Generation and validation of novel adeno-associated viral vectors for the analysis of Ca 2+ homeostasis in motor neurons

A finely tuned Ca homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and...

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Bibliographic Details
Published in:Scientific reports 2017-07, Vol.7 (1), p.6521
Main Authors: Norante, Rosa Pia, Massimino, Maria Lina, Lorenzon, Paolo, De Mario, Agnese, Peggion, Caterina, Vicario, Mattia, Albiero, Mattia, Sorgato, Maria Catia, Lopreiato, Raffaele, Bertoli, Alessandro
Format: Article
Language:English
Subjects:
Animals
Calcium - metabolism
Dependovirus - genetics
Fluorescent Dyes - analysis
Genes, Reporter
Genetic Vectors
Homeostasis
Mice
Motor Neurons - physiology
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Summary:A finely tuned Ca homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.
ISSN:2045-2322
DOI:10.1038/s41598-017-06919-0