Fatty acids increase neuronal hypertrophy of Pten knockdown neurons

Fricano, Catherine J.; DeSpenza, Tyrone; Frazel, Paul W.; Li, Meijie; O’Malley, A. James; Westbrook, Gary L.; Luikart, Bryan W.

doi:10.3389/fnmol.2014.00030

Cited by 21 publications

(20 citation statements)

References 28 publications

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“…Injected animals were transcardially perfused with 4% paraformaldehyde and 4% sucrose for immunohistochemical analysis at varying days-post injection (DPI) utilizing previously described methods (Luikart et. al., 2011; Fricano et. al., 2014).…”

Section: Methodsmentioning

confidence: 99%

Rapamycin prevents, but does not reverse, aberrant migration in Pten knockout neurons

Getz

DeSpenza

et al. 2016

Neurobiology of Disease

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Phosphatase and tensin homolog (PTEN) is a major negative regulator of the Akt/mammalian target of rapamycin (mTor) pathway. Mutations in PTEN have been found in a subset of individuals with autism and macrocephaly. Further, focal cortical dysplasia (FCD) has been observed in patients with PTEN mutations prompting us to examine the role of Pten in neuronal migration. The dentate gyrus of PtenFlox/Flox mice was injected with Cre- and non-Cre-expressing retroviral particles, which integrate into the dividing genome to birthdate cells. Control and Pten knockout (KO) cell position in the granule cell layer was quantified over time to reveal that Pten KO neurons exhibit an aberrant migratory phenotype beginning at 7.5 days-post retroviral injection (DPI). We then assessed whether rapamycin, a mTor inhibitor, could prevent or reverse aberrant migration of granule cells. The preventative group received daily intraperitoneal (IP) injections of rapamycin from 3–14 DPI, before discrepancies in cell position have been established, while the reversal group received rapamycin afterward, from 14–24 DPI. We found that rapamycin prevented and reversed somal hypertrophy. However, rapamycin prevented, but did not reverse aberrant migration in Pten KO cells. We also find that altered migration occurs through mTorC1 and not mTorC2 activity. Together, these findings suggest a temporal window by which rapamycin can treat aberrant migration, and may have implications for the use of rapamycin to treat PTEN-mutation associated disorders.

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Section: Methodsmentioning

confidence: 99%

Rapamycin prevents, but does not reverse, aberrant migration in Pten knockout neurons

Getz

DeSpenza

et al. 2016

Neurobiology of Disease

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“…Moen, Fricano, and colleagues were interested in factors affecting the soma size of neurons in vivo. 10,26 They examined the effect of (1) knocking down the phosphatase and tensin homolog (Pten) gene, (2) infusion of fatty acids (FA) via osmotic minipumps, and (3) the interaction of Pten knockdown and FA infusion on the soma size. Only a subset of the data are used here to highlight the key points with a simpler analysis.…”

Section: Fatty Acid Datasetmentioning

confidence: 99%

A Bayesian predictive approach for dealing with pseudoreplication

Lazic

Mellor

Ashby

et al. 2019

Preprint

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Pseudoreplication occurs when the number of measured values or data points exceeds the number of genuine replicates, and when the statistical analysis treats all data points as independent and thus fully contributing to the result. By artificially inflating the sample size, pseudoreplication contributes to irreproducibility, and it is a pervasive problem in biological research. In some fields, more than half of published experiments have pseudoreplicationmaking it one of the biggest threats to inferential validity. Researchers may be reluctant to use appropriate statistical methods if their hypothesis is about the pseudoreplicates and not the genuine replicates; for example, when an intervention is applied to pregnant female rodents (genuine replicates) but the hypothesis is about the effect on the multiple offspring (pseudoreplicates). We propose using a Bayesian predictive approach, which enables researchers to make valid inferences about biological entities of interest, even if they are pseudoreplicates, and show the benefits of this approach using two in vivo data sets.

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“…6. Following stereotaxic injection, the mice can be euthanized for slice prep electrophysiology 1 or perfused and their brains removed, sliced, and stained via immunohistochemistry for analysis 10 .…”

Section: Stereotaxic Injectionmentioning

confidence: 99%

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Fricano-Kugler¹,

Williams²,

Salinaro³

et al. 2016

JoVE

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Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto-or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

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Fatty acids increase neuronal hypertrophy of Pten knockdown neurons

Cited by 21 publications

References 28 publications

Rapamycin prevents, but does not reverse, aberrant migration in Pten knockout neurons

Rapamycin prevents, but does not reverse, aberrant migration in Pten knockout neurons

A Bayesian predictive approach for dealing with pseudoreplication

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

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