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Looking to get a better or clearer understanding of the many confusing processes involved I have gone back through the postings and online documents today.
There are still some confusing results with questions needing answering....
There seems to be two partially separate processes at work concerning the ANG gene and angiogenin which is produced by ANG or at least how they have been described?
On one hand we have specific mutations in the ANG gene, most of which cause loss of function but not all.
Reminder:
'What is the normal function of the ANG gene?
The ANG gene provides instructions for making a protein called angiogenin '
Also the ANG gene has to be in the right place to function correctly.................
'We next examined whether these mutant ANG undergo nuclear translocation, another essential requirement for mediating angiogenesis
Some of the mutations retained the ability to undergo nuclear translocation others could not.'
(A merge of the two panels show that WT and K17I ANG are able to translocate to the nucleus, but that S28N and P112L ANG cannot. Because nuclear translocation is essential for ANG to induce angiogenesis, S28N and P112L are unlikely to be angiogenic even though they retain 14 and 9%, respectively, of the ribonucleolytic activity .)
In the above it looks like ANG has to be able to translocate to the nucleus but if mutations S28N and P112L ANG cannot then they would not be able to help with angiogenesis.
Does that mean that increasing the product of the ANG gene (angiogenin) would not help those with these specific mutations as the ANG gene is not in the right location?
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Secondary effect of ANG gene?
The role of ANG in neuroprotection probably extends beyond its effect on endothelial cells, as significant immunostaining of ANG was observed in ventral horn motor neurons of both normal human fetal and adult spinal cords.
We have recently shown that ANG may have functions independent of angiogenesis, as it is involved more broadly in ribosomal biogenesis. ANG binds to the promoter region of rDNA and stimulates rRNA transcription.
Looking at the above in ribosomal biogenesis does ANG gene alone or the product of ANG (angiogenin) do this after ANG is bound to the promoter region of rDNA?
Does angiogenin stimulate rRNA transcription or does ANG have to bind first followed by angiogenin release?
As such, ANG function in motor neurons may be related to ribosomal biogenesis and protein translation.
A defect in this pathway, as a consequence of ANG mutation in ALS, may lead to insufficient synthesis of ribosomes, thereby affecting motor neuron viability. Efforts to create and characterize ANG transgenic and knockout mice and to determine the expression and function of ANG in motor neurons and glia cells are under way.
Mechanistic studies indicate that ANG undergoes nuclear translocation in endothelial cells where it binds to the promoter region of rDNA, stimulates rRNA transcription, and is essential for cell proliferation. ANG-mediated rRNA transcription has been shown to be required for angiogenesis induced by vascular endothelial cell growth factor (VEGF), an essential angiogenic protein that has also been implicated in ALS. In a mouse model of ALS, disruption of the promoter element of VEGF results in selective motor neuron degeneration. In SOD1G93A rats, treatment with intraventricular VEGF results in substantially improved motor function, delayed disease onset, and extended survival. In humans, VEGF was shown to be a modifier of ALS by protecting motor neurons from ischemic injury and death. A potential role of ANG in ALS is thus envisioned from its involvement in VEGF-mediated angiogenesis.
Our data suggest that ANG is the first gene in which loss-of-function mutations are documented in ALS patients.
So what are they saying in the above, in plain English.
ANG moves to the nuclear in endothelial cells where it binds a to a region of rDNA which then stimulates rRNA transcription. Vascular endothelial cell growth factor (VEGF) requires ANG rRNA transcription to function correctly to protect neurons. VEGF protects motor neurons from ischemic injury, death and if disrupted motor neurons degenerate.
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Hints that ANG mutations may be involved in protein inclusions?
A K17I mutation in the ANG gene encoding angiogenin has been identified in a case that we previously published as ALS with neuronal intranuclear protein inclusions (Seilhean et al. in Acta Neuropathol 108:81-87, 2004).
These inclusions were immunoreactive for smooth muscle alpha-actin but not for angiogenin. Moreover, they were not labeled by anti-TDP-43 antibodies, while numerous cytoplasmic inclusions immunoreactive for ubiquitin, p62 and TDP-43 were detected in both oligodendrocytes and neurons in various regions of the central nervous system. In addition, expression of smooth muscle alpha-actin was increased in the liver where severe steatosis was observed.
This is the first neuropathological description of a case with an ANG mutation. Angiogenin is known to interact with actin. Like other proteins involved in ALS pathogenesis, such as senataxin, TDP-43 and FUS/TLS, it plays a role in RNA maturation.
