inflammation in experimental models of alzheimer’s disease ...sunnyvale, ca) in human sk-n-sh...

Small molecule modulator of sigma 2 receptor is neuroprotective and reduces cognitive deficits and neuro-inflammation in experimental models of Alzheimer’s disease

Bitna Yia,#, James J. Sahnb,#, Pooneh Memar Ardestania,#, Andrew K. Evansa, Luisa Scottc, Jessica Z. Chanb, Sangeetha Iyerc, Ashley Crispc, Gabriella Zunigac, Jonathan Pierce-Shimomurac, Stephen F. Martinb, and Mehrdad Shamlooa

aStanford University School of Medicine, Department of Neurosurgery, 1050 Arastradero Road, Building A, Palo Alto, CA 94304

bDepartment of Chemistry, The University of Texas at Austin, 105 E. 24th Street, Stop A5300, Austin, TX 78712, USA

cWaggoner Center for Alcohol and Addiction Research, Institute of Neuroscience, Center for Learning and Memory, Center for Brain, Behavior, and Evolution, and Department of Neuroscience, The University of Texas at Austin, Texas 78712

Abstract

Accumulating evidence suggests that modulating the sigma 2 receptor (Sig2R) can provide

beneficial effects for neurodegenerative diseases. Herein, we report the identification of a novel

class of Sig2R binding ligands and their cellular and in vivo activity in experimental models of

Alzheimer’s disease (AD). We report that SAS-0132 and DKR-1051, selective ligands of Sig2R,

modulate intracellular Ca2+ levels in human SK-N-SH neuroblastoma cells. The Sig2R antagonists

SAS-0132 and JVW-1009 are neuroprotective in a C. elegans model of amyloid precursor protein-

mediated neurodegeneration. Since this neuroprotective effect is replicated by genetic knockdown

and knockout of vem-1, the ortholog of progesterone receptor membrane component-1

(PGRMC1), it indicates that Sig2R ligands modulate a PGRMC1-related pathway. Last, we

demonstrate that SAS-0132 improves cognitive performance both in the Thy-1 hAPPLond/Swe+

transgenic mouse model of AD and in healthy wild-type mice. These results demonstrate that

Sig2R is a promising therapeutic target for neurocognitive disorders including AD.

Keywords

Alzheimer’s disease; AD; sigma 1 receptor; Sig1R; sigma 2 receptor; Sig2R; progesterone receptor membrane component-1; PGRMC1

*Corresponding Author: Mehrdad Shamloo, Stanford University School of Medicine, Department of Neurosurgery, 1050 Arastradero Road, Building A, Palo Alto, CA 94304. Tel: (650) 725-3152; Fax: (650) 723-4147; [email protected].#Indication of co-first author status

DisclosuresThe authors have no conflicts of interest to declare.

HHS Public AccessAuthor manuscriptJ Neurochem. Author manuscript; available in PMC 2017 February 16.

Published in final edited form as:J Neurochem. 2017 February ; 140(4): 561–575. doi:10.1111/jnc.13917.

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INTRODUCTION

Alzheimer’s disease (AD) is currently the sixth leading cause of death in the US (Reitz &

Mayeux 2014, James et al. 2014). The hallmarks of AD include neuronal loss,

neuroinflammation, and the accumulation of amyloid plaques and neurofibrillary tangles

(Mattson 2004, Citron 2010, Huang & Mucke 2012). The multifaceted pathology of AD

makes it difficult to develop effective treatments. Current therapies for AD rely upon

transiently mitigating some of its symptoms. However, these medications do not alter

pathology underlying the disease (Cummings et al. 2014), and there remains an unmet need

for therapeutic approaches to treat AD.

It is notable that modulating sigma receptors, for which the two known subtypes are sigma 1

(Sig1R) and sigma 2 (Sig2R) (Nguyen et al. 2014a, Matsumoto et al. 2007), has only

recently been explored as a possible strategy for treating neurological disorders. Both sigma

receptor subtypes are expressed in the central nervous system (CNS) and distinguished from

one another based upon their affinity for different ligands and biological profiles. Sig1Rs,

which have been cloned, are chaperone proteins that reside in the endoplasmic reticulum-

mitochondrion interface as well as in nuclear and plasma membranes (Su et al. 2010,

Hashimoto 2013, Su et al. 2016); the structure of a trimer of Sig1R has been characterized

by X-ray crystallography (Schmidt et al. 2016). Although Sig2Rs have not been cloned, they

are widely distributed in the CNS and are implicated in cellular processes relevant to cancer

and CNS disorders (Bowen 2000, Mach et al. 2013, Huang et al. 2014, Guo & Zhen 2015).

Recent photo affinity and fluorescent-probe experiments suggest that the putative Sig2R

binding site is the progesterone receptor membrane component-1(PGRMC1) protein

complex (Xu et al. 2011). PGRMC1 is a cytochrome-like single-transmembrane protein that

has been structurally characterized as a heme-bound dimer (Kabe et al. 2016) and is

involved in neuroprotection and axonal migration (Rohe et al. 2009, Runko & Kaprielian

2004, Cahill 2007). However, other reports conclude PGRMC1 and Sig2R are distinct

molecular entities (Abate et al. 2015, Chu et al. 2015). These two views are not necessarily

mutually exclusive, and further work is needed to resolve this issue. Notwithstanding this

controversy, we originally identified the compounds described herein as Sig2R binding

ligands and found they modulate a PGRMC1-related pathway, thus we will refer to the

biological target by the descriptor Sig2R/PGRMC1.

We became interested in Sig2R/PGRMC1 several years ago as a possible target for drug

discovery in neurodegenerative diseases because of its widespread occurrence in the CNS

and its putative involvement in calcium homeostasis. Within the CNS, Sig2R/PGRMC1 is

highly expressed in the cortex and hippocampus, two regions known to be important for

cognitive function (Izzo et al. 2014b, French & Pavlidis 2011). Moreover, Sig2R/PGRMC1

signaling has been implicated in cellular processes relevant to CNS diseases, including

neuroprotection, axonal migration, and mitochondrial protection (Cahill 2007, Qin et al. 2015). For example, progesterone, which binds to both Sig2R and PGRMC1 (Chu et al. 2015), exerts neurogenic and neuroprotective effects in neural progenitor cells derived from

adult rat brain (Liu et al. 2009). In the context of AD-related pathology, afobazole, which is

a pan-selective Sig1R-Sig2R ligand, reduces neurotoxic microglia stimulation and apoptosis

induced by fragments of amyloid beta (Aβ) (Behensky et al. 2013). Recent work conducted

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by Cognition Therapeutics also show that Aβ oligomers are Sig2R/PGRMC1 ligands and

that blocking the binding of these oligomers to Sig2R/PGRMC1 mitigates the synaptotoxic

effects of Aβ oligomers (Izzo et al. 2014a, Izzo et al. 2014b). Moreover, exposure of

cultured neurons to Aβ oligomers produces progressive upregulation of Sig2R/PGRMC1

(Izzo et al. 2014b). This study also revealed that density of Sig2R/PGRMC1 remains

unchanged in severely demented AD patients compared to the age-matched normal

individuals despite a high degree of cell loss, suggesting that Sig2R/PGRMC1 may be

upregulated in AD patients. These findings suggest Sig2R/PGRMC1 is involved in AD

pathology. Thus, we hypothesized that ligands that modulate Sig2R/PGRMC1 may hold

significant promise as medicinal agents to treat AD.

