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 patients. 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.

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 impairments (e.g. intellectual
disability and microcephaly), as well as muscle weakness and impaired movement.

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As the condition is a progressive disorder, many symptoms of triple-A syndrome
may present later in life and worsen over time. 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 named 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 WD-repeat
regions. This finding was particularly interesting to the investigators because
this repeat motif is known to form b-propeller structures involved in protein-protein
interactions and proper protein folding. Defects in WD-repeat proteins have
been implicated in the pathogenesis of several diseases such as Cockayne
syndrome and dactylaplasia. 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 base 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, as they are involved in a diverse
array of cellular processes such as signal transduction, RNA processing, and
vesicular trafficking. Thus, after the discovery of the AAAS gene, researchers next examined its pattern of expression to
better understand the alterations in the gene are involved in triple-A

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 technique. 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. 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. Using the anti-CNE19 antibody specific for ALADIN, western blots
showed that the protein was only expressed in pancreas, adrenal and pituitary
glands but not in the kidney, skeletal muscle, and placenta. Thus, the specific
expression of ALADIN in these tissues may explain why disruption of the protein
results in some or all of the triple A 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-ALADIN. These cells were then fixed, labelled with antibodies
against Nup358 and Tpr to visualize nuclear pore complexes (NPCs), and
visualized using deconvoluted microscopy.  Imaging results revealed that NPC and ALADIN
fluorescence signals co-localized, however the ALADIN and Nup358 signals
overlapped more closely than the ALADIN and Tpr signals. While Tpr is localized
to the nuclear basket, Nup358 (also known as RanBP2) is present on the
cytoplasmic face of NPCs.  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.  As many of the
triple-A mutations result in the C-terminal truncation of ALADIN, subcellular
localization of ALADINR478X, the most severe of these C-terminally
truncated mutants, was analyzed. 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, which suggests that C-terminus of ALADIN is
necessary for the targeting of the nucleoporin to NPCs. However, alone the
C-terminus of ALADIN was 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. 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 (GFP-ALADIN100-546) 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 due to experimental design.

Interestingly, one triple-A linked point mutation in the
N-terminus (Q15K) did not affect ALADIN NPC localization. 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 proper protein folding leading to ALADIN mislocalization (ex: GFP-ALADINH160R,
mutants do localize to NPCs and (like Q15K) may also disrupt the ability of
ALADIN to interact with proteins or exist within a critical protein complex. 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 proper 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 function of NPCs. The
authors investigated if failure of ALADIN to localize to the NPC disrupts
general nucleocytoplasmic transport or NPC structure and assembly. First, the
structure of the NE and NPCs in fibroblasts derived from a patient possessing non-functional
ALADIN (due to an AAAS splice-site
mutation) was examined via electron microscopy. Compared with a normal
fibroblast cell line, the nuclei, NEs, and NPCs displayed a normal morphology.

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. Localization of these
proteins were unchanged compared to control cells suggesting that the
selectivity barrier is unaffected. Thus, ALADIN mutations result in 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.

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. 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. 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. 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. 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). This interaction was independently
confirmed through co-immunoprecipitation and FRET techniques. FTH1 is a well-known
nuclear protein, so it was thought that ALADIN was necessary for its nuclear
import. To test this, SK-N-SH 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. 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 (cite). Thus, the inability of this protein to
localize to the nucleus when ALADIN is absent from NPCs may lead to increased
levels of oxidative stress, resulting in increased cell death and contributing
heavily to the triple-A phenotype. To test if increased oxidative stress could
be involved in triple-A pathogenesis, 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 the
activity of the antioxidant enzymes glutathione reductase and glutathione
peroxidase and is commonly used indicator of the intensity of oxidative stress.

A lower GSH/GSSG ratio compared to control implies that a greater amount of
glutathione is present in its oxidized (GSSG) form, which is indicative of
oxidative stress. 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 staining. 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 other cell types.