Triple in triple-A syndrome, Huebner et. al.

            Triple A Syndrome
is an inherited autosomal recessive disorder defined by three features: alacrima
(absence of tear secretion), achalasia (inability of the lower
esophageal sphincter to relax), and adrenal insufficiency, though this
last feature fails to manifest in select patientsi.

In addition to these hallmark features, this disease may impact the autonomic
nervous system, which controls several diverse and involuntary processes such
as blood pressure and body temperature i. Consequently, this disease
is highly variable in terms of severity, age of onset, and number of symptoms observed.

Interestingly, triple-A syndrome has been associated with other neurological deficiencies
(e.g. intellectual disability and microcephaly), as well as impaired motor
functions. As the condition is a progressive disorder, many symptoms of triple-A
syndrome may present later in life and worsen over timeii.

Currently, there is no cure and available treatments are tailored to manage individual
signs and symptoms of the disease.

            To find the
dysfunctional gene implicated in triple-A syndrome, Huebner et. al. investigated 47 affected
families using a genome-wide systematic scan and identified a gene of interest
on chromosome 12q13 which they termed AAAS

Sequence analysis revealed that this gene contains 16 exons and encodes a
protein of 546 residues with a molecular mass of ~60 kDa. This protein, referred
to as ALADIN (alacrima achalasia adrenal insufficiency
neurologic disorder protein), was also shown to contain four tryptophan-aspartic
acid (WD)-repeat regions. This discovery of particular interest to the
investigators because this repeat motif is known to form b-propeller structures involved
in protein-protein interactions and proper protein foldingiv.

Defects in WD-repeat proteins have been implicated in the pathogenesis of several
diseases such as Cockayne syndrome and dactylaplasiav.

While the presence of these repeat regions in the protein sequence provides a
clue on how this protein functions in normal cells, it is insufficient to make
conclusions on the precise activity of ALADIN, or how mutations could affect
its function, based on this evidence alone. This is partly due to the diversity
of WD-repeat proteins, which are involved in a diverse array of cellular
processes such as signal transduction, RNA processing, and vesicular
trafficking iv. Thus, after the discovery of the AAAS gene, scientists aimed to determine
its pattern of expression to better understand the alterations in the gene are
involved in triple-A pathogenesis.

Since triple-A syndrome is characterized by a specific set
of abnormalities, it was suspected that AAAS
might be expressed exclusively in affected tissues involved in the disease. Cho
et. al. first determined the
expression levels of the wild-type AAAS
allele in human tissues using the multiple human tissue northern (MTN) blot

Labelled DNA probes consisting of exon 1 or spanning exons 4-16 of the gene
were used to detect AAAS mRNA in 16
different human tissues, including those unaffected by the disease. Interestingly,
MTN blot results showed that the gene was expressed in all tissues tested, but
more highly expressed in the placenta, testis, pancreas, kidneys, cerebellum, gastrointestinal
tract, and the adrenal and pituitary glands vi. To examine if the AAAS mRNA is translationally repressed
in unaffected tissues, ALADIN levels were probed by western blot analysis. However,
in this line of experiments, ALADIN expression was only probed in adrenal, pituitary,
pancreatic, kidney, placental, and skeletal muscle samples due to low tissue
availability. Western blots showed that the protein was only expressed in
pancreas, adrenal and pituitary glands but not in the kidney, skeletal muscle,
and placenta vi. Since the triple-A syndrome is associated with
defects in tissues in which ALADIN is expressed, it is highly likely that the
protein performs a crucial function that when absent results (at least somewhat)
in the disease phenotype.    

After it was shown that AAAS
is ubiquitously transcribed but only translated in select tissues, the
subcellular localization of wild type and mutant ALADIN was investigated to provide
insight on the normal function of the protein and its role in triple-A syndrome.

To elaborate on previous cell fractionation assays, which had demonstrated that
ALADIN is associated with the nuclear membrane, Cronshaw and Matunis examined
the subcellular localization of the wild-type protein by transfecting HeLa
cells with GFP-ALADINvii.