As part of a broad effort to identify novel compounds with possible applications in

neuroscience (Sahn et al. 2014, Martin 2013), we screened collections of substituted

heterocyclic compounds against a variety of CNS proteins in the Psychoactive Drug

Screening Program (PDSP) (Besnard et al. 2012). We thus identified a subclass of

substituted norbenzomorphans that bind to Sig2R with high affinity (Sahn & Martin 2012,

Sahn et al. 2016). This is an important discovery because norbenzomorphans have compact

molecular scaffolds and may be easily modified for structure-activity-relationship studies.

Moreover, they are structurally distinct from all other known Sig2R binding ligands (Mach,

Zeng, and Hawkins 2013; Huang et al. 2014; Guo and Zhen 2015). Of the initial set of

compounds, SAS-0132 emerged as an attractive candidate for further study because it

exhibits 9-fold selectivity for Sig2R over Sig1R, and has low off-target affinity for most

other CNS-relevant targets. With this newly identified Sig2R/PGRMC1 ligand in our hands

as a pharmacological tool, we tested our hypothesis that modulation of Sig2R/PGRMC1

might lead to beneficial effects in the treatment of AD.

Here, we report that the Sig2R/PGRMC1 antagonist SAS-132 produces neuroprotective,

cognitive enhancing, and anti-inflammatory effects in animal models of neurodegeneration.

These results suggest that Sig2R/PGRMC1 represents a promising target to treat

neurodegenerative diseases.

MATERIALS AND METHODS

Intracellular calcium assay

Calcium responses were measured using a Calcium 6-QF assay kit (Molecular Devices,

Sunnyvale, CA) in human SK-N-SH neuroblastoma cells as described in the Appendix

supplemental information. Effects of DKR-1051 or SAS-0132 were tested at final

concentrations of 0.3, 3, 10, 30, or 100 µM. Effects of SAS-0132 on the calcium response

induced by DKR-1051 (100 µM) were tested at final concentrations of 0.1, 0.3, 3, 10, or 30

µM.

C. elegans strains, maintenance, and transgenesis

C. elegans were grown and maintained according to the standard method (Brenner 1974).

Transgenic animals were generated through the MOSSCI technique as previously described

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(Frokjaer-Jensen et al. 2008). Details are described in the Appendix supplemental

information.

C. elegans pharmacological and RNAi treatments

For pharmacological treatment alone, plates contained either vehicle or compound dissolved

in DMSO (final concentration of 200 µM and 0.2% DMSO). For pharmacological and RNAi

co-treatment, the vem-1 (C. elegans homolog of PGRMC1) RNAi colony was selected from

the Ahringer library (Genesequence) and grown overnight in LB with 50 µg/mL of

carbenicillin. After 24 h, this culture was seeded onto carbenicllin-containing NGM-agar

plates with or without 1 mM isopropylthiogalactoside (IPTG) for RNAi treatment. These

plates also contained either vehicle or the Sig2R/PGRMC1 ligands dissolved in DMSO

(final concentration of 200 µM and 0.2% DMSO). Plates that contained vehicle but did not

contain IPTG were considered as sham control plates. The RNAi bacteria were induced

overnight at room temperature for dsRNA expression.

C. elegans neurotoxicity assay

Well-fed, age-synchronized larvae 4 (L4) were picked onto seeded plates with the sterility

compound FUdR (0.12 mM) to prevent progeny from hatching. Larvae were then allowed to

age at 20°C on seeded plates for ~24 h (D1 adult), ~72 h (D3 adult) or ~116 h (D5 adult) to

assess age-related neurodegeneration. Animals were immobilized on 2% agar pads and

scored for neuronal health at 40× within 1 h of 0.7 mM sodium azide treatment. GFP-filled

neurons were scored for neuronal health by an observer blind to condition, and were

considered degenerated if they displayed shrinking and/or dimming of the soma (see Figure

3B: the VC5 neuron in the D3 animal is considered degenerated). The same observer scored

neurodegeneration in each dataset. The percentage degeneration of VC4 and VC5 neurons

was measured out of the total number of neurons scored (>60 neurons). The percentage that

succumbed to APP-induced degeneration was compared for different groups (strain, drug

condition) using planned Fisher’s exact tests (Zar 1999).

Experimental designs for in vivo studies

All animals were single-housed at the start of drug administration and kept under a reverse

light-dark cycle with lights off at 8:30 AM and on at 8:30 PM in a temperature- and

humidity-controlled environment and given food and water ad libitum. All experiments were

in accordance with protocols approved by the Stanford University Administrative Panel for

Laboratory Animal Care and conformed to the U.S. National Institutes of Health Guide for

the Care and Use of Laboratory Animals. For pharmacokinetic (PK) study, male C57Bl/6J

mice at 2–3 month age were used (Jackson Laboratory). For efficacy studies, male Thy1-

hAPPLond/Swe+ (APPLond/Swe+) and their age-matched (4.5 to 6.7 month-old) wild-type

(WT) littermates were used as a mouse model of AD (Rockenstein et al. 2001). This

transgenic line was maintained by crossing heterozygous Thy1-hAPPLond/Swe+ mice with

C57BL/6J breeders. The genotype of all mice was determined by PCR using tail-tip DNA.

For the PK study, a total of 9 C57Bl/6J mice (n=3 per route) were used. For the efficacy

studies, two separate cohorts of APPLond/Swe+ mice were used in two independent studies. In

study I, a total of 43 mice were used in 4 experimental groups (n=9–12 mice per group) to

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assess the chronic daily dosing effect of SAS-0132 at 10mg/kg. In study II, a total of 38

mice were used in 8 experimental groups (n=4–5 mice per group) to assess the dose-

response effects of chronic daily dosing of SAS-0132 at 3, 10, and 30mg/kg. Since the

maximum feasible number of mice to be tested during a testing day is 35–45, we had to

reduce the group size to 4–5 animals per group in the dose response study. For details, see

Supplemental table 1.

