Another angiogenic gene linked to amyotrophic lateral sclerosis.

Abstract

A new study by Greenway and colleagues links mutations in the angiogenin gene to patients with amyotrophic lateral sclerosis (ALS)--a progressive and fatal motoneuron disease. This is an unexpected finding because angiogenin was originally identified as a molecule involved in the formation of blood vessels (angiogenesis). Angiogenin bears striking similarity to vascular endothelial growth factor (VEGF), which is the prototypic angiogenic factor that has recently emerged as a molecule with important neuroprotective activities. Besides VEGF, angiogenin is the second so-called angiogenic factor implicated in ALS, raising the question of whether additional angiogenic factors might have a role in ALS. Overall, these findings identify angiogenin as a novel candidate gene in the pathogenesis of ALS--a discovery that ultimately might lead to the development of new therapeutic strategies.

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Another angiogenic gene linked to amyotrophic
lateral sclerosis
Diether Lambrechts, Peggy Lafuste, Peter Carmeliet and Edward M. Conway
The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology (VIB), KU Leuven,
Leuven, B-3000, Belgium
A new study by Greenway and colleagues links
mutations in the angiogenin gene to patients with
amyotrophic lateral sclerosis (ALS) – a progressive and
fatal motoneuron disease. This is an unexpected finding
because angiogenin was originally identified as a mole-
cule involved in the formation of blood vessels (angio-
genesis).Angiogeninbearsstrikingsimilarity tovascular
endothelial growth factor (VEGF), which is the proto-
typic angiogenic factor that has recently emerged as a
molecule with important neuroprotective activities.
Besides VEGF, angiogenin is the second so-called angio-
genic factor implicated in ALS, raising the question of
whether additional angiogenic factors might have a role
in ALS. Overall, these findings identify angiogenin as a
novel candidate gene in the pathogenesis of ALS – a
discovery that ultimately might lead to the development
of new therapeutic strategies.
Uncovering the mysteries of ALS
Amyotrophic lateral sclerosis (ALS), also known as Lou
Gehrig’s disease, is a devastating neurodegenerative dis-
order in which the rapidly progressive loss of upper and
lower motoneurons paralyses patients, causing death
within a few years. In spite of its notoriety, the mechan-
isms underlying ALS remain obscure and therapies with
long-term benefit are lacking. Recently and unexpectedly,
a hypoxia-induced angiogenic signal –vascular endothelial
growth factor (VEGF) – was shown to contribute to the
pathogenesis of ALS [1]. Now, Greenway et al. [2] report
that mutations in the angiogenin gene (ANG), which
encodes another angiogenic factor that is upregulated by
hypoxia, might increase the risk of developing ALS [2],
further supporting the hypothesis of a neurovascular link
to ALS.
VEGF: the first angiogenic factor linked to ALS
As its name implies, angiogenin is considered to have
angiogenic properties (Box 1) (Figure 1) and is, in this
respect, similar to VEGF. Although VEGF is a pleiotropic
growthfactor,itiswidelyrecognizedasthemostimportant
molecule in angiogenesis and, indeed, has been the basis
for the first anti-angiogenic cancer therapy to be success-
fully used in the clinic [3]. The link between VEGF and
ALS,whichwasuncoveredafewyearsago,hasbeenatotal
surprise. Transgenic mice with a deletion in the hypoxia-
responsive element of the VEGF gene, which results in low
VEGF levels, develop an adult-onset, progressive neuro-
degenerative disorder similar
intriguing connection has been confirmed in humans by
demonstrating that haplotypes producing low levels of
VEGF increase the risk of ALS in several populations
(Sweden [4], England [4], Belgium [4] and New England
USA [5]), but not in all [6,7]. Further mechanistic studies
in ALS animal models indicated that VEGF exerts
neuroprotective activities on motoneurons not only as an
angiogenic factor by increasing neurovascular perfusion,
but also via direct effects on the neurons themselves [8].
