Astrocytes Exposed to Chronic High Glucose Promotes Neuronal Synaptic Loss Through

Impaired Nutrient Support: Implications for Alzheimer’s Disease Progression

Hirra A. Arain*1, 2,

1. Department of Biology, Hofstra University, Hempstead New York 11549, U.S.A;

*corresponding author harain1@pride.hofstra.edu

2. Biomedical Research, NYU Winthrop Hospital, 101 Mineola Blvd, Mineola New York,

11501, U.S.A

Abstract

The literature has revealed a strong prevalence of metabolism dysfunction in the

pathogenesis of Alzheimer’s disease (AD). Regulation of metabolism is controlled by astrocytes,

glial cells that are vital to forming the blood brain barrier and providing nutrients under

energetically strenuous conditions. Due to the prevalence of Type II diabetes (T2DM) associated

with AD later in life, coupled with the similar metabolic abnormalities associated with AD, it

was relevant to examine the dynamics of how T2DM milieu may promote AD. As astrocytes are

the main metabolic cells in the brain, we investigated the ability of T2DM to promote AD

through astrocytes. Using a cell culture system, differentiated neurons were exposed to

astrocyte-conditioned media from astrocytes exposed to chronic hyperglycemia. Neurite density

was both visualized and quantified using scanning electron microscopy. Our data revealed that

neurons, exposed to conditioned media from astrocytes contained significantly higher neurites.

Though when the neurons were exposed to conditioned media from the astrocytes treated with

high glucose, decrease neuronal extensions was revealed. In addition, we also observed the

absence of synaptic spines present in the control differentiated neuroblastoma cells. Overall, our

data suggests that the astrocytes exposed to the diabetic condition lose their ability to support

neurite outgrowth and promote neurodegeneration. Furthermore, our data establishes a new

cellular mechanism of T2DM promoted AD neurodegeneration and reveal astrocytes as the

driver of such pathology.

Significant Statement: An association between Type II diabetes and Alzheimer’s disease has

been observed in the clinic, though no cellular mechanism has elucidated such association to

date. Here we report that conditioned media from astrocytes exposed to a hyperglycemia

decreases neurite density. This data thus demonstrates that astrocytes are affected by the

hyperglycemic induced by Type II diabetes and further this data implicates astrocytes in

promoting the neurodegeneration observed in Alzheimer’s disease. These findings have

additional implications for understanding both the etiology of Alzheimer’s disease and also

demonstrate therapeutic potentials of treatments that target astrocytes and Type II diabetes.

Keywords: Alzheimer’s Disease, Astrocytes, Dementia, Hyperglycemia, Synaptic Loss,

Neurodegeneration

Introduction

Alzheimer’s Disease (AD) is a neurodegenerative disease and to date is the most

common form of acquired dementia later in life (Masters et al., 2015). Pathologically, AD is

defined by the buildup of extracellular amyloid-beta monomers called senile plaques as well as

the presence of intracellular neurofibrillary tangles within dying neurons (Masters et al., 2015).

Currently, there are no known definitive diagnostic tests or viable disease treatments for AD.

Research for the past 20 years has centered mainly on the amyloid cascade hypothesis which

implicates amyloid as the driver of AD through neurotoxicity (Masters et al., 2015). However,

recent failures from clinical trials targeting the amyloid plaques indicates that new investigations

into other mechanisms that may contribute to AD progression are needed (Green et al., 2009;

Salloway et al., 2014).

Research has revealed a link between metabolic dysfunction and AD pathogenesis. For

example, patients who have type II diabetes mellitus (T2DM) are at a higher risk for developing

AD later in life (Li et al., 2015; Walker and Harrison, 2015). In accordance with such an

association, AD patients additionally demonstrate a progressive suppression of cerebral glucose

uptake in brain regions (e.g., hippocampus) associated with AD (de Leon et al., 2001; Mosconi et

al., 2008). In addition, it has been demonstrated that patients with sporadic AD, a non inheritable

form that accounts for 95% of all AD cases, have single nucleotide polymorphism (SNP) in two

cholesterol transporting proteins, Apolipoprotein E–ε4 (APOE-ε4) and Clutsterin (APOJ) (Farrer

et al., 1997; Harold et al., 2009).

