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The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent.

ABSTRACT: Angiogenin (ANG), a member of the RNase superfamily (also known as RNase 5) has neurotrophic, neuroprotective and angiogenic activities. Recently it has also been shown to be important in stem cell homeostasis. Mutations in ANG are associated with neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal dementia (FTD). ANG is a secreted protein which is taken up by cells and translocated to the nucleus. However, the import pathway/s through which ANG is taken up is/are still largely unclear. We have characterised the uptake of ANG in neuronal, astrocytic and microglial cell lines as well as primary neurons and astrocytes using pharmacological agents as well as dominant negative dynamin and Rab5 to perturb uptake and intracellular trafficking. We find that uptake of ANG is largely clathrin/dynamin independent and microtubule depolymerisation has a marginal effect. Perturbation of membrane ruffling and macropinocytosis significantly inhibited ANG uptake suggesting an uptake mechanism similar to RNase A. Our findings shed light on why mutations which do not overtly affect RNase activity but cause impaired localization are associated with neurodegenerative disease.

SUBMITTER: Ferguson R

PROVIDER: S-EPMC5828446 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent.

Ferguson Ross R Subramanian Vasanta V

PloS one 20180227 2

Angiogenin (ANG), a member of the RNase superfamily (also known as RNase 5) has neurotrophic, neuroprotective and angiogenic activities. Recently it has also been shown to be important in stem cell homeostasis. Mutations in ANG are associated with neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal dementia (FTD). ANG is a secreted protein which is taken up by cells and translocated to the nucleus. However, the import pathway/s through which ANG is taken up ...[more]

PMID: 29486010

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Project description:Development of nonviral particles for gene delivery requires a greater understanding of the properties that enable gene delivery particles to overcome the numerous barriers to intracellular DNA delivery. Linear poly(beta-amino) esters (PBAE) have shown substantial promise for gene delivery, but the mechanism behind their effectiveness is not well quantified with respect to these barriers. In this study, we synthesized, characterized, and evaluated for gene delivery an array of linear PBAEs that differed by small changes along the backbone, side chain, and end group of the polymers. We examined particle size and surface charge, polymer molecular weight, polymer degradation rate, buffering capacity, cellular uptake, transfection, and cytotoxicity of nanoparticles formulated with these polymers. Significantly, this is the first study that has quantified how small differential structural changes to polymers of this class modulate buffering capacity and polymer degradation rate and relates these findings to gene delivery efficacy. All polymers formed positively charged (zeta potential 21-29 mV) nanosized particles (?150 nm). The polymers hydrolytically degraded quickly in physiological conditions, with half-lives ranging from 90 min to 6 h depending on polymer structure. The PBAE buffering capacities in the relevant pH range (pH 5.1-7.4) varied from 34% to 95% protonatable amines, and on a per mass basis, PBAEs buffered 1.4-4.6 mmol of H(+)/g. When compared to 25 kDa branched polyethyleneimine (PEI), PBAEs buffer significantly fewer protons/mass, as PEI buffers 6.2 mmol of H(+)/g over the same range. However, due to the relatively low cytotoxicity of PBAEs, higher polymer mass can be used to form particles than with PEI and total buffering capacity of PBAE-based particles significantly exceeds that of PEI. Uptake into COS-7 cells ranged from 0% to 95% of cells and transfection ranged from 0% to 93% of cells, depending on the base polymer structure and the end modifications examined. Five polymers achieved higher uptake and transfection efficacy with less toxicity than branched-PEI control. Surprisingly, acrylate-terminated base polymers were dramatically less efficacious than their end-capped versions, in terms of both uptake (1-3% for acrylate, 75-94% for end-capped) and transfection efficacy (0-1% vs 20-89%), even though there are minimal differences between acrylate and end-capped polymers in terms of DNA retardation in gel electrophoresis, particle size, zeta potential, and cytotoxicity. These studies further elucidate the role of polymer structure for gene delivery and highlight that small molecule end-group modification of a linear polymer can be critical for cellular uptake in a manner that is largely independent of polymer/DNA binding, particle size, and particle surface charge.

Dataset Information

The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent.

Publications

The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent.

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