PK study

Mice were dosed with 10 mg/kg of SAS-0132 intravenously, subcutaneously, or via oral

gavage. Retro-orbital blood samples were collected before drug administration, and 5 min,

30 min, 1 h after drug administration. Three hours post-dose, mice were sacrificed, and

blood samples were collected by cardiac puncture. Subsequently, brains were collected after

perfusion with PBS. Plasma was immediately separated by centrifugation and stored at

−80°C until analysis. The brain tissue samples were homogenized using distilled water and

the homogenates were stored at −80°C until analysis. The concentration of SAS-0132 was

then determined using LC/MS. The bioavailability was calculated as the ratio of

subcutaneous or oral to intravenous area under the curve calculated up to the last measured

time point (AUC(0–3h)).

Drug Treatment and Behavioral Testing

SAS-0132 (purity > 95%; HPLC) was synthesized as previously described (Sahn et al.

2016). For the in vivo studies, solutions of SAS-0132 were freshly prepared daily before

administration as described in the Appendix supplemental information. In the in vivo efficacy studies, APPLond/Swe+ and WT littermates were pseudo-randomly assigned to

vehicle or drug groups based on the body weight and the locomotor activity determined in

the pre-drug administration activity chamber test so that experimental groups were balanced

for the body weight and locomotor activity. In the chronic daily dosing study, APPLond/Swe+

and WT mice were administered vehicle or SAS-0132 at a dose of 10 mg/kg once a day for

64 days. In the chronic daily dosing dose-response study, APPLond/Swe+ and WT mice were

administered vehicle or SAS-0132 at doses of 3, 10, or 30 mg/kg daily for 60 days. Our

previous studies have shown 4 weeks of treatment is sufficient to detect chronic effects of

drugs with a diverse range of mechanisms in this mouse model (Nguyen et al. 2014b). Thus,

all animals from the in vivo study cohorts were evaluated in a battery of cognitive and

behavioral tests in the following order during the animal’s active cycle: Activity chamber,

Social discrimination, Morris Water Maze (MWM, only in the dose-response study), and Y-

maze tests as previously described during the last 4–9 weeks of drug administration

(Coutellier et al. 2014, Faizi et al. 2012). See the Appendix supplemental information for

behavioral testing procedure. All behavior tests were performed blindly by investigators

being blind to the treatment groups and genotypes. More specifically, the animals were

housed singly and were coded with animal IDs corresponding to the earmarks. All data

collections were done using these codes and the data were processed and analyzed blindly

based on group codes independent of treatment and genotype. The dosing was performed by

dosing personnel who were not collecting experimental data from the animals. All results

were re-analyzed and confirmed by a second investigator who was unware of the

experimental groups. In both efficacy studies, mice were tested in two cohorts divided

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between morning and afternoon sessions with treatment groups balanced between sessions.

SAS-0132 was subcutaneously administered in the late afternoon except on those days

behavioral testing occurred in which mice were dosed after testing to minimize acute effects

of the drug administration on behavior. No exclusion was made for the behavior test

analyses with the exception of MWM. In MWM, a total of 6 mice from WT-vehicle,

APPLond/Swe+-3 mg/kg, APPLond/Swe+-10 mg/kg, and APPLond/Swe+-30 mg/kg groups were

excluded based on extensive floating and swimming inability.

Tissue collection and biochemical analysis

After the behavioral testing was completed, mice were sacrificed and perfused with PBS,

and the brains were collected. The brains were sagittally bisected; half of the brain was

frozen and stored at −80°C until use; the other half was placed overnight in 4% formalin and

transferred and kept at 4°C in 30% sucrose until processed. To determine if chronic

administration of SAS-0132 leads to any toxic effects, blood and organs were collected and

used for blood chemistry and histopathology studies. To determine the effects of SAS-0132

on AD-related pathology, quantitative RT-PCR (qRT-PCR), ELISA and

immunohistochemical analyses were performed following the previously reported methods

(Schmittgen & Livak 2008, Maier et al. 2008, Johnson-Wood et al. 1997). Details are

described in Appendix supplemental information.

Statistics

In order to obtain 80% power in our in vivo efficacy studies, we conducted a power-analysis

using our historical data to detect a statistically significant difference of 20% in one single

behavioral task and determined to use 9–12 mice per group when feasible.

Statistical analyses were performed with the IBM SPSS Statistical Package 22.0 and Prism

5.0 (GraphPad Software). The calcium assay and qRT-PCR data were analyzed by one-way

analyses of variance (ANOVA), followed by Dunnett’s test for post-hoc analysis. C. elegans neurodegeneration data analyses were performed with Fisher's exact tests. Behavioral and

biochemical data analyses were performed with either t-test (Y-maze and social

discrimination tests, immunohistochemistry data), repeated measures of ANOVA, or

ANOVA followed by Fisher’s LSD or Dunnett’s post-hoc analyses (MWM). In all cases,

outliers were excluded according to Grubbs’ test and p < 0.05 was considered to be

significant.

RESULTS

Biological properties of SAS-0132 and related Sig2R/PGRMC1 binding ligands in vitro

Sigma receptor binding affinities of some norbenzomorphans—SAS-0132

(Figure 1) is a member of a novel family of Sig2R binding ligands with a 9-fold preference

for Sig2R (Ki 90 nM) over Sig1R (Ki 841 nM) (Sahn et al. 2016). It also exhibits low off-

target affinity (Ki > ~0.5 to 10 µM) for most CNS-relevant targets, including opioid,

adrenergic, nACR, mAChR, and NMDA receptors, as well as the neurotransmitter

transporters and ion channels (Supplemental table 2). Two structurally related compounds,

DKR-1005 and DKR-1051 also show similar degree of selectivity for Sig2R over Sig1R,

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whereas another structural analog JVW-1009 is approximately equipotent for both SigR

subtypes (Figure 1).

Effects of Sig2R binding ligands on intracellular calcium—Sig2R agonists have

been reported to modulate intracellular Ca2+ levels (Vilner & Bowen 2000, Cassano et al. 2009). Thus, we tested the effects of DKR-1051 and SAS-0132 on intracellular Ca2+ levels

in human SK-N-SH neuroblastoma cells. DKR-1051 induces concentration-dependent

increases in intracellular Ca2+ concentration, suggesting that it has agonist properties (Figure

2A). SAS-0132 fails to produce a significant calcium response at concentrations up to 30

µM, but induces significant increases in intracellular Ca2+ at 100 µM (Figure 2B). At 100

µM, the effects of SAS-0132 on intracellular Ca2+ are weaker than the effects of DKR-1051.

As SAS-0132 has no effect on intracellular Ca2+ at concentrations up to 30 µM, we

examined whether these lower concentrations of SAS-0132 can antagonize the calcium

response induced by DKR-1051. To evaluate the antagonistic effects of SAS-0132,

SAS-0132 was applied prior to adding the putative Sig2R/PGRMC1 agonist DKR-1051 in

order to allow SAS-0132 to first interact with the receptor in the absence of DKR-1051. To

maximize the signal window to detect antagonistic effects, we also used the high

concentration of DKR-1051 (i.e., 100 µM), which produces a robust calcium response.

