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Location and Number of Astrocytes Determine Dopaminergic Neuron Survival and Function Under 6-OHDA Stress Mediated Through Differential BDNF Release

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Abstract

While astrocytes throughout the CNS share many common traits, they exhibit significant differences in function and number among brain regions. The aim of the present study is to assess the effect of region-specificity and number of astrocytes on the survival of dopaminergic neurons under stress, and to understand the possible mechanism by which these astrocytes extend neuroprotection to dopaminergic neurons. Purified astrocytes obtained from forebrain, midbrain, and hindbrain region were characterized through FACS and immunofluorescence. Co-culture experiments (using trans-wells) were then performed to measure the effect of region-specificities and numbers of astrocytes on primary midbrain culture under 6-OHDA stress. Cell survival augmented with an increase in astrocyte seeding number and total cell survival was comparable among the different region-specific astrocytes for all numbers. However, striking differences were observed in dopaminergic neuronal (TH) cell survival in the presence of midbrain astrocytes in comparison to forebrain and hindbrain astrocytes at all seeding numbers. At 75 μM 6-OHDA insult, while cell survival was comparable in purified astrocytes from the different brain regions, a distinct increase in BDNF secretion (significantly higher than its constitutive release) was noted for midbrain astrocytes compared to forebrain and hindbrain astrocytes. The TH immunopositive population decreased when TrkB inhibitor was added to the co-culture under 6-OHDA toxicity, suggesting that BDNF released by co-cultured astrocytes plays a key role in the survival of dopaminergic neurons. This BDNF release decreased in presence of NO inhibitor and increased in the presence of NO donor (DETA/NO). We conclude that the BDNF released from astrocytes under 6-OHDA toxicity is mediated through NO release through both autocrine and paracrine signaling, and this BDNF release is primarily responsible for the differential effect of region-specific astrocytes on TH neuron survival under these conditions.

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Funding

This work is supported by the “Innovative Young Biotechnologist Award” achieved by I.D. from the Department of Biotechnology (DBT), Government of India, New Delhi; contract grant no. BT/07/IYBA/2013-7. K.G. is funded by Manipal University.

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Author notes

  1. Indrani Datta and Kavina Ganapathy contributed equally to this work.

Authors and Affiliations

  1. Department of Biophysics, National Institute of Mental Health and Neurosciences, an Institute of National Importance, P.B. No 2900, Hosur Road, Bengaluru, Karnataka, 560029, India

    Indrani Datta

  2. Department of Pharmacology, Al-Ameen College of Pharmacy, Bengaluru, Karnataka, India

    Kavina Ganapathy & Ramesh Bhonde

  3. School of Regenerative Medicine, Manipal University, Bengaluru, Karnataka, India

    Rema Razdan

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  1. Indrani Datta
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Correspondence to Indrani Datta.

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Electronic Supplementary Material

Supplementary Fig. 1

Representative FACS histograms showing the percentage immunopositive β-tubulin III neuronal population, GFAP astrocyte population, and TH dopaminergic neuronal population in primary MB culture in control and 6-OHDA-treated conditions. (GIF 102 kb)

High resolution image (TIFF 273 kb)

Supplementary Fig. 2

Representative dot plots of FACS analysis for annexin V/PI staining showing percentage of live cells, necrotic cells, early and late apoptotic cells in primary MB cells co-cultured with different numbers of astrocytes from three regions of the brain. (GIF 153 kb)

High resolution image (TIFF 480 kb)

Supplementary Fig. 3

Characteristic FACS analysis of GFAP, β-tubulin III, and TH immunopositive population in primary MB culture under 6-OHDA stress and co-cultured with astrocytes (80,000) from three regions of the brain. (GIF 133 kb)

High resolution image (TIFF 337 kb)

Supplementary Fig. 4

Representative FACS analysis of population immunopositive for VMAT2, α-synuclein (αSyn) and phosphorylated α-synuclein serine129 (pSyn) in primary MB cells co-cultured with 80,000 MB astrocytes under 6-OHDA stress with respect to primary MB culture in control and 6-OHDA-treated conditions. (GIF 148 kb)

High resolution image (TIFF 451 kb)

Supplementary Fig. 5

Characteristic FACS histograms of the percentage immunopositive GFAP, TH, and β-tubulin III in primary MB cells co-cultured with midbrain astrocytes at the number of 80,000 under 6-OHDA stress with and without the TrkB inhibitor (ANA12) to demonstrate the effect of BDNF on the survival of dopaminergic neurons in primary MB culture. (GIF 185 kb)

High resolution image (TIFF 434 kb)

Supplementary Fig. 6

Representative FACS histogram of population immunopositive for TH in primary midbrain cells co-cultured with 80,000 astrocytes from FB, MB, and HB. (GIF 138 kb)

High resolution image (TIFF 311 kb)

Supplementary Fig. 7

Ethidium bromide-stained gel showing the expression of nNOS and iNOS in the FB, MB, and HB astrocytes under control and 6-OHDA stress. (GIF 43 kb)

High resolution image (TIFF 151 kb)

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Datta, I., Ganapathy, K., Razdan, R. et al. Location and Number of Astrocytes Determine Dopaminergic Neuron Survival and Function Under 6-OHDA Stress Mediated Through Differential BDNF Release. Mol Neurobiol 55, 5505–5525 (2018). https://doi.org/10.1007/s12035-017-0767-0

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