The metabolism of the central nervous system (CNS) is tightly regulated by astrocytes,

the most abundant glial cell in the CNS that contributes to forming the blood brain barrier

(BBB). Astrocytes couple neuron energy demand to glucose and metabolite transport by forming

projections and monitoring activity between synapses of communicating neurons and the

surrounding blood vessels (Bélanger et al., 2011). Astrocytes additionally transport glycolytic

metabolites such as glucose and glycogen derived lactate under energetically strenuous

conditions, a disruption of which has been implicated in impairment of memory (Bélanger et al.,

2011; Gandhi et al., 2009; Newman et al., 2011; Pellerin et al., 1998; Tarczyluk et al., 2013;

Tekkök et al., 2005). Furthermore, astrocytes are also the only source of brain glycogen and

additionally are the main source of de novo cholesterol, lipids products of which are transported

from the glial cells to neurons through lipoproteins (Chen et al., 2013; Gruetter, 2003; Hayashi,

2011).

Due to the prevalence of T2DM promoting AD later in life, coupled with the similar

metabolic abnormalities associated with AD, it is relevant to examine the dynamics of how

T2DM milieu may promote AD. Further, as astrocytes play a major role in aiding neuronal

metabolism, which is proposed to directly impact memory formation, we sought to investigate if

chronic hyperglycemia, a diabetic induced event, can affect astrocyte metabolite support to

neurons. Such alterations in astrocyte derived nutrient support may be detrimental not only to

memory formation but also to overall neuronal function. This disruption could possibly be the

mediator for synaptic loss observed in AD, thus pointing to a possible link between AD and

T2DM.

In this study, we investigate if prolonged exposure of human astrocytes to high levels of

glucose disrupt the ability of astrocytes to support neurons nutritionally, and as a result lead to

the synaptic loss and neurodegeneration observed in AD. To implement this, a human neuronal

cell culture model with conditioned media from astrocyte was used. Analysis of synaptic density

was visualized and quantified using scanning electron microscopy. Our results reveal that

astrocytes exposed to hyperglycemia promote neuronal degeneration in cell culture. Thus, our

data is one of the first that demonstrates an association between T2DM in AD and additionally

implicates astrocytes in AD pathogenesis.

Materials and Methods

Cell Culture and Treatment Human hippocampal astrocytes (hHA, ScienCell) were cultured at 37°C, 5% CO2 in

Astrocyte Basal Medium (ABM, ScienCell) supplemented with provided components from the

basal media (3% fetal bovine serum, 1% Astrocyte growth serum, 1% PennStrep; ScienCell). For

experimental conditions, once cells became 80% confluent the monolayer was trypsinized using

0.25mg Trypsin/0.05% EDTA (ScienCell) according to the manufacture’s instructions and plated

in 60mm dishes (Falcon) at a density of 70,000 per well. After 24hrs of plating, the cells were

treated with varying levels of glucose conditions (5mM and 40mM) supplemented to the basal

media for six days, with the media being changed every two days. On the sixth day, the astrocyte

conditioned media (ACM) was collected and stored at -20°C until further use.

Human SH-SY5Y (CRL-2266_SHSY5Y_RRID: CVCL_U924) were cultured at 37°C,

5% CO2 in DMEM: F12 (50:50; Corning) supplemented with 10% FBS, 1% GlutMax, and 1%

PennStrep. Once cells reached 80% confluence, the cells were sub-cultured using 0.25mg/0.05%

EDTA (Gibco) and seeded on 25mm coverslips (Corning). After two days of plating, the media

was swapped with DMEM: F12 (50:50) with 1% FBS and 10µM all trans-retinoic acid. The cells

were differentiated for four days, with media supplemented with fresh retinoic acid added every

two days. After differentiation, the cells were switched to a serum free media (DMEM: F12

50:50 with no FBS) and 35% ACM from each treatment was added to each neuron. The ACM

was incubated with the differentiated neurons for 24 hrs and the cells were analyzed.