Notably, SAS-0132 significantly attenuated the DKR-0151-induced calcium response at the

lower concentrations which are not associated with significant calcium response (Figure

2C).

Sig2R/PGRMC1 antagonists are neuroprotective—Given that Sig2R/PGRMC1

appears to be involved in neuroprotection (Izzo et al. 2014b, Cahill 2007, Rohe et al. 2009),

we queried whether any of our compounds might improve the survival of neurons in a C. elegans model of neurodegeneration. This novel transgenic model has a single copy insertion

of human amyloid precursor protein 695 (SC_APP) under a pan-neuronal promoter in

addition to the endogenous worm APP-like protein, apl-1. VC class neurons along the

ventral cord are genetically manipulated to express a fluorescent label, so neurodegeneration

in the transparent bodies of live worms can be easily monitored (Figures 3A,B).

Degeneration in two cholinergic neurons, VC4 and VC5, is characterized by shrinking

somas visible in both fluorescence and phase-contrast images, and dimming GFP-labeling

(Figure 3B). The presence of SC_APP in this worm model increases the rate of degeneration

(% degeneration out of the total number of neurons scored) of these cholinergic neurons in

an age-dependent manner (Figure 3C). Notably, neuronal degradation in the SC_APP strain

was significantly diminished by two different loss-of-function alleles of vem-1, a C. elegans ortholog of PGRMC1 (Figure 3D; Fisher’s exact test (n = 194–230), SC_APP strain vs. each

SC_APP; vem-1(null) strain, p < 0.01 and 0.001, respectively). Neurodegeneration was

similarly diminished by pharmacological modulation of VEM-1 with Sig2R/PGRMC1

binding ligands (Figure 3E). A 5-day treatment of SC_APP worms with SAS-0132 and the

structural analog JVW-1009 (200 µM) decreased neurodegeneration on day 5 of adulthood

when compared to the vehicle treated group (Figure 3E; Fisher’s exact test (n = 116–378),

SAS-0132 or JVW-1009 vs. vehicle, p < 0.01). In contrast, neurodegeneration was

exacerbated in SC_APP worms treated with DKR-1005 relative to the control group (Figure

3E; Fisher’s exact test (n = 110–378), DKR-1005 vs. vehicle, p < 0.01). Importantly,

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JVW-1009 and SAS-0132 both reduced neurodegeneration in SC_APP C. elegans but

provided no further neuroprotection in animals with vem-1 (PGRMC1) expression knocked-

down by RNAi (Figure 3F; Fisher’s exact test (n = 110–142), sham vs. RNAi, p < 0.001;

RNAi vs. JVW-1009 or double treatment, n.s., Figure 3G; Fisher’s exact test (n = 130–138),

sham vs. RNAi, p<0.01; RNAi vs. SAS-0132 or double treatment, n.s.). This suggests that

neuroprotection occurs via inhibition of a PGRMC1-mediated pathway. As the effects of the

Sig2R/PGRMC1 null mutations are redundant to the effects of JVW-1009 and SAS-0132

and contrary to the effects of DKR-1051, we have classified JVW-1009 and SAS-0132 as

Sig2R/PGRMC1 antagonists and DKR-1005 as a Sig2R/PGRMC1 agonist.

SAS-0132 is bioavailable and crosses the blood-brain barrier

The finding that the Sig2R antagonists SAS-0132 and JVW-1009 have neuroprotective

effects suggested that they might mitigate neuronal damage in neurodegenerative diseases

such as AD. Toward evaluating this intriguing possibility, we determined the PK properties

of SAS-0132. Detectable levels of SAS-0132 were measured in plasma up to 3 h post-

administration of 10 mg/kg through intravenous, subcutaneous, and oral administration

(Figure 4A). The bioavailability of SAS-0132 was 45% and 7% after subcutaneous and oral

administration, respectively (Figure 4B). Consistent with its favorable ClogD (3.5; pH=7.4),

SAS-0132 readily crosses the blood-brain-barrier and achieves a brain concentration of 3.8

µM (subcutaneous administration) and brain/plasma ratio above 7 through all routes of

administration at 3 h post-administration (Figures 4C,D). The finding that only a trace (1.6

ng/g) of SAS-0132 could be detected in brain homogenates 24 h after a single injection of

SAS-0132 (10 mg/kg, subcutaneous administration) suggests that it does not accumulate in

the brain.

SAS-0132 improves cognitive performance in APPLond/Swe+ mice

Having confirmed the bioavailability and brain permeability of SAS-0132, we assessed

whether chronic treatment with SAS-0132 might alleviate the cognitive deficits associated

with AD in an animal model of the disease. We selected the APPLond/Swe+ transgenic mouse

model, which expresses human APP751 cDNA containing the London (V717I) and Swedish

(K670M/N671L) mutations under the regulatory control of the murine Thy1 gene

(Rockenstein et al. 2001, Rockenstein et al. 2007); these animals exhibit cognitive deficits in

several behavioral tests (Nguyen et al. 2014b, Faizi et al. 2012, Coutellier et al. 2014). Two

separate cohorts of mice were used to assess chronic daily dosing and chronic daily dosing

dose-response effects of SAS-0132. Importantly, all behavioral tests were conducted prior to

administration of the daily dose of SAS-0132 when SAS-0132 from the previous day’s

dosing is expected to be cleared from the brain.

In the chronic daily dosing study, daily dosing of APPLond/Swe+ mice and their WT

littermates with SAS-0132 (10 mg/kg) was initiated at 4.5 months of age and continued for

64 days. In the sociability test performed after 35 days of dosing, vehicle-treated WT

animals showed a normal sociability by showing preference for the cup containing a mouse

over the empty cup; this preference was not significantly altered by SAS-0132 treatment

(Figure 5A, t-test, mouse versus empty cup, * p < 0.05). In contrast, vehicle-treated

APPLond/Swe+ mice exhibited impaired sociability and showed no preference between the

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mouse and empty cup (Figure 5A, t-test, mouse vs. empty cup, n.s.). The SAS-0132 treated

APPLond/Swe+ mice showed a distinct preference for the cup containing a mouse over the

empty cup (Figure 5A, t-test, mouse vs. empty cup, * p < 0.05). In the Y-maze test

performed after 50 days of dosing, vehicle-treated APPLond/Swe+ mice were impaired as

assessed by low spontaneous alternation behavior in contrast to the WT mice (Figure 5B,

one-sample t-test vs. 50% theoretical mean, ** p < 0.01, *** p < 0.001). APPLond/Swe+ mice

treated with SAS-0132 showed an alternation level comparable to vehicle-treated WT mice

(Figure 5B, one-sample t-test vs. 50% theoretical mean, * p < 0.05). Total arm entries were

not different among the four groups (Figure 5B).