Most promising is the fact that delivery of VEGF, as a
gene or a protein, into the central nervous system
prolongs survival in rodent ALS models [8,9]. These find-
ings have prompted the development of VEGF-based
therapies for humans and have also stimulated interest
in evaluating the role that other so-called angiogenic fac-
tors might have in modifying the risk of developing this
devastating illness.
toALS [1]. This
Angiogenin: the second angiogenic factor linked to ALS
Greenway et al. [2] have studied angiogenin predicting
that, similar to VEGF, its functional abnormalities would
enhance the risk of ALS. To test this hypothesis, these
authors led an impressive Euro–American collaborative
effort in which the coding and flanking regions of the ANG
gene were sequenced in almost 3000 individuals, half of
whom had ALS. At least seven different heterozygous
missense mutations were identified in the ANG gene of
those patients with ALS. Five of the ANG mutations were
locatedinresiduesoftheevolutionarilyconservedcatalytic
site, whereas one mutation was located in the region that
encodesthenucleartranslocationsignal,whichisessential
for angiogenin trafficking into the nucleus. Unfortunately,
the functional consequence of these ANG mutations was
not studied and, therefore, a direct causative link between
ANG mutations and ALS is still lacking. Thus, the means
by which these mutations might reduce, modify or perhaps
even increase the angiogenic or neuroprotective activity of
angiogenin remain a mystery.
Putative role of ANG mutations in ALS
The most direct approach that is generally used to define
the function of a gene or a mutation is to generate a
transgenic animal model. Gene-targeting studies in mice
might provide important insights. Unfortunately, this
approach has been confounded by the fact that there
are six ANG genes and three pseudogenes in mice,
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Corresponding author: Carmeliet, P. (peter.carmeliet@med.kuleuven.be)
Available online 14 July 2006.
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whereas only a single ANG gene exists in humans [10].
As a result, definitive studies that delineate the biological
properties of angiogenin are lacking, and this imposes
limitations for the identification of the molecular mechan-
isms by which ANG mutations might contribute to ALS.
An exciting hypothesis is that angiogenin, similar to
VEGF, might be neuroprotective via overlapping path-
ways(Figure1).Recentevidence suggeststhat angiogenin
is expressed by motoneurons and glial cells, especially
when stressed by hypoxia, and is neuroprotective by
stimulating the survival of stressed motoneurons in
culture [11]. Endothelial cells in the spinal cord might
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TRENDS in Molecular MedicineVol.12 No.8
Box 1. Biological activities of angiogenin
? In
culture medium as an angiogenic factor. It is however unclear whether
angiogenin is as potent as other angiogenic factors. In vitro, the
mitogenic activity of angiogenin on endothelial cells is only minimal
compared with that of basic fibroblast growth factor or VEGF [15], but
in vivo angiogenin has angiogenic activities in several experimental
models [16].
? Angiogenin is expressed by and also affects endothelial cells,
vascular smooth muscle cells and many tumour cells.
? Putative receptors for angiogenin on the cell surface have been
identified, but these have not been shown to induce signalling.
? Angiogenin shares structural homology with the pancreatic RNase
superfamily. Its RNase activity is low compared with that of the
prototypic pancreatic RNase A. Limited evidence suggests that the
RNase activity of angiogenin might mediate its angiogenic activity.
? Hypoxic conditions stimulate the expression of angiogenin.
? The angiogenic effects of VEGF are dependent, to someextent, upon
theactivityofangiogenin(i.e.angiogeninseemstobeadownstream
effector of VEGF, at least in endothelial cells [17]).
? Circulating angiogenin levels have been associated with the
progression of various tumours, whereas angiogenin inhibitors
1985, angiogeninwas isolated fromtumour-conditioned
efficiently suppress tumour growth in mice, partially by inhibiting
tumour angiogenesis (but also possibly by inducing tumour
necrosis).
? Angiogenin mRNA levels are low in the developing foetus,
increase in the neonate and are maximal in the adult. This expres-
sion pattern is not temporally related to vascular development,
implying that angiogenin might also function in processes other
than angiogenesis.
? Angiogenin is translocated to the nucleus, where it enhances rRNA
transcription via binding to specific DNA elements in rRNA genes
and thereby stimulates ribosomal biogenesis [18].
? Angiogenin also exhibits microbicidal activity against bacterial and
fungal pathogens, suggesting that it also contributes to systemic
responses to infection [12].