Scanning Electron Microscopy

Cells were fixed in 2.5% glutaraldehyde (Scanning Electron Microscopy) for two hrs at

room temperature. Once fixed, the cells were incubated twice in phosphate-buffered saline (PBS,

Corning) for fifteen min each. After the incubations, the cells were washed in distilled water

twice for two min. The cells were then gradually dehydrated in ethanol ranging from 30-90% in

ten percent increments for 10 min each and then twice at 100% ethanol for 10 min each. Critical

point drying was performed in a SAMDRI-795. The coverslips were removed and mounted on

25mm aluminum specimen stubs using double-sided sticky tape. Samples were gold coated using

the EMS-550 sputter coater for 45 sec at 45mA and imaged using a FEI Quanta 250 SEM.

Statistical Analysis

Statistical analysis was performed using VassarStats (www.Vasssarstats.net). All

normally distributed data were analyzed with student’s independent t-tests to test for difference

in means between samples. Probability values less than 0.05 were regarded as significant. For

neurite quantification, each independent sample had two pictures randomly taken. Neurites were

categorized as thin extensions from the cell body and quantification was performed by manually

counting each extension from each cell body. In each sample, neurite quantification was

performed for ten randomly selected cells and the count of the neurites from the selected sample

of cells was averaged. This was repeated three independent times for each treatment type and a

representative image was selected for representation. Errors bars represent the standard deviation

from three independent experiments.

Results

Astrocytes Support and Increase Neurite Density in vivo

Before we could probe the effects of hyperglycemia on astrocyte nutrition support, we

first investigated if astrocytes were excreting supportive factors, which support neurite health.

Neurons that received the ACM had a higher abundance of neurites extended from the cell body

as revealed through the scanning electron micrographs (Figure 1). Quantification of the neuronal

extensions supported these findings as the data demonstrated a significant increase in neurites in

neurons treated with the ACM (t(3)=4.9;p=0.008, Figure 2). In addition, our data revealed the

absence of synaptic spines present on the projections from the cell body in the control neuron

(Figure 3). Overall, this data demonstrates that ACM increases neuronal extensions.

Hyperglycemia Decreases Astrocyte Support and Increases Synaptic Loss

We next sought to investigate if hyperglycemia could disrupt astrocytes nutritional

support and lead to neurodegeneration. The scanning electron micrographs revealed that the

neurons that received the ACM from the hyperglycemic condition had significantly less neurites

when compared to the neurons exposed to control ACM (Figure 4). Quantification of the neurites

supported these results as neurons exposed to ACM from the high glucose condition had

significantly less neurites with an average amount of three extensions per cell body

(t(3)=5.2;p=0.010, Figure 5). Overall, our data demonstrated that ACM from the high glucose

treatment decreases neuronal extensions.

Discussion

Patients with AD have perturbations in glucose and lipid metabolism and transport

(Farrer et al., 1997; Morrow et al., 2002; Mosconi et al., 2008). Further, an association between

type II diabetes and Alzheimer’s has been established clinically though no cellular and molecular

relationship has been demonstrated (Li et al., 2015). In this study, we investigated if astrocytes,

supportive glial cells vital to neurons metabolism, may be consequently affected by the diabetic

induced hyperglycemia and induce the synaptic loss observed in AD. Using scanning electron

microscopy, we both visualized and quantified neuronal extensions in differentiated

neuroblastoma cells treated with ACM to investigate the effects of hyperglycemia on astrocyte

promoted neurite loss through decrease nutrition support. Our data demonstrated that neurons in

the presence of ACM from astrocytes exposed to high glucose revealed less neuronal extensions.

Thus our data suggests that astrocytes exposed to a diabetic hyperglycemic environment might

lose their ability to support neurite outgrowth and as a result induce neurodegeneration.

In the context of aging, astrocytes have emerged as a key player in its progression.

Reactive astrocytes, A1 astroglia that respond to inflammatory signals and are neurotoxic, are

observed in AD brains postmortem (González-Reyes et al., 2017; Rodríguez-Arellano et al.,

2016; Verkhratsky et al., 2010). Further, in the aging mouse brain, astrocytes have been

demonstrated to polarize to the toxic A1 phenotype as demonstrated by morphological and

genetic analyses (Boisvert et al., 2018; Clarke et al., 2018). As our data demonstrated the

unsupportive nature of the astrocytes in response to the hyperglycemia, it is plausible that such

observations may be a result of phenotype switching from a neutral state to a toxic reactive state.

