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Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures.

ABSTRACT: Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedited technique for separation of neurons from co-cultured astrocytes. Our newly established method makes use of cold jet, which exploits different adhesion characteristics of subpopulations of cells (Jirsova etal., 1997), and is rapid, performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this purification method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis determined that many astrocytic mRNAs and biological processes are regulated by neuronal interaction. Our results validate the cold jet as an efficient method to separate astrocytes from neurons in co-culture, and reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

SUBMITTER: Goudriaan A

PROVIDER: S-EPMC3906515 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures.

Goudriaan Andrea A Camargo Nutabi N Carney Karen E KE Oliet Stéphane H R SH Smit August B AB Verheijen Mark H G MH

Frontiers in cellular neuroscience 20140130

Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedited technique for separation of neurons ...[more]

PMID: 24523672

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Project description:Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease caused by loss of motor neurons. ALS patients experience rapid deterioration in muscle function with an average lifespan of 3-5 years after diagnosis. Currently, the most effective therapeutic only extends lifespan by a few months, thus highlighting the need for new and improved therapies. Neurotrophic factors (NTFs) are important for neuronal development, maintenance, and survival. NTF treatment has previously shown efficacy in pre-clinical ALS models. However, clinical trials using NTFs produced no major improvements in ALS patients, due in part to the limited blood brain barrier (BBB) penetration. In this study we assessed the potential neuroprotective effects of a novel class of compounds known as MicroNeurotrophins (MNTs). MNTs are derivatives of Dehydroepiandrosterone (DHEA), an endogenous neurosteroid that can cross the BBB and bind to tyrosine kinase receptors mimicking the pro-survival effects of NTFs. Here we sought to determine whether MNTs were neuroprotective in two different models of ALS. Our results demonstrate that BNN27 (10 ?M) attenuated loss of motor neurons co-cultured with astrocytes derived from human ALS patients with SOD1 mutations via the reduction of oxidative stress. Additionally, in the G93A SOD1 mouse, BNN27 (10 mg/kg) treatment attenuated motor behavioral impairment in the paw grip endurance and rotarod tasks at postnatal day 95 in female but not male mice. In contrast, BNN27 (10 mg/kg and 50 mg/kg) treatment did not alter any other behavioral outcome or neuropathological marker in male or female mice. Lastly, BNN27 was not detected in post-mortem brain or spinal cord tissue of treated mice due to the rapid metabolism of BNN27 by mouse hepatocytes relative to human hepatocytes. Together, these findings demonstrate that BNN27 treatment failed to yield significant neuroprotective effects in the G93A SOD1 model likely due to its rapid rate of metabolism in mice.

Dataset Information

Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures.

Publications

Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures.

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