? Angiogenin is a liver-derived component of normal serum and
expression is upregulated in the acute-phase response suggesting
that it has a role in the host response to injury.
? An Asian leaf-eating monkey, the douc langur, does not express
angiogenin because of a homozygous missense mutation in the
single ANG gene. No vascular or neurological phenotype has been
detected but extensive studies have not been performed.
Figure 1. Angiogenin binds to the endothelial (or motoneuron) cell surface and interacts with a 170-kDa putative receptor and/or a 42-kDa binding protein, after which it is
internalized and goes to the nucleus. Once there, angiogenin binds to ribosomal DNA to enhance rRNA transcription and contribute to cell proliferation, angiogenesis and,
possibly, neuroprotection. The vessel-forming properties of VEGF and other angiogenic factors are, in part, also dependent upon the nuclear activity of angiogenin.
However, the crosstalk pathways between VEGF and angiogenin and their respective receptors have not been delineated (dashed lines). It is possible that angiogenin
functions at the nuclear crossroads of angiogenic and/or neurotrophic growth-factor activity.
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also be a source of angiogenin for motoneurons, but it is
unknown whether angiogenin crosses the blood–brain
barrier or whether it is present in the cerebrospinal fluid.
These questions have only minimally been addressed
experimentally and it has not been shown that angiogenin
is required for VEGF to exert neuroprotective signals.
The alternative or additional possibility that angiogenin
might have VEGF-independent modes of action that
increase susceptibility to ALS (e.g. by regulating innate
immunity [12] or rRNA synthesis [13]) has not been
explored nor excluded.
Whatever the mechanism, the association of ALS with
ANG mutations is of great interest and relevance. The
search to identify genes and factors that impact on the
pathogenesis or progression of ALS continues to be chal-
lenging, especially for the sporadic forms of the disease
where little progress has been made. Intriguingly, Green-
way et al. [2] have reported that ANG mutations are
present in patients with both sporadic and familial forms
of ALS. In four families, ANG mutations were found in
affected but not in unaffected siblings. Unfortunately,
proof that ANG mutations were inherited in these pedi-
grees was not provided because of the unavailability of
DNA from some family members. It is surprising that ANG
mutations caused both the sporadic and familial form of
ALS. This might be explained by implicating modifying
(genetic or environmental) factors that might influence the
penetrance of ALS in individuals with ANG mutations.
Indeed, a variable contribution of these modifying factors
might explain why ANG mutations might cause clinically
apparent ALS in some but not in other members within a
single family (affected members in such a family might be
referred to as ‘sporadic ALS’ cases, even though ANG
mutations are inherited). This notion is supported by
the finding of an apparently healthy 65-year-old individual
with an ANG mutation [2]. Similar observations have been
made in Parkinson’s disease where mutations in the
LRKK2 gene cause both sporadic and familial forms of
the disease [14].
An additional striking observation made by Greenway
etal.[2]isthat12of15patientswithANGmutationswereof
Irish or Scottish descent. These individuals all shared simi-
larancienthaplotypes,whichmostprobablyoriginatedfrom
an Irish and Scottish ancestral population and are likely to
have been spread by emigration. This suggests that ANG
mutations are largely restricted to patients from Irish and/
or Scottish ancestry and that ANG mutations in ALS are
probably rare; in this study population, they accounted for
?1% of the patients with ALS.
Remaining challenges
The identification of ANG mutations in patients with ALS
marksasignificantmilestoneinALSresearchbyproviding
additional intriguing evidence that supports a link
between ALS and angiogenesis. But challenges remain:
a priority is to unravel the physiological function of angio-
genin in more detail, and this will require sophisticated
methodology to overcome the obstacle of the existence of
multiple ANG genes. The second and most crucial
challenge will be to elucidate the molecular mechanisms
underlying the links between angiogenin and ALS and to
translate these into effective treatments for ALS. Seeking
a cure for this terrible disease is, however, worth all the
effort.
Conflict of interest
The author, PC, is an inventor of patent applications
related to the use of VEGF to treat neurological disorders,
including ALS.
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