These cells were then fixed, labelled with fluorophore-conjugated antibodies
against Nup358 and Tpr to visualize nuclear pore complexes (NPCs), and observed
by deconvoluted microscopy. NPC and ALADIN fluorescence signals co-localized,
however the ALADIN signal was shown to overlap more closely with the Nup358
signal than that of the Tpr signal. While Tpr is localized to the nuclear
basket, Nup358 (also known as RanBP2) is present on the cytoplasmic face of
NPCs, where it carries out essential functions in nuclear transport.  Therefore, these imaging results implicate ALADIN
as a nucleoporin and pinpoint its localization to the cytoplasmic face of
nuclear pores.

Cronshaw and Matunis examined the specific domains of the protein essential to
target ALADIN to the NPC.  Many of the
triple-A mutations result in the C-terminal truncation of ALADIN, so the subcellular
localization of ALADINR478X, the most severe of these C-terminally
truncated mutants, was analyzed iv, vii. HeLa cells were transfected
with GFP-tagged ALADINR478X and the NPCs were visualized as before
(with antibodies against Nup358 and Tpr). Unlike the wild-type protein, GFP-ALADINR478X
was found dispersed in the cytoplasm, suggesting that the C-terminus of ALADIN
is necessary for the targeting of the nucleoporin to NPCs. However, the
C-terminus alone is insufficient to target the protein to NPCs because when
HeLa cells were transfected with the C-terminal domain of the protein (GFP- ALADIN317-546),
the fragment localized to the cytoplasm vii. To find other domains necessary
for targeting ALADIN to NPCs, the authors created a series of N-terminal deletion
mutants. When transfected into HeLa cells, a fluorescently tagged ALADIN mutant
lacking the first 100 residues was found distributed throughout the cell,
including the nucleus, indicating that the N-terminal domain is also needed to
target ALADIN to the NPC. As N-terminally truncated ALADIN was found in the
nucleus, this domain may also contain a cytoplasmic retention signal, however
there is not strong evidence to support this claim and this result may be due
to experimental design vii.

Interestingly, one triple-A linked point mutation in the
N-terminus (Q15K) did not affect ALADIN localization iv. This residue
may be involved in interactions with other proteins or factors essential for
ALADIN function, such as transport cargo or structural proteins. Analysis of
mutations in the WD-repeats of ALADIN yielded similar results. While some WD-mutations
do disrupt protein folding leading to ALADIN mislocalization, some WD-ALADIN
mutants do localize to NPCs and (like Q15K) may disrupt the ability of ALADIN
to interact with proteins or exist within a critical protein complex iv.

In conclusion, these sets of experiments by Cronshaw and Matunis show that triple-A
syndrome-linked AAAS mutations either
result in mislocalization of ALADIN to the cytoplasm by affecting protein
structure (i.e.  C-terminal truncation)
or interfere with the ability of ALADIN to interact with factors essential for
its correct function. These types of mutations could cause defects in NPC
structure and/or nucleocytoplasmic transport.

            Nucleoporins like ALADIN play roles essential to the
structure and/or function of NPCs. Cronshaw and Matunis attempted to refine the
role of ALADIN in general
nucleocytoplasmic transport or NPC structure and assembly in patient fibroblast
cells with non-functional ALADIN (due to an AAAS
splice-site mutation). First, the structure of the NE and NPCs in patient-derived
fibroblasts were examined via electron microscopy. Compared to control
fibroblasts, the nuclei, NEs, and NPCs of these cells displayed a normal
morphology vii. These results were confirmed through
immunofluorescence microscopy using nucleoporin specific antibodies. To detect
if these ALADIN mutants affected the selectivity barrier of NPCs, cells were
also immunostained with antibodies against importin b and
transportin vii. Localization of these proteins were unchanged
compared to control cells suggesting that the selectivity barrier is
unaffected. Based on these results, ALADIN mutations must cause functional
rather than structural defects. This makes sense in the context of the disease,
as disruption of normal NPC structure and general nucleocytoplasmic transport would
almost certainly be lethal while triple-A syndrome itself is not lethal and
most tissues are unaffected i,ii.

