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

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

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

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