In the chronic daily dosing dose-response study, APPLond/Swe+ mice and their WT

littermates were aged to 6.7 months prior to initiating the administration of SAS-0132 at

doses of 3, 10, and 30 mg/kg for a total of 60 days. In a social discrimination task performed

after 30 days of dosing, all groups show normal sociality except for WT and APPLond/Swe+

mice treated with 10 mg/kg of SAS-0132 (Data not shown). We attribute this observation to

animal variations because the higher dose of 30 mg/kg did not impair sociability. The social

discrimination test was performed 1 day after the sociability test, and only APPLond/Swe+

mice treated with 3 mg/kg of SAS-0132 showed a significant preference for a novel mouse

over a familiar one (Figure 6A, t-test, familiar vs. novel). In the Y-maze test, which was

performed after 45 days of dosing, vehicle-treated APPLond/Swe+ mice showed a deficit in

spontaneous alternation behavior (Figure 6B, one-sample t-test vs. 50% theoretical mean,

n.s.), whereas those treated with 3 and 10 mg/kg of SAS-0132 showed normal spontaneous

alternation behavior (Figure 6B, one-sample t-test vs. 50% theoretical mean, n.s.).

Administration of 3 and 10 mg/kg of SAS-0132 to WT mice did not affect their

performance, but those dosed with 30 mg/kg showed impaired alternation behavior,

suggesting that high doses of SAS-0132 might have adverse effects on memory (Figure 6B,

one-sample t-test vs. 50% theoretical mean, n.s.). The number of total arm entries was not

different among groups (Figure 6B). The MWM test was initiated after 35 days of dosing.

During the hidden platform learning period, WT mice treated with 3 and 30 mg/kg of

SAS-0132 found the escape platform faster than the vehicle-treated WT mice (Figure 6C,

repeated measures of ANOVA, * p < 0.05, Dunnett’s post-hoc analysis, * p < 0.05). When

the platform was removed, vehicle-treated WT mice did not show target quadrant

preference, whereas WT mice treated with 3 mg/kg of SAS-0132 showed a clear preference

for the target quadrant (Figure 6C. t-test, target vs. non-target quadrant, * p < 0.01).

Administration of SAS-0132 had no effect on the cognitive functions of APPLond/Swe+

animals during the hidden platform learning phase (Figure 6D). After the spatial learning

phase, the reversal learning hidden platform trial was performed. On day 2 of this training,

WT mice treated with 10 and 30 mg/kg of SAS-0132 found the escape platform faster than

WT mice treated with vehicle (Figure 6E, one-way ANOVA, * p < 0.05, Fisher’s LSD, * p ≤

0.05). With the exception of the group treated with 3 mg/kg of SAS-0132, all WT groups

demonstrated recall and showed target quadrant preference during the reversal learning

probe trial (Figure 6E, paired t-test, target versus non-target quadrant, * p < 0.05, *** p <

0.001). There were no significant effects of SAS-0132 treatment in APPLond/Swe+ mice

during the reversal learning trials (Figure 6F). During the reversal learning probe trial,

vehicle-treated APPLond/Swe+ mice did not show a preference for the target quadrant over the

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non-target quadrants, whereas APPLond/Swe+ mice treated with 10 mg/kg of SAS-0132

showed target quadrant preference, indicating that SAS-0132 treatment reversed the reversal

training probe deficits in the APPLond/Swe+ group (Figure 6F, paired t-test, target vs. non-

target quadrant, * p < 0.05).

In both chronic daily dosing and chronic daily dosing dose-response studies, animals treated

with SAS-0132 remained healthy and exhibited no detectable abnormalities. Locomotor

activity measured in the activity chamber test after 24 days of treatment was not significantly

different between vehicle- and SAS-0132-treated mice, indicating that general locomotor

activity were not affected by SAS-0132 (data not shown). Comprehensive blood chemistry

and histopathology studies conducted using plasma and organs obtained from WT mice

treated with vehicle and SAS-0132 (10 mg/kg/d for 64 days) revealed no signs of SAS- 0132

induced toxicity (Supplemental tables 3–4).

Effects of SAS-0132 treatment upon Aβ pathology, synaptic loss, and neuroinflammation

Upon completion of the behavioral tests, we queried whether the cognitive-enhancing effects

of SAS-0132 might be attributable to its effects on AD-related pathological features. In

vehicle treated APPLond/Swe+ mice from the chronic daily dosing study (6.5 months old at

tissue collection), increased intracellular Aβ immunoreactivity was observed in the cortex,

hippocampus, and amygdala, relative to vehicle-treated WT group (Supplemental figure 1A,

*** p < 0.001 vs. WT groups, independent samples t-test). APPLond/Swe+ animals treated

with SAS-0132 displayed Aβ immunoreactivity similar to that seen in the vehicle-treated

APPLond/Swe+ groups, indicating that chronic treatment with SAS-0132 does not affect levels

of intracellular Aβ (Supplemental figure 1A). Similarly, ELISA analysis using brains of

these animals revealed no significant difference in soluble and insoluble Aβ40 and Aβ42

between vehicle- and SAS-0132-treated APPLond/Swe+ mice (Supplemental figure 1B,

independent samples t-test). Collectively, these data indicate that SAS-0132 does not affect

Aβ burden in the APPLond/Swe+ mouse model of AD.

Because Aβ is known to be toxic to synapses and to induce a neuroinflammatory response

(Shankar et al. 2007, Koffie et al. 2009, Meyer-Luehmann et al. 2008, Halle et al. 2008), we

determined whether SAS-0132 affects synaptic pathology and the neuroinflammatory

response. Quantifying the immunoreactivity of the pre-synaptic marker synaptophysin

revealed a significant reduction in synaptophysin levels in the CA3 region of APPLond/Swe+

mice compared to WT mice (chronic daily dosing study cohort, 6.5 months old at tissue

collection), but chronic treatment with SAS-0132 (10 mg/kg/d) did not alter the extent of

synapse loss (Supplemental figure 2). With respect to neuroinflammation, quantitative

immunohistochemistry with iba1 did not reveal any differences in microglia/monocyte-

derived macrophages in vehicle-treated APPLond/Swe+ mice relative to vehicle-treated WT

mice (chronic daily dosing study cohort, 6.5 months old at tissue collection).There were no

drug-related effects on immunoreactivity for iba1 in either WT or APPLond/Swe+ mice

(Supplemental figure 2). On the other hand, qRT-PCR analysis revealed that APPLond/Swe+

animals had elevated inflammatory markers TNFα, CD14, and IL1β at 8.75 months (dose-

response cohort), although elevation of the IL1β was not significant at 8.75 months (dose-

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response cohort) (Figure 7). Notably, treatment with SAS-0132 decreased the expression of

the inflammatory cytokine IL1β (Figure 7).