As the inquiry into triple-A syndrome
progressed, studies began to reveal some rare cases of triple-A syndrome that
are not associated with mutations in AAAS,
suggesting that other modifying genes/factors must play a role in pathogenesis.

This finding synergizes with the thought that mutations in the 15th amino
acid or WD-repeat domains of ALADIN interrupt interactions between ALADIN and
essential protein partners. While studying the transmembrane nucleoporin NDC1,
which is involved in NPC assembly, Yamazumi et.

al. demonstrated that this protein interacted with ALADIN viii.

This interaction was first discovered through co-immunoprecipitation assays in 293T
cells transfected with FLAG-NDC1. When lysates were immunoprecipitated with an
anti-FLAG antibody, ALADIN was one of the proteins identified by LC-based
tandem mass spectrometry (MS/MS).  This
interaction was confirmed to occur in living cells as when HeLa
cells were transfected with FLAG-NDC1 and GFP-ALADIN, the two fusion proteins
were observed to co-localize at the nuclear rim via confocal microscopy viii.

These sets of experiments were important to show that not only do NDC1 and
ALADIN bind to each other in living cells, but they do so at the NE.

This heavily implies that NDC1 is essential to the function of ALADIN.  

Since failure of ALADIN to localize to the
NPC is known to at least partially cause the triple-A phenotype, the authors
investigated the role of NDC1 in this process. HeLa cells were transfected with
GFP-ALADIN and shRNA against NDC1 to knock down expression of NDC1 and
subjected to fluorescence microscopy. Confocal imaging revealed that while GFP-ALADIN
localized to the NPCs in control co-transfected cells, the fusion protein was
found dispersed in the cytoplasm in NDC1 knockdown cells viii. These
results strongly imply that NDC1 is important in ALADIN localization to the
NPCs and suggests a mechanism by which it acts to tether the protein at the
cytoplasmic face of the NPC through interactions with WD-repeats and Q15 of
ALADIN. These results also suggest that the genetic cause of triple-A syndrome
in patients without mutations in AAAS
may be the disruption of NDC1.

If impairment of NDC1 is responsible for the
manifestation of triple-A syndrome in some patients, then examining how loss of
NDC1 affects nuclear transport may shed light on the disease-causing mechanism
of mutated ALADIN. Yamazumi et. al.

examined the nuclear import of the NLS of SIV40 large T antigen and XRCC1 in NDC1
knockdown cells viii. HeLa cells were co-transfected with either Dronpa-tagged
NLSSV40 or Dronpa-XRCC1 and visualized via confocal imaging. Dronpa-NLSSV40
mislocalized to the cytoplasm while Dronpa-XRCC1 still localized to the nucleus,
which shows that NDC1 is required for selective nuclear import of NLSSV40.

Importantly, this may indicate that NDC1-mediated anchoring of ALADIN to NPCs
is essential for the nuclear import of essential proteins whose absence in the
nucleus contribute to the triple-A phenotype.

            The work of Storr et. al. elaborated on this conclusion by attempting to find protein
cargos whose transport is mediated by ALADIN. Through bacterial two-hybrid
screens, in which constructs containing the full-length ALADIN coding sequence were
used as “bait” for “prey” cDNA libraries constructed from a HeLa cell line or human
cerebellar tissue, ALADIN was found to interact with ferritin heavy-chain
protein (FTH1) ix. This
interaction was independently confirmed through co-immunoprecipitation and FRET
techniques. FTH1 is a well-known nuclear protein, so it was thought that ALADIN
could be necessary for its nuclear import. To test this, SK-N-SH neuroblastoma cells were co-transfected with FTH1-V5-HIS and EGFP-AAAS constructs (either wild-type or
mutant) and imaged through immunofluorescence microscopy. FTH1-V5-HIS localized to the nucleus in cells co-transfected with wild-type
AAAS constructs, but was aberrantly
localized to the cytoplasm when co-transfected with the EGFP-mutant AAAS constructs
ix. This result shows that ALADIN is needed at NPCs to mediate the import
of FTH1 into the nucleus.

