by the manufacturer in triplicate. After normalization to the number of cells/well
cr
(crystal violet staining), the data were expressed as the mean ratio of NGF-treated
cells/untreated cells + the SEM (n= 4-5) and a t-test (GraphPad Prism, La Jolla, CA) was used
us
to determine the significance (p
