Angiogenin mutations that disrupt the protein’s RNase action or cellular location have been associated with both familial and sporadic forms of ALS (see ARF related news story on Greenway et al., 2006) as well as Parkinson’s disease (see ARF related news story on van Es et al., 2011), but it remains unproven as an ALS gene, wrote John Hardy of the University College London, U.K., in an e-mail to ARF (see also Schymick et al., 2007). However, as Prehn noted in an e-mail, biological evidence suggests the protein does play a role in the disease. Angiogenin concentrations are abnormally reduced in the plasma and cerebrospinal fluid of people with ALS (McLaughlin et al., 2010), while it protects motor neurons from excitotoxic insults and serum starvation in vitro. Moreover, treating ALS model mice with angiogenin delays their death (Kieran et al., 2008; see ARF related news story on Li et al., 2011).

In the current work, joint first authors Alexandra Skorupa and Matthew King and colleagues figured out how angiogenin might protect neurons. In immunofluorescence experiments with mouse primary mixed motor neuron cultures, they observed that, while motor neurons possessed endogenous angiogenin, they did not internalize recombinant angiogenin added to the media. Conversely, astrocytes that did not make much angiogenin on their own did take it up, shunting it first into vesicles and then into the nucleus. Based on these data, the team hypothesized that motor neurons produce angiogenin and astroglia internalize it.

In further experiments, the researchers showed that conditioned medium from stressed NSC34 motor neuron-like cells contained angiogenin, and that purified astrocytes took it up. This confirmed the neuron-to-astrocyte signal. To examine the astrocyte-to-neuron response, the team treated mixed cultures with recombinant angiogenin for six hours, washed away the exogenous RNase, then collected the media 18 hours later and tested it on a different mixed culture. They found that the conditioned media protected neurons from toxicity caused by the glutamate agonist AMPA. The authors concluded that angiogenin-treated cultures produce something that protects motor neurons, but they did not investigate what confers that protection.

The researchers did pursue the mechanism of angiogenin uptake. They reasoned that there were two possible entryways: clathrin-mediated or lipid raft-mediated endocytosis. They found that only a clathrin pathway blocker affected angiogenin uptake. The researchers suspected the angiogenin receptor might be syndecan 4, which binds similar angiogenic, neuroprotective factors (Tkachenko et al., 2004). Finding that syndecan 4 and angiogenin colocalize in astrocytes supported their suspicion. That RNA interference for syndecan 4 blocked angiogenin uptake reinforced it.

What does angiogenin do once inside astrocytes? Since the protein cleaves RNAs, Prehn and colleagues examined astrocyte nucleic acids, finding that angiogenin fragmented them. However, the ALS-linked angiogenin mutation lysine-401-isoleucine failed to mediate the nuclease effect. “Angiogenin regulates astroglia function by targeting novel subsets of RNAs,” the researchers concluded in the paper, noting that “future studies will need to identify and characterize these novel angiogenin substrates.” The researchers are currently exploring the relevant RNAs, Prehn wrote in an e-mail to ARF. They are also partnering with a company to develop angiogenin as a therapy, he wrote.—Amber Dance.

Reference:
Skorupa A, King MA, Aparicio IM, Dussmann H, Coughlan K, Breen B, Kieran D, Concannon CG, Marin P, Prehn JH. Motoneurons secrete angiogenin to induce RNA cleavage in astroglia. J Neurosci. 2012 Apr 11;32(15):5024-38. Abstract