Astroglia in Thick Tissue with Super Resolution and Cellular Reconstruction

<div><p>We utilized the recently published method of passive CLARITY to explore brain astrocytes for the first time with our optimized method. Astrocytes are the fundamental cells in the brain that act to maintain the synaptic activity of neurons, support metabolism of all neurons, and communicate through extensive networks throughout the CNS. They are the defining cell that differentiates lower organisms from humans. From a disease vantage point they are the principal cause of brain tumors and the propagator of neurodegenerative diseases like amyotrophic lateral sclerosis. New methods to study these cells is paramount. Our modified use of CLARITY provides a new way to study these brain cells. To reduce cost, speed up tissue clearing process, reduce human handling error, and to retrieve quantifiable data from single confocal and pseudo-super resolution microscopy we modified and optimized the original protocol.</p></div

cleared cortex shows highly detailed astroglia processes and interactions.

<p>A) astroglia processes interact with other astroglia and cerebral blood vessels, b) computational zoom shows highly branched astroglia processes interacting with other astroglia, c) magnified image of cortical astroglia wrapping a local cerebral blood vessel.</p

Pseudo super resolution uncovers intimate branching and interactions covering nearby cells.

<p>A) 3d-rendering of a single astroglia at super resolution shows overlap with local non-astroglia cells, b) automated morphological reconstruction shows minor and major processes with detailed contextual resolution.</p

automated morphological reconstruction of cleared astroglia.

<p>A) overview of single photon image of grey matter astroglia, b) automated morphological reconstruction of cleared astroglia shows great diversity, c) magnified reconstruction of grey matter astroglia shows highly branched processes, d) magnified reconstruction of grey matter astroglia shows volume diversity amongst astroglia.</p

Astroglial transcriptome dysregulation in early disease of an ALS mutant SOD1 mouse model

<p>Astroglia are a morphologically diverse and highly abundant cell type in the CNS. Despite these obvious observations, astroglia still remain largely uncharacterized at the cellular and molecular level. In disease contexts such as amyotrophic lateral sclerosis (ALS), it has been widely shown that astroglia downregulate crucial physiological functions, become hypertrophied, reactive, and toxic to motor neurons. However, little is known about the astroglia-specific transcriptomic changes that occur during ALS disease progression, especially early in disease. To address this, we FACS-isolated pure astroglia from early and mid-symptomatic superoxide dismutase 1 (SOD1) G93A spinal cord and performed microarray sequencing, in hopes to uncover markers and pathways driving astroglia dysfunction in ALS. After extensive analyses, we uncovered genes selectively enriched and downregulated in both control and SOD1 astroglia at both disease points. In addition, we were able to identify genes and pathways differentially expressed that may have relevance with other neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease, suggesting a common theme among astroglial dysfunction in neurodegenerative disease. In aggregate, this study sheds light on the common and unique themes of dysfunction that astroglia undergo during neurodegenerative disease progression and provides candidate targets for therapeutic approaches.</p

fALS-iPSC lines.

<p>Note: NA, not applicable/not known; ND, not determined; M, male; F, female; W, white; AA, African American.</p><p>fALS-iPSC lines.</p

Differentiation of SOD1-iPSCs to astroglia.

<p>Representative pictures of control (006) and SOD1-iPSC (008) derived astrocyte after 15-week differentiation showing astroglial marker expression. (A) CD44 (red) and GFAP (green) expression by differentiated cells. Thin arrows indicate GFAP+ only cells. Arrowheads indicate CD44+ only cells. Thick arrows indicate CD44+/GFAP+ cells. (B) Quantification of CD44+ and GFAP+ cells at different time points. (C) EAAT1 (red) expression by GFAP+ (green) astrocytes. Arrows indicate double positive cells. (D) Aquaporin 4 (AQ4, red) and EAAT2 (green) expression. Nuclei were stained with DAPI (blue). Size bar, 20μm.</p

Differentiation of SOD1-iPSCs to NPCs.

<p>(A, B) NPCs were generated via EB formation. (A) Rosettes and neural tube structures were formed and the cells expressed Pax6 and Sox1. Representative pictures were taken from 006 control and 002 SOD1 lines. Nuclei were stained with DAPI. (B) Quantification of colonies with rosette structures. (C, D) NPCs were induced by inhibition of SMAD pathway. Most cells expressed Pax6, Sox2 and Sox1 at week 2. Nuclei were stained with DAPI. (D) Dynamic examination of Pax6, Sox1 and Sox2 expression by NPCs after 2–5 week. (E) Time line of neural induction by the inhibition of SMAD pathway and astrocyte differentiation. Size bar, 50μm.</p

Some mRNA species are more susceptible to oxidative damage in SOD1^G93A mice.

<p>(A) Semi-quantitative RT-PCR analysis confirmed that the identified mRNA species by DNA microarray were present in the oxidized mRNA pool. MBP, cytochrome c, cytochrome c oxidase Va and ribosome protein S6 mRNAs, which had strong signal intensities on the arrays, were present in the oxidized mRNA pool. MAP2 and PCM1 mRNAs, which had very weak signal intensities on the arrays, were hardly detected in the oxidized mRNA pool. These oxidized mRNA species are significantly decreased in vitamin E treated SOD1^G93A mice (<i>G93A+vitE</i>). (B) Semi-quantitative RT-PCR analysis showed that the oxidized mRNA species are not upregulated in the whole spinal cord of SOD1^G93A mice compared to their non-transgenic littermates. SOD1 mRNA, which includes endogenous mouse SOD1 and transgenic human SOD1, was used as a control. n = 3.</p

RNA oxidation is an early event preceding motor neuron degeneration.

<p>Lumbar spinal cords from indicated age of SOD1^G93A mice (<i>G93A</i>) and non-transgenic littermates (<i>WT</i>) were examined. (A) In the ventral horn, motor neurons with strong 15A3 immunoreactivity had normal nuclear and chromatin morphology by Hoechst 33342 staining; the dying motor neurons showed abnormal nuclear and chromatin morphology but less 15A3 immunoreactivity. <i>Arrows</i> point to the same neuron. Scale bar, 25 µm. (B) Motor neurons with strong 15A3 immunoreactivity had only minor mitochondrial vacuolization as examined by electron microscopy. Scale bar, 0.5 µm. (C) Motor neurons with strong 15A3 immunoreactivity did not have ubiquitinated protein aggregation.</p