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Generation and validation of novel adeno-associated viral vectors for the analysis of Ca²⁺ homeostasis in motor neurons.

ABSTRACT: A finely tuned Ca²⁺ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.

SUBMITTER: Norante RP

PROVIDER: S-EPMC5529510 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Generation and validation of novel adeno-associated viral vectors for the analysis of Ca2+ homeostasis in motor neurons.

Norante Rosa Pia RP Massimino Maria Lina ML Lorenzon Paolo P De Mario Agnese A Peggion Caterina C Vicario Mattia M Albiero Mattia M Sorgato Maria Catia MC Lopreiato Raffaele R Bertoli Alessandro A

Scientific reports 20170726 1

A finely tuned Ca2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca2+ 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 Ca2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, ...[more]

PMID: 28747684

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Project description:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

Dataset Information

Generation and validation of novel adeno-associated viral vectors for the analysis of Ca²⁺ homeostasis in motor neurons.

Publications

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

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