DISCUSSION

In this study, we report the identification of a novel norbenzomorphan class of Sig2R/

PGRMC1 ligands and their cellular and in vivo activity. The Sig2R/PGRMC1 ligands

reported here have Ki values in the low nanomolar range, which are comparable to the Ki

values of other known Sig2R/PGRMC1 ligands (Guo & Zhen 2015, Huang et al. 2014,

Vilner & Bowen 2000). The three ligands (i.e. SAS-0132, DKR-1005, and DKR-1051)

display selectivity for Sig2R/PGRMC1 over the Sig1R, with selectivity ratios greater than 9.

The discovery of this novel class of Sig2R/PGRMC1 ligands is of great importance, as these

norbenzomorphans have favorable pharmacokinetic attributes required for CNS indications.

In addition, they have compact molecular scaffolds and are amenable to structure-activity

relationship studies. Thus, norbenzomorphan analogs may be promising candidates for

further development into drug leads.

Regulation of intracellular Ca2+ is one important cellular process mediated by Sig2R/

PGRMC1, and structurally diverse Sig2R agonists are known to promote the release of

calcium from the endoplasmic reticulum and mitochondria (Cassano et al. 2009, Vilner &

Bowen 2000, Brent et al. 1996). We have shown that DKR-1051 produces concentration-

dependent increases in intracellular Ca2+ levels, suggesting that DKR-1051 has agonist

properties (Figure 2A). The other Sig2 binding ligand SAS-0132 failed to produce a

significant calcium response at up to 30 µM. At 100 µM, however, SAS-0132 increased

intracellular Ca2+ levels, but its effects on intracellular Ca2+ levels were weaker than the

effects of DKR-1051 (Figures 2A–2B). Interestingly, SAS-0132 attenuated the calcium

response induced by DKR-1051 at the lower concentrations that have no effect on

intracellular Ca2+ levels alone (Figure 2C). Collectively, this observation suggests that

SAS-0132 is an antagonist of Sig2R/PGRMC1 that has partial agonist activity. At the

molecular level, Sig2R/PGRMC1 could potentially modulate intracellular Ca2+ by indirectly

influencing the activity of ion-channels expressed in the endoplasmic reticulum,

mitochondria, and cell membrane (Vilner & Bowen 2000). As Sig2R/PGRMC1 is localized

in high density to subcellular fractions known to store calcium (Basile et al. 1992), another

possibility is that Sig2R/PGRMC1 directly gate the release of calcium. Further studies will

be needed to determine the exact mechanisms by which Sig2R agonists increase intracellular

calcium concentrations. Sig2R/PGRMC1-mediated calcium mobilization has important

clinical implications because intracellular Ca2+ plays a critical role in learning and memory,

and is strongly implicated in neuronal health (Bezprozvanny 2009). Perturbed Ca2+

homeostasis also appears to be involved in the dysfunction and death of neurons (Demuro et al. 2010, Mattson 1994), and its probable role in AD pathogenesis is supported by studies of

animal models of AD as well as of patients (Bezprozvanny & Mattson 2008, Zhang et al. 2015, Chakroborty et al. 2012). Because Ca2+ dyshomeostasis is a pathological feature of

AD and other neurodegenerative diseases (Berridge 2010), restoring Ca2+ homeostasis via

modulation of Sig2R/PGRMC1 may be a promising new approach to treat

neurodegenerative disorders.

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C. elegans is well-suited as a model organism for human diseases because it has clear

orthologs for two-thirds of all human genes (Hulme & Whitesides 2011, Consortium 1998,

Sonnhammer & Durbin 1997, Lai et al. 2000, Kuwabara & O'Neil 2001). Relevant to our

study, C. elegans expresses vem-1, an ortholog of PGRMC1, as well as other genes involved

in AD-related pathways (Link 2006, Simonsen et al. 2012, Safra et al. 2013, Haynes & Ron

2010, Runko & Kaprielian 2004). Our results demonstrate that single copy insertion of the

human APP gene into the C. elegans genome leads to age-dependent degeneration of

cholinergic neurons. While previous studies have not examined age-dependent

neurodegeneration, multi-copy overexpression of the C. elegans ortholog of APP (apl-1) or

human APP has been shown to cause behavioral dysfunction and partial lethality in C. elegans (Hornsten et al. 2007, Ewald et al. 2012). Importantly, the neurodegeneration we

observed in transgenic APP worms was diminished by genetic manipulation of PGRMC1 or

by pharmacological inhibition with SAS-0132 and JVW-1009. Conversely,

neurodegeneration was exacerbated by treatment with the agonist DKR-1005 (Figure 3).

These findings provide in vivo genetic evidence that PGRMC1 is critically involved in APP-

mediated neurodegeneration, and the Sig2R antagonists SAS-0132 and JVW-1009 exert

neuroprotection via a PGRMC1-related pathway.

In chronic daily dosing and chronic daily dosing dose-response studies, significant cognitive

enhancing effects of SAS-0132 were observed in both transgenic and WT animals. In the

chronic daily dosing study, SAS-0132 rescued AD-related sociability deficits and spatial

memory deficits (Y-maze-test). In the chronic daily dosing dose-response study, SAS-0132

rescued deficits in the social discrimination test (3 mg/kg) as well as deficits in spatial

memory in the Y-maze test (3 mg/kg and 10 mg/kg). SAS-0132 also led to improvements in

spatial and long-term memory in both WT (3, 10 and 30 mg/kg) and APPLond/Swe+ mice (10

mg/kg) in the MWM test. Although the cognitive enhancing effects of SAS-0132 shown in

the chronic daily dosing dose-response study should be interpreted cautiously due to the

small sample size, it is noteworthy that the effects of SAS-0132 were sufficiently large to be

detected despite the low statistical power. Importantly, cognitive performance of the animals

was evaluated prior to administration of the daily dose of SAS-0132. Because SAS-0132 is

largely cleared from the brain within 24 hours, the observed improvements in cognitive

performance likely arose from sustained modulation of Sig2R/PGRMC1 rather than from an

acute effect from treatment with SAS-0132.

There are several mechanisms that might explain the cognition-enhancing effects of

SAS-0132. First, SAS-0132 could produce pro-cognitive effects by being neuroprotective

against APP-mediated neuronal toxicity. For example, the Sig2R binding ligands CT01344

and CT01346 were found to block binding of Aβ oligomers to Sig2R/PGRMC1 and restore

AD-related behavioral deficits in the APPLond/Swe+ mouse model of AD (Izzo et al. 2014a).

