Neuroprotective Activities of Heparin, Heparinase ... - ACS Publications

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Tissue Engineering and Regenerative Medicine

Neuroprotective Activities of Heparin, Heparinase III, and Hyaluronic Acid on the A#42-treated Forebrain Spheroids Derived from Human Stem Cells Julie Bejoy, Liqing Song, Zhe Wang, Qing-Xiang Sang, Yi Zhou, and Yan Li ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00021 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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ACS Biomaterials Science & Engineering

Neuroprotective Activities of Heparin, Heparinase III, and Hyaluronic Acid on the Aβ42treated Forebrain Spheroids Derived from Human Stem Cells

Julie Bejoy1, Liqing Song1, Zhe Wang2, Qing-Xiang Sang2, 3, Yi Zhou4, Yan Li*, 1, 3

1

Department of Chemical and Biomedical Engineering; FAMU-FSU College of Engineering;

Florida State University; Tallahassee, Florida, USA 2

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida,

USA 3

Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, USA

4

Department of Biomedical Sciences, College of Medicine, Florida State University,

Tallahassee, Florida, USA

*Corresponding author Dr. Yan Li; Tel: 850-410-6320; Fax: 850-410-6150; email: [email protected]. Mailing address for Julie Bejoy, Liqing Song, and Yan Li: 2525 Pottsdamer St., A131, Tallahassee, FL 32310, USA Mailing address for Zhe Wang and Qing-Xiang Sang: 95 Chieftan Way, Tallahassee, Florida, 32306-4390, USA Mailing address for Yi Zhou: 1115 W Call St., Tallahassee, Florida, 32306-4340, USA Submitted to ACS Biomaterials Science and Engineering

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Extracellular matrix (ECM) components of the brain play complex roles in neurodegenerative diseases. The study of microenvironment of brain tissues with Alzheimer’s disease revealed co-localized expression of different ECM molecules such as heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and hyaluronic acid. In this study, both cortical and hippocampal populations were generated from human induced pluripotent stem cell-derived neural spheroids. The cultures were then treated with heparin (competes for Aβ affinity with HSPG), heparinase III (digests HSPGs), chondroitinase (digests CSPGs), hyaluronic acid, and a MMP-2/9 inhibitor (SB-3CT) together with amyloid β (Aβ42) oligomers. The results indicate that inhibition of HSPG binding to Aβ42 using either heparinase III or heparin reduces Aβ42 expression and increases the population of β-tubulin III+ neurons, whereas the inhibition of MMP2/9 induces more neurotoxicity. The results should enhance our understanding of the contribution of ECMs to the Aβ-related neural cell death.

Keywords: human induced pluripotent stem cells, heparan sulfate proteoglycans, chondroitin sulfate proteoglycan, matrix metalloproteinases, hyaluronic acid, neuroprotective


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Introduction International estimate for the number of Alzheimer's patients reached around 48 million worldwide in 2015 1. Since there are no disease-modifying therapies available, the improved disease modeling is necessary for evaluating disease pathology and therapeutic interventions. Transgenic animal models do not fully recapitulate microenvironment of endogenous proteins from human, thus, it is essential to create complementary models from human cells. Alzheimer’s disease (AD) is a slowly progressing neurodegenerative disorder characterized by the misfolding, aggregation and the gain of toxicity due to amyloid-β (Aβ) and tau in the brain 2. Induced pluripotent stem (iPS) cells have emerged as the most promising tools for disease modeling since they can be derived from the patient’s own body

3-6

and have the capacity to self-organize into

mini-brain like organoids 7, 8. The pathological mechanism underlying AD is found to be the conformational changes in the Aβ peptides and hyperphosphorylated tau proteins that assemble to form β-sheeted plates resulting in forming neuritic fibrillatory tangles (NFT).