Furthermore, as our study did not exclude additional molecules other than metabolites, such as

soluble protein factors, it does not exclude that the disruption in synaptic density by astrocytes

may be due to neurotoxic factors that may promote neurodegeneration.

To understand how the hyperglycemia may affect astrocytes, it is possible that the high

glucose could change the levels of post-translation modification of sugars on important proteins

require for neuronal support. In diabetic hyperglycemia, studies point to protein modification as

the contributor to hyperglycemia induced neurotoxicity (Parween et al., 2017; Wang et al.,

2016). O-glucose-N-acetyl (O-GlcNAc) is a post-translational modification where a sugar

monomer is added to serine and threonine residues within proteins (Bond and Hanover, 2015).

During hyperglycemia, increased O-GlcNAc addition to neuronal proteins have been

documented (Wani et al., 2017). Thus it is relevant to examine the levels of O-GlcNAc in

astroglia proteins important in metabolism, as this post-transcriptional regulation may be

disrupted in AD as a result of hyperglycemia.

Our data did not reveal synaptic spines along the neurites, suggesting that the use of SH-

SY5Y as in previous research regarding neuronal toxicity and synaptic loss may not be a proper

model to study neurodegeneration (Nampoothiri et al., 2014; Petratos et al., 2008). Synaptic

spines are actin extensions on dendrites of neurons that increase surface area to facilitate the

acquiring of chemical signals to initiate an action potential (Nimchinsky et al., 2002). The

presence of these actin extensions on dendrites have been demonstrated to be a critical structure

in fully differentiated adult neurons (Nimchinsky et al., 2002). SH-SY5Y has been demonstrated

in the literature to not biochemically identify with a specific set of neurotransmitters required in

truly differentiated neurons (Kovalevich and Langford, 2013). Thus the literature has prompted a

discussion on the legitimacy of all trans-retinoic acid differentiated SH-SY5Y as a model to

study neurodegeneration in AD and Parkinson’s disease (Kovalevich and Langford, 2013). Our

findings of no synaptic spines support many in the literature that argue that the retinoic

differentiated neurons do not display characteristics of fully differentiated neurons, and in

additions our findings further support the use of other alternative cell culture models for

neurodegenerative models.

Overall, our data supports a link between the hyperglycemia induced by diabetes and

neuronal degeneration through astrocytes. Furthermore, our study supports treatment measures,

which suppress or prevent type II diabetes as a plausible treatment to combat AD. In addition,

out data reveals additional therapeutic potentials regarding astrocytes to treat AD.

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Figure 1. Astrocyte conditioned media increases overall neuronal extensions in culture. Scanning electron micrographs of retinoic-acid differentiated SH-SY5Y treated without (A, B) or with (C, D) astrocyte-conditioned media. Scale bars represent 20µM (A, C) or 50 µM (B, D). Figure 2. Astrocyte conditioned media increases neurite density around the cell body. Average quantity of neurites extended from the cell body from either retinoic-acid differentiated SH-SY5Y treated without (A, B) or with astrocyte conditioned media (C, D). Errors bars represent the standard deviation. n=3 replicates, *P<0.05. Figure 3. No presence of synaptic spines on neuronal extensions from differentiated SH-SY5Y. Scanning electron micrograph of a representative neuronal extension from a control retinoic-acid differentiated SH-SY5Y. Scale bars represent 50µM. Arrows point to the neurites extended from the cell body. Figure 4. Astrocytes exposed to a hyperglycemic condition decreases neuronal neurites. Scanning electron micrographs of differentiated SH-SY5Y exposed to astrocyte-conditioned media from astrocytes exposed to either (A, C) 5mM or (B, D) 40mM for 24hrs. Scale bars represent 20µM (A, C) or 50 µM (B, D). Figure 5. Astrocytes exposed to a hyperglycemic condition decreases neurite density around the cell body. Average quantity of neurites extended from the cell body of differentiated SH-SY5Y exposed to astrocyte-conditioned media (ACM) from astrocytes exposed to the following treatments (5mM, 40mM) for 24hrs. n=3 replicates, *P<0.05.

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