Second, by regulating intracellular calcium levels, SAS-0132 treatment may counteract Aβ related dysregulation of calcium homeostasis. For example, application of Aβ oligomers to

cultured neurons induces an unregulated flux of Ca2+ through the plasma membrane via

various mechanisms that include activating endogenous receptors coupled to Ca2+ influx,

disrupting membrane lipid integrity, and forming Ca2+-permeable channels (Arispe et al. 1993, Rovira et al. 2002, Kayed et al. 2004, Demuro et al. 2010). Dysregulated Ca2+

homeostasis can trigger detrimental processes such as apoptosis and free radical formation,

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and these adverse events can eventually lead to cognitive deficits. Thus, with its ability to

modulate intracellular Ca2+ concentration, SAS-0132 may lead to cognitive enhancing

effects. Last, the benefits of SAS-0132 might not be exclusively related to its effects on APP

related pathways, but result from more generalized mechanisms that are not specific to APP.

For example, in our dose-response study, we discovered that chronic treatment of WT mice

with SAS-0132 improved spatial and long-term memory in the MWM test. Although further

studies are required to confirm this finding, these cognitive enhancing effects of SAS-0132

in healthy animals certainly warrant further study.

In contrast to its significant effect on cognition, SAS-0132 had no discernible effects upon

several markers of AD pathology, including Aβ burden and synaptic loss. As

neuroinflammation is one of the main pathological features of AD, we also sought to

determine the effects of SAS-0132 on neuroinflammation. In the chronic daily dosing study,

we were unable to evaluate the effects of SAS-0132 on AD-related neuroinflammation, as

APPLond/Swe+ mice showed little evidence of neuroinflammation. Although this finding is

interesting as high levels of Aβ and behavioral deficits associated with AD are present in

this APPLond/Swe+ mice at this age, it is in line with a previous study that reported the lack of

signs of neuroinflammation in this mouse model at 8 months of age (Lull et al. 2011).

Multiple lines of transgenic models of AD have been shown to develop AD-related

neuropathology in an age-dependent manner (Abbas et al. 2002, Sly et al. 2001, Oakley et al. 2006, Rockenstein et al. 2001). Thus, it is possible that the APPLond/Swe+ mice model

may show evidence of neuroinflammation at older age. Indeed, we observed significant

increases in expression of neuro-inflammatory genes in APPLond/Swe+ mice compared to WT

mice in the chronic daily dosing dose-response study (8.7 months of age at tissue

collection). Chronic treatment with SAS-0132 was shown to decrease AD-related

neuroinflammatory responses as measured by reduced brain levels of the inflammatory

cytokine IL1β. Because chronic inflammation in response to neuronal damage is thought to

accelerate and potentially underlie disease progression in AD (Heneka et al. 2015), the anti-

inflammatory effects of SAS-0132 might be partly responsible for its cognitive enhancing

effects.

In this study, we report that pharmacological and genetic inhibition of Sig2R is

neuroprotective, cognitive enhancing, and anti-inflammatory in experimental models of AD

and excessive amyloidosis. Regulation of intracellular calcium concentration by Sig2R

modulation could be responsible for these beneficial effects. Further mechanistic studies will

be needed to investigate the role of the Sig2R/PGRMC1 in modulation of

neuroinflammation and cellular signaling, leading to neuroprotection and modulation of

intracellular calcium homeostasis. The fact that the Sig2R/PGRMC1 antagonist SAS-0132

has significant benefits when it is chronically administered to symptomatic animals inspires

the intriguing question of whether treatment of presymptomatic animals could prevent the

development of AD and/or cognitive deficits. Our findings clearly demonstrate the

significant potential of modulating pathways mediated by Sig2R/PGRMC1 as a novel

mechanism to treat AD. This hypothesis is supported by recent work conducted by Izzo and

coworkers, who have shown that CT01344 and CT01346: (1) block binding of Aβ oligomers

to Sig2R/PGRMC1 on neurons (Izzo et al. 2014a, Izzo et al. 2014b); (2) prevent and reverse

the effects of Aβ oligomers on membrane trafficking and synapse loss in neuronal culture

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(Izzo et al. 2014a); and (3) reverse cognitive deficits in a mouse model of AD (Izzo et al.

2014a). These collective discoveries suggest that modulating pathways mediated by Sig2R/

PGRMC1 is meritorious as a new approach to study neurodegenerative conditions and, if

shown to be safe and well tolerated, should be further evaluated.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

Acknowledgments and dedication

Receptor binding profiles was performed by the National Institute of Mental Health's Psychoactive Drug Screening Program (NIMH PDSP). We thank the director of NIMH PDSP, Bryan L. Roth MD, PhD, at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. For the C. elegans study, some strains were provided by CGC, which is supported by the NIH Office of Research Infrastructure Programs (P40 OD010440). This manuscript is dedicated to Dr. Steven Alan Sahn: a brilliant physician, a dedicated and passionate coach, and a loving and devoted father.

Funding

This study was supported by National Institutes of Health (GM 24539, 1R01AG041135, NS069375), Robert A. Welch Foundation (F-0652), Alzheimer’s Association, and Lumind Research Foundation. We are also grateful to Dr. Richard Pederson (Materia, Inc.) for catalyst support.

Abbreviations used

AD Alzheimer’s disease

Sig1R sigma 1 receptor

Sig2R sigma 2 receptor

PGRMC1 progesterone receptor membrane component-1

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Figure 1. Sig2R binding ligands with neuromodulatory (JVW-1009, SAS-0132, DKR-1005, and DKR-1051) and cognition enhancing activity (SAS-0132).

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Figure 2. Effects of the Sig2R ligands on intracellular calcium in SK-N-SH neuroblastoma cells(A) DKR-1051 induces concentration-dependent increases in intracellular calcium (### p <

0.001 versus DKR-1051 (0 µM), one-way ANOVA followed by Dunnett’s multiple

comparison test). (B) SAS-0132 does not induce significant increases in intracellular

calcium up to 30 µM, but produces a significant calcium response at 100 µM (### p < 0.001

versus SAS-0132 (0 µM), one-way ANOVA followed by Dunnett’s multiple comparison

test). (C) SAS-0132 attenuated the calcium response induced by DRK-1051 (### p < 0.001

versus DKR-1051 (0 µM); * p < 0.05 and ** p < 0.01 versus DKR-1051 (100 µM), one-way

ANOVA followed by Dunnett’s multiple comparison test). Data are presented as mean ±

SEM from 3–4 independent experiments performed in duplicate or triplicate.