These tangles together with the

cytoplasm of neurons form the plaques called senile plaques (SP). The detailed evaluation of the plaques and the surrounding microenvironment of AD brain revealed prominent expression of different ECM molecules such as proteoglycans (PGs), matrix metalloproteinases (MMPs), and hyaluronic acid (HA) etc. 9. Compared to normal brain, immunohistochemical analysis revealed co-localized expression of proteoglycans, including heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs), with SPs and NFTs both in the brains of AD patients and in the transgenic animal AD models 10, 11. Proteoglycans with high negative charge affect the early stages of fibril formation and enhance the lateral aggregation of fibrils at later stages 12. Distribution of sulfate groups on the



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proteoglycan is the important feature for the Aβ-PG interaction 12. Perlecan, one type of HSPG and a part of basement membrane, accelerates Aβ fibril formation and maintains Aβ fibril stability by using Perlecan’s HS glycosaminoglycan chains

13

. Whereas agrin, another kind of

HSPG, binds to Aβ, accelerates fibril formation, and protects fibrillar Aβ from proteolysis

14

.

Different types of sulfated CSPGs are also associated with SPs and NFTs and responsible for the decreased neurite growth both in in vitro cultures and in response to injury in the central nervous system

15

. Studies suggest that CSPGs support the formation of amyloid precursor proteins

(APP) that produce Aβ peptide

16

. The neuritic density of the AD cells is found to decrease in

the CSPG-containing areas compared to other areas, supporting the CSPG-controlled neurite growth 11. Amyloid plaques in the AD patients also showed prominent expression of other ECMrelated molecules such as HA

17

and matrix metalloproteinase MMP-9

18, 19

. HA positive

neurons in both rat and human brain were found to be resistive to the alteration caused by the AD pathology, indicating the attenuating effect of HA on AD neurons

17, 20

. MMP-9 was detected

near the extracellular amyloid plaques, which when activated, was capable of degrading Aβ peptides

18, 21

. The enzyme cleaves at three different sites on the Aβ peptide and eliminates the

neurotoxic β-sheet-forming capacity of the amyloid peptide 22. Taken together, these studies suggest the positive impacts of HA and MMP, but the negative effects of HSPGs and CSPGs on neural degeneration and the needs for evaluating ECMs to identify potential therapeutic targets 9. The objective of this study is to evaluate the impact of these ECM molecules on Aβ42-induced neurotoxicity and thereby on the neural growth and differentiation using human iPS cell-derived neural spheroids.


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Forebrain regions of the brain are sensitive to the neural degeneration, suggesting that in vitro forebrain-like models derived from iPS cells could be used for studying AD-related degeneration

3, 4, 23-26

. In this study, two neural differentiation paradigms from human iPS cells

through 3-D spheroid formation were investigated to obtain a forebrain-dominant culture. The first type of spheroids used dual Smad inhibition followed by sonic Hedgehog (Shh) pathway inhibition to enrich cortical neurons

27, 28

. The second type of spheroids used inhibitors of Wnt,

Shh, and SMAD signaling followed with Wnt activation to enrich hippocampal dentate gyrus neurons

29, 30

. Exogenous Aβ42 oligomers were added to different spheroid outgrowth

27, 28, 31

.

The impacts of ECM enzymes (to digest proteoglycans), HA, heparin, and the MMP inhibitor on the Aβ-induced neuropathology were examined. The results of this study should enhance our understanding of the contribution of these ECM-related molecules to neuronal degeneration. Materials and Methods Undifferentiated hiPSC culture Human iPSK3 cells were derived from human foreskin fibroblasts transfected with plasmid DNA encoding reprogramming factors OCT4, NANOG, SOX2 and LIN28 (kindly provided by Dr. Stephen Duncan, Medical College of Wisconsin)

32, 33

. Human iPSK3 cells

were maintained in mTeSR serum-free medium (StemCell Technologies, Inc., Vancouver, Canada) on growth factor reduced Geltrex (Life Technologies)