has an antioxidant activity in the nucleus, where it helps to prevent DNA
damage. In the presence of FTH1, the ability of free iron present in the
nucleus to convert reactive oxygen species into free radicals and to induce DNA
damage is markedly reduced ix. Thus, the inability of this protein
to localize to the nucleus may lead to increased levels of oxidative stress, which
in turn could result in increased cell death and contribute heavily to the
triple-A phenotype. To test this hypothesis, Prasad et. al. assayed the effect of AAAS
knockdown on redox homeostasis in the adrenocortical cell line H295R by
measuring the levels of glutathione and glutathione disulfide (also known as
oxidized glutathione). The GSH/GSSG ratio represents the redox level and reflects
the activity of the antioxidant enzymes glutathione reductase and glutathione
peroxidase. A decreased GSH/GSSG ratio implies that a greater amount of
glutathione is present in its oxidized/GSSG form and therefore indicates increased
oxidative stress x. When
AAAS was knocked down via shRNA in
H295R, the GSH/GSSG ratio was significantly decreased compared to that of cells
transfected with control shRNA. This increased oxidative stress was shown to
induce apoptosis, evidenced by heightened levels of cleaved PARP, and reduce
the viability of H295R adrenal cells, evidenced by reduced propidium iodide
(PI) staining x. These events were confirmed to be caused by
oxidative stress as treating these cells with the antioxidant N-acetylcysteine
(NAC) returned cell viability levels back to that of controls. This data
supports the conclusion that the absence of ALADIN at the NPCs results in an
increase in oxidative stress and cell death in adrenal cells, most likely due
to the failure to import FTH1 into the nucleus. It is unclear if this effect is
specific to adrenal cells or if other cells have protective or redundant
mechanisms since conflicting results were found in neuroblastoma cells and other
cell types.

study of triple-A syndrome has yielded significant results as, for the first
time, a nucleoporin has been implicated as the cause of a hereditary disease in
humans. Mutations in the AAAS gene prevent
the localization of ALADIN to the NPCs and / or its interaction with essential
factors, including transport machinery and/or cargo. This prevents the nuclear
import of FTH1 in adrenal cells and severely increases oxidative stress ix.

Among the features of triple-A syndrome is adrenal insufficiency which, based
on these results, may be caused by a massive wave of oxidative stress-induced cell
death. Indeed, ATCH-resistant adrenal insufficiency can when more than 90% of adrenal
glands are destroyed xi.

However, this oxidative stress mechanism does not explain the triple-A phenotype
in other tissues. Inconsistent results in other cell lines suggest may hint that
adrenal cells are more susceptible to oxidative stress. Conversely, cells of other
tissues may possess compensatory FTH1 transport mechanisms,
sparing them from oxidative stress. Puzzlingly, no studies have addressed the
mechanism by which ALADIN mediates the nuclear import of FTH1. This is a salient
question as ALADIN has not been shown to contain a NLS and there is no
evidence that it can cross the NE ix.  One popular hypothesis is that ALADIN is needed
for the assembly of an essential transport complex at the cytoplasmic face of
the NPC that is necessary for the nuclear import of FTH1, but further research
is needed to support this idea. Finally, due to the extreme variation in the
disease phenotype, it is very likely that other ALADIN associated proteins or
triple-A associated genes that are compromised in this disease. These
additional factors may be involved in the manifestation of alacrima and
achalasia in those affected by triple-A syndrome.

i Triple A syndrome. Genetic
and Rare Diseases Information Center.

Published September 24, 2015. Accessed December 18, 2017.


ii Prpic, I.,
Huebner, A., Persic, M., Handschug, K. and Pavletic, M. (2003), Triple A
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iii Huebner A, Yoon SJ, Ozkinay
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iv Huebner A,
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v Handschug K, Sperling S,
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vi Cho A-R, Yang K-J,
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vii Cronshaw JM, Matunis
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viii Yamazumi
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ix Storr HL, Kind B,
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x Prasad R, Metherell
LA, Clark AJ, Storr HL. Deficiency of ALADIN Impairs Redox Homeostasis in Human
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xi ACTH resistance.

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