Looking at the role of Angiogenin......
There is some confusion in the papers because the following talks of angiogenin moves to the nucleus where it stimulates the production of ribosomal RNA (rRNA) but earlier we had a report from another paper that ANG has to be able to do that same?
This may just be sloppy write up by one of the authors, or by my reading of one of the paper but is confusing?
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Angiogenin is found in a variety of cells throughout the body and circulates in the bloodstream. When angiogenin binds to receptors on the surface of endothelial cells, it triggers a series of reactions that brings angiogenin inside the cell.
Once inside endothelial cells, angiogenin moves to the nucleus where it stimulates the production of ribosomal RNA (rRNA), a chemical cousin of DNA. Ribosomal RNA is required for assembling protein building blocks (amino acids) into functioning proteins.
Angiogenin stimulates the production of additional ribosomal RNA when there is an increased demand for proteins, such as for the growth and division of endothelial cells. Angiogenin may also be involved in other steps of angiogenesis, such as helping to break down the tissue that surrounds existing blood vessels to allow room for the growth of new blood vessels
Angiogenin is a liver-derived component of normal serum the concentration of which can increase in various disease states and its expression is regulated in vivo in a manner that is characteristic of acute phase proteins (Olson et al, 1998).
In the current work, Li and Hu lay out the biochemical pathway linking angiogenin and apoptosis inducing factor (AIF). AIF is normally located in mitochondria, but when it moves to the nucleus—as it does in ALS model mice (Oh et al., 2006)—it chews up DNA, contributing to apoptosis.
The following indicates that angiogenin stops AIF moving to the nucleus by up regulating Bcl-2 which inhibits caspase-3 ......Therefore giving an explanation of the death or destructive pathway.
Critically is this common in most ALS cases albeit by slightly different but related pathways and the cause of neuron death?
(In addition to angiogenin and AIF, the players include the apoptosis dampener Bcl-2 and the pro-apoptosis proteins caspase-3 and serum polymerase-1 (PARP-1).
The researchers delineate a pathway in which apoptosis normally proceeds from activation of caspase-3, to cleavage of PARP-1, to nuclear translocation of AIF.
Angiogenin alters the situation by upregulating Bcl-2, which inhibits caspase-3 and thus the rest of the downstream pathway. Much of this pathway was already known, noted Piera Pasinelli of Thomas Jefferson University in Philadelphia, Pennsylvania, but “they really teased out the mechanism by which angiogenin can be anti-apoptotic…in a Bcl-2-dependent manner,” she said.
To study apoptosis, the researchers starved P19 cells of serum. This treatment normally causes PARP-1 cleavage within a few hours and nuclear translocation of AIF by 24. But with angiogenin in the culture media, PARP-1 stayed whole and AIF remained mitochondrial, confirming that angiogenin blocks apoptosis via this pathway. In a previous study, the authors confirmed that angiogenin also prevents activation of caspase-3 (Li et al., 2010).
The researchers already knew Bcl-2 was upregulated by angiogenin (Li et al., 2010), and suspected it would be crucial to angiogenin’s effects. If so, then removing Bcl-2 should allow apoptosis to proceed unhindered, even in the presence of angiogenin. Li used RNA interference to knock down Bcl-2. In the knocked down, serum-starved cell cultures, angiogenin was less effective at preventing caspase-3 activation, PARP-1 cleavage and AIF translocation.)
Notice in the above the researchers said, in the last paragraph, that angiogenin was less effective which implies that it did not totally stop caspase-3 activation so is there another factor at work?
Angiogenin upregulates anti-apoptotic genes, including Bag1, Bcl-2, Hells, Nf-κb and Ripk1, and downregulates pro-apoptotic genes, such as Bak1, Tnf, Tnfr, Traf1 and Trp63.
Knockdown of Bcl-2 largely abolishes the anti-apoptotic activity of angiogenin, whereas the inhibition of Nf-κb activity results in a partial, but significant, inhibition of the protective activity of angiogenin.
Thus, angiogenin prevents stress-induced cell death through both the Bcl-2 and Nf-κb pathways.
From the above it looks like upregulating Bcl-2 and Nf-kb through some other method may also help us besides increasing angiogenin levels?
Into the heart, an air that kills, from yon far country blows.
What are those blue remembered hills, what sphires what farms are those.
That is the land of lost content,I see it shining plain,
The happy highways where I went and cannot come again
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