34

. The cells were passaged by

Accutase every 5-6 days and seeded at 1×106 cells per well of 6-well plate in the presence of 10 µM Y27632 (Sigma) for the first 24 hours 34-36. Cortical and hippocampal differentiation of hiPSCs Human iPSK3 cells were seeded into Ultra-Low Attachment (ULA) 24-well plates (Corning Incorporated, Corning, NY) at 3×105 cells/well in differentiation medium composed of



Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) plus 2% B27 serumfree supplement (Life Technologies). Y27632 (10 µM) was added during the seeding and removed after 24 hours. At day 1, the cells formed embryoid bodies (EBs) and were treated with dual SMAD signaling inhibitors 10 µM SB431542 (Sigma) and 100 nM LDN193189 (Sigma) 3739

. After 8 days, the cells were treated with fibroblast growth factor (FGF)-2 (10 ng/mL, Life

Technologies) and cyclopamine (a Shh inhibitor, 1 µM, Sigma) for cortical differentiation until day 27 27, 28. After 27 days, cells were harvested or replated for characterizations or treatments. For hippocampal differentiation 29, at day 1, the EBs were treated with 10 µM SB431542, 100 nM LDN193189, cyclopamine (1 µM), and IWP4 (a Wnt inhibitor, 2 µM, Stemgent, Cambridge, MA). At day 20, the growth factors were changed to CHIR99021 (a Wnt activator, 5 µM, STEMCELL Technologies Inc.) and brain-derived neurotrophic factor (BDNF) (5 ng/mL, R&D Systems). The cells were treated for another 7 days and then harvested or replated for characterizations or biomolecule treatments. Aβ42 oligomer treatment and culture characterizations To prepare oligomers of the Aβ42 peptide, biotinylated Aβ42 (Bachem) was fully dissolved at 0.5 mg/mL in hexafluor-2-propanole (HFIP, Sigma) 27, 28. 10 µL of HFIP Aβ(1-42) solution was dispensed into a siliconized Snap-Cap microtube, put in a desiccator to completely evaporate HFIP and thereafter stored at -80oC. Oligomer solutions were prepared freshly for each experiment. The stock was dissolved in 10 µL of DMSO (to 105 µM) and incubated for 3 hours at room temperature. Oligomers of Aβ42 were added to the neural cultures derived from human iPSK3 cells at 0 or 1 µM. Electrophysiology


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Whole-cell patch-clamp recordings were conducted on the cells after 48 days of cortical differentiation. Patch electrodes with resistances of 4-8 MΩ were pulled from borosilicate glass and fire-polished. Current traces were digitized at 20 kHz and filtered at 1 kHz respectively with an Axopatch 200B amplifier. Data acquisition and analysis were performed with pCLAMP10 software (Molecular Devices). The bath solution contained 2 mM KCl, 148 mM NaCl, 2 mM MgCl2, 10 mM HEPES and 1 mM EGTA, pH 7.4. The pipette solution contained 130 mM KCl, 10 mM HEPES and 5 mM EGTA, pH 7.4. Spontaneous post-synaptic currents were recorded in voltage-clamp configuration at a holding potential of -80 mV. Treatment with heparin, heparinase III, hyaluronic acid, and Chondroitinase ABC The cultures were treated with different molecules in presence of Aβ42 oligomers: (1) 100 Units/mL heparin (Sigma, H3149), which compete with heparan sulfate proteoglycans for Aβ42 binding to reduce Aβ42 uptake; (2) 0.05 Units/mL heparinase III (Sigma, H8891), which degrades heparan sulfate proteoglycans, (3) 0.2 mg/mL hyaluronic acid (Sigma), a type of ECM in brain perineuronal net; (4) 0.05 U/mL Chondroitinase ABC (Chabc, Sigma), which degrades chondroitin sulfate proteoglycans, for 72-96 hours, respectively. The culture without treatment was used as the control. The treated samples were evaluated by Live/Dead assay, MTT assay, and immunocytochemistry etc..