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Figure 3. Neuroprotective effects of SAS-0132(A) In our C. elegans model of neurodegeneration, transgenic worms carry a single copy

insertion of human amyloid precursor protein (SC_APP). VC class cholinergic neurons

express GFP. (B) Representative fluorescence and phase contrast images of VC4 and 5

neurons in transgenic SC_APP C. elegans (on day 1 and 3 of adulthood). (C) Transgenic C. elegans expressing SC_APP display age-dependent degeneration of VC4 and 5 compared to

wild-type (WT) controls (*** p < 0.001 versus WT control, planned comparisons with

Fisher’s exact test, n=60–128). (D) Neurodegeneration in these transgenic C. elegans is

significantly diminished with two different null alleles of vem-1, an ortholog of PGRMC1

(** p < 0.01 and *** p < 0.001 versus transgenic hAPP animals with WT PGRMC1,

planned comparisons with Fisher’s exact test, n=194–230) (E) DKR-1005 exacerbates the

neurodegeneration shown in the transgenic SC_APP worm, while JVW-1009 and SAS-0132

decrease the neurodegeneration in the transgenic SC_APP worm (** p < 0.01 versus vehicle

group, planned comparisons with Fisher’s exact test, n=110–378) (F) Knockdown of

PGRMC1 or pharmacological modulation of Sig2R/PGRMC1 with JVW-1009 ameliorates

the neurodegeneration shown in the transgenic C. elegans (*** p < 0.001 versus

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JVW-1009(−)/PGRMC1 RNAi (−) group, planned comparisons with Fisher’s exact test,

n=110–142). JVW-1009 provides no further effects in animals with reduced PGRMC1

expression. (G) Knockdown of PGRMC1 or pharmacological modulation of Sig2R/

PGRMC1 with SAS-0132 ameliorates the neurodegeneration shown in the transgenic C. elegans (** p < 0.01 versus SAS-0132(−)/PGRMC1 RNAi (−) group, planned comparisons

with Fisher’s exact test, n=130–138). SAS-0132 provides no further effects in animals with

reduced PGRMC1 expression.

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Figure 4. Pharmacokinetics of SAS-0132(A) Plasma concentration of SAS-0132 in mice plasma collected over 3 h post-dose after a

single injection of SAS-0132 (10 mg/kg) via intravenous (IV), subcutaneous (SC), and oral

(PO) administration (n = 3 per route). (B) Bioavailability of SAS-0132 by SC and PO dosing

(n = 3 per route). (C,D) Brain concentration and brain/plasma ratio of SAS-0132 3 h after

the administration of SAS-0132 (n = 3 per route). Data are represented as mean ±SEM.

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Figure 5. Effects of SAS-0132 on cognitive functions in the chronic daily dosing study(A) In the sociability test, vehicle-treated wild-type (WT) mice explored a cup containing a

C57Bl/6 mouse more than an empty cup, showing normal sociability. APPLond/Swe+ mice

treated with vehicle showed lack of sociability, while APPLond/Swe+ mice treated with SAS-

0132 (10 mg/kg) showed normal sociability (t-test, mouse versus empty cup, *p < 0.05, ** p < 0.01, n =9–12 per group).(B) In the Y-maze, WT mice showed spontaneous alternation

behavior, indicating a normal spatial memory. The vehicle-treated APPLond/Swe+ mice

showed impaired spontaneous alternation performance. The APPLond/Swe+ mice treated with

SAS-0132 (10 mg/kg) showed spontaneous alteration behavior to a level comparable to the

WT mice (One-sample t-test vs 50% theoretical mean, *p < 0.05, ** p < 0.01, *** p <

0.001, n=9–12 per group). The number of total entries were not significantly different

among groups (one-way ANOVA). Data represent mean ± SEM.. Wild-type, WT;

APPLond/Swe+, APP.

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Figure 6. Effects of SAS-0132 on cognitive function in the chronic daily dosing dose-response study(A) In the social recognition test, only the APPLond/Swe+ mice group treated with 3 mg/kg of

SAS-0132 distinguished between a novel and familiar mouse, preferring a novel mouse (t-

test, familiar versus novel, ***p < 0.001, n = 3 – 5 per group). (B) In the Y-maze test, the

APPLond/Swe+ mice showed impaired spontaneous alternation behavior, which was restored

by chronic administration of SAS-0132. Both wild-type (WT) and APPLond/Swe+ mice dosed

with 30 mg/kg showed impaired spontaneous alternation behavior (One-sample t-test vs

50% theoretical mean, *p < 0.05, ** p < 0.01, n = 3 – 5 per group). The number of total

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entries was not significantly different among groups (One-way ANOVA). (C) WT mice

groups treated with 3 and 30 mg/kg SAS-0132 showed faster escape latency compared to

vehicle-treated WT mice during the hidden platform training (Repeated measures of

ANOVA, * p < 0.01 versus vehicle-treated WT, n = 3 – 5 per group). During the probe trial,

WT mice treated with 3 mg/kg of SAS-0132 showed target quadrant preference(t-test, target

versus non-target quadrant, * p < 0.01), while the other three groups did not show this

preference (n = 3 – 5 per group). (D) APPLond/Swe+ mice showed signs of learning as

indicated by preference for the target over the non-target quadrant during the probe trial,

although not significant (n = 3 – 5 per group). Three of the five mice in the 30 mg/kg

APPLond/Swe+ group did not perform the task. Thus, this group was excluded from the

analyses. (E) On the second day of the reversal learning training, WT mice treated with 10

and 30 mg/kg SAS-0132 found the escape platform significantly faster relative to vehicle-

treated WT mice (one-way ANOVA, * p < 0.05 post-hoc, n = 3 – 5 per group). During the

reversal probe trial, all WT groups showed target quadrant preference with the exception of

the 3 mg/kg dose group (paired t-test, * p < 0.05, *** p < 0.001, n = 3 – 5 per group). (F)

The reversal learning training revealed no main effect of drug on learning in APPLond/Swe+

mice (repeated measures of ANOVA), although a non-significant decreased escape latency

was observed for APPLond/Swe+ mice treated with 3 and 10 mg/kg SAS-0132 compared to

the vehicle-treated APPLond/Swe+ mice (n = 3 – 5 per group). During the reversal probe trial,

APPLond/Swe+ vehicle-treated mice did not show target quadrant preference. APPLond/Swe+

mice treated with 10 mg/kg SAS-0132 showed preference for the target over the non-target

quadrant (paired t-test, ** p < 0.01, n = 3 – 5 per group). Three of five mice in the 30 mg/kg

APPLond/Swe+ group did not pass the swimming performance criteria. Thus, this group was

excluded from the analyses. Three additional mice in 3 other groups (WT-vehicle,

APPLond/Swe+-3 and-10 mg/kg), were also excluded for the same reason. Data represent

mean ± SEM. Wild-type, WT; APPLond/Swe+, APP.

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Figure 7. Effects of SAS-0132 on neuroinflammation in the chronic daily dosing dose-response studyTNFα, CD14, and IL1β expression was elevated in the vehicle-treated APPLond/Swe+ mice

compared to wild-type (WT) counterparts, although not significantly. SAS-0132 treatment

attenuated increases in IL1β expression. Bar graphs depict mRNA expression in cortical

tissue from WT and APPLond/Swe+ mice. mRNA expression was normalized relative to

vehicle-treated WT mice for each gene. Data represent mean ± SEM. n= 3–5 per group.

Wild-type, WT; APPLond/Swe+, APP.

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inflammation in experimental models of alzheimer’s disease ...sunnyvale, ca) in human sk-n-sh...

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