Triple onset, and number of symptoms observed. Interestingly, triple-A

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 iii. 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

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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 techniquevi. 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.

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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.

Next, 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,

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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

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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.

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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”

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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.

FTH1 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

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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.

The 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.

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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.

References

i Triple A syndrome. Genetic and Rare Diseases Information Center.
https://rarediseases.info.nih.gov/diseases/457/triple-a-syndrome. Published September 24, 2015. Accessed
December 18, 2017.

ii

iii Huebner A, Yoon SJ, Ozkinay F, et al. (Nov 2000). Triple A syndrome–clinical aspects and molecular genetics.
Endocr. Res. 26 (4): 751–759. doi:10.3109/07435800009048596

iv

v Handschug K, Sperling S, Yoon SK, Hennig S, Clark AJL, Huebner A. Triple A syndrome is caused by mutations
in AAAS, a new WD-repeat protein gene. Hum Mol Genet 2001; 10:283–290.

vi

vii

viii

ix

x

xi ACTH resistance. CheckOrphan. http://www.checkorphan.org/diseases/acth-resistance. Accessed December 18,
2017.