The culture supernatants were collected for lactate

dehydrogenase (LDH) activity assay. Biochemical assays: Live/Dead, MTT, Caspase, and LDH assay The cells were evaluated for viability using Live/Dead® staining kit (Molecular Probes). After 72 hours, the cells were incubated in DMEM-F12 containing 1 µM calcein-AM (green) and 2 µM ethidium homodimer I (red) for 30 min. The samples were imaged under a fluorescent microscope (Olympus IX70, Melville, NY). Using ImageJ software, the viability was analyzed



and calculated as the percentage of green intensity over total intensity (including both green cells and red cells). For MTT assay, the replated neural cells were incubated with 5 mg/mL 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) solution. The absorbance was measured at 500 nm using a microplate reader (Biorad, Richmond, CA). Image-iT™ Live Green Poly Caspase Detection kit (Molecular Probes) was used to detect the expression of caspases. The replated cells were incubated for one hour with the fluorescent inhibitor of caspases reagent and analyzed by a fluorescence microscope 40, 41. The cytotoxicity of cells was assessed using LDH activity assay kit (Sigma, MAK066). Briefly, a total volume of 100 µL of spent medium and LDH reaction mixture was mixed well and the initial absorbance at 450 nm was measured using a microplate reader (Bio-Rad iMarkTM). The mixture was incubated at 37°C and taken a measurement every 5 min. The LDH activity was calculated through the subtraction of final and initial measurements in comparison to the standard curve. Immunocytochemistry Briefly, the samples were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2-0.5% Triton X-100 for intracellular markers

42

.

The samples were then blocked and

incubated with various mouse or rabbit primary antibodies (Supplementary Table S1). After washing, the cells were incubated with the corresponding secondary antibody: Alexa Fluor® 488 goat anti-Mouse IgG or Alexa Fluor® 594 goat anti-Rabbit or donkey anti-goat IgG (Life Technologies). The samples were stained with Hoechst 33342 and visualized using a fluorescent microscope (Olympus IX70, Melville, NY). The proportion of positive cells was calculated based on the area of a marker of interest normalized to the nuclei using ImageJ analysis, indicating the relative expression among different conditions. Flow cytometry


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To quantify the levels of various marker expression, the cells were harvested by trypsinization and analyzed by flow cytometry. Briefly, 1×106 cells per sample were fixed with 4% PFA and washed with staining buffer (2% fetal bovine serum in phosphate buffer saline). The cells were permeabilized with 100% cold methanol for intracellular markers, blocked, and then incubated with primary antibodies against Aβ42, β-tubulin III, tau, or MAP2 followed by the corresponding secondary antibody (Supplementary Table S1). The cells were acquired with BD FACSCanto™ II flow cytometer (Becton Dickinson) and analyzed against isotype controls using FlowJo software. Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from neural cell samples using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol followed by the treatment of DNA-Free RNA Kit (Zymo, Irvine, CA). Reverse transcription was carried out using 2 µg of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA) (according to the protocol of the manufacturer).

Primers specific for target genes

(Supplementary Table S2) were designed using the software Oligo Explorer 1.2 (Genelink, Hawthorne, NY). The gene β-actin was used as an endogenous control for normalization of expression levels. Real-time RT-PCR reactions were performed on an ABI7500 instrument (Applied Biosystems, Foster City, CA), using SYBR1 Green PCR Master Mix (Applied Biosystems). The amplification reactions were performed as follows: 2 min at 50oC, 10 min at 95oC, and 40 cycles of 95oC for 15 sec and 55oC for 30 sec, and 68oC for 30 sec. Fold variation in

gene

expression

was

quantified

by

means

of

the

comparative

Ct

method:

2( ) , which is based on the comparison of expression of the target gene



(normalized to the endogenous control β-actin) between the cortical and the hippocampal samples. Statistical analysis Each experiment was repeated three times and the representative results were presented. To assess the statistical significance, one-way ANOVA followed by Fisher’s LSD post hoc tests were performed. A p-value

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