Triple A Syndromeis an inherited autosomal recessive disorder defined by three features: alacrima(absence of tear secretion), achalasia (inability of the lower esophagealsphincter to relax), and adrenal insufficiency, though this last feature failsto manifest in select patients. In addition to these hallmark features, thisdisease may impact the autonomic nervous system, which controls several diverseand involuntary processes such as blood pressure and body temperature.Consequently, this disease is highly variable in terms of severity, age ofonset, and number of symptoms observed. Interestingly, triple-A syndrome hasbeen associated with other neurological impairments (e.g. intellectualdisability and microcephaly), as well as muscle weakness and impaired movement.

As the condition is a progressive disorder, many symptoms of triple-A syndromemay present later in life and worsen over time. Currently, there is no cure andavailable treatments are tailored to manage individual signs and symptoms ofthe disease.            To find thedysfunctional gene implicated in triple-A syndrome, Huebner et. al. investigated 47 affectedfamilies using a genome-wide systematic scan and identified a gene of intereston chromosome 12q13 which they named AAAS.Sequence analysis revealed that this gene contains 16 exons and encodes aprotein of 546 residues with a molecular mass of ~60 kDa. This protein, referredto as ALADIN (alacrima achalasia adrenal insufficiencyneurologic disorder protein), was also shown to contain four WD-repeatregions. This finding was particularly interesting to the investigators becausethis repeat motif is known to form b-propeller structures involved in protein-proteininteractions and proper protein folding.

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Defects in WD-repeat proteins havebeen implicated in the pathogenesis of several diseases such as Cockaynesyndrome and dactylaplasia. While the presence of these repeat regions in theprotein sequence provides a clue on how this protein functions in normal cells,it is insufficient to base conclusions on the precise activity of ALADIN, orhow mutations could affect its function, based on this evidence alone. This ispartly due to the diversity of WD-repeat proteins, as they are involved in a diversearray of cellular processes such as signal transduction, RNA processing, andvesicular trafficking.

Thus, after the discovery of the AAAS gene, researchers next examined its pattern of expression tobetter understand the alterations in the gene are involved in triple-Apathogenesis. Since triple-A syndrome is characterized by a specific setof abnormalities, it was suspected that AAASmight be expressed exclusively in affected tissues involved in the disease. Choet. al. first determined theexpression levels of the wild-type AAAS allele in human tissues using themultiple human tissue northern (MTN) blot technique. Labelled DNA probesconsisting 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 resultsshowed that the gene was expressed in all tissues tested, but more highlyexpressed in the placenta, testis, pancreas, kidneys, cerebellum, gastrointestinaltract, and the adrenal and pituitary glands. To examine if the AAAS mRNA is translationally repressedin 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 tissueavailability. Using the anti-CNE19 antibody specific for ALADIN, western blotsshowed that the protein was only expressed in pancreas, adrenal and pituitaryglands but not in the kidney, skeletal muscle, and placenta. Thus, the specificexpression of ALADIN in these tissues may explain why disruption of the proteinresults in some or all of the triple A phenotype.   After it was shown that AAASis ubiquitously transcribed but only translated in select tissues, thesubcellular localization of wild type and mutant ALADIN was investigated to provideinsight on the normal function of the protein and its role in triple-A syndrome.To elaborate on previous cell fractionation assays, which had demonstrated thatALADIN is associated with the nuclear membrane, Cronshaw and Matunis examinedthe subcellular localization of the wild-type protein by transfecting HeLacells with GFP-ALADIN.

These cells were then fixed, labelled with antibodiesagainst Nup358 and Tpr to visualize nuclear pore complexes (NPCs), andvisualized using deconvoluted microscopy.  Imaging results revealed that NPC and ALADINfluorescence signals co-localized, however the ALADIN and Nup358 signalsoverlapped more closely than the ALADIN and Tpr signals. While Tpr is localizedto the nuclear basket, Nup358 (also known as RanBP2) is present on thecytoplasmic face of NPCs.  Therefore,these imaging results implicate ALADIN as a nucleoporin and pinpoint itslocalization to the cytoplasmic face of nuclear pores.             Next,Cronshaw and Matunis examined the specific domains of the protein essential totarget ALADIN to the NPC.

  As many of thetriple-A mutations result in the C-terminal truncation of ALADIN, subcellularlocalization of ALADINR478X, the most severe of these C-terminallytruncated mutants, was analyzed. HeLa cells were transfected with GFP-tagged ALADINR478Xand the NPCs were visualized as before (with antibodies against Nup358 andTpr). Unlike the wild-type protein, GFP-ALADINR478X was founddispersed in the cytoplasm, which suggests that C-terminus of ALADIN isnecessary for the targeting of the nucleoporin to NPCs. However, alone theC-terminus of ALADIN was insufficient to target the protein to NPCs because whenHeLa 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 targetingALADIN to NPCs, the authors created a series of N-terminal deletion mutants. Whentransfected into HeLa cells, a fluorescently tagged ALADIN mutant lacking thefirst 100 residues (GFP-ALADIN100-546) was found distributedthroughout the cell, including the nucleus, indicating that the N-terminaldomain is also needed to target ALADIN to the NPC. As N-terminally truncatedALADIN was found in the nucleus, this domain may also contain a cytoplasmic retentionsignal, however there is not strong evidence to support this claim and thisresult may due to experimental design. Interestingly, one triple-A linked point mutation in theN-terminus (Q15K) did not affect ALADIN NPC localization. This residue may beinvolved in interactions with other proteins or factors essential for ALADINfunction, such as transport cargo or structural proteins. Analysis of mutationsin the WD-repeats of ALADIN yielded similar results.

While some WD-mutations dodisrupt proper protein folding leading to ALADIN mislocalization (ex: GFP-ALADINH160R,GFP-ALADINS263P, and GFP-ALADINV313A), some WD-ALADINmutants do localize to NPCs and (like Q15K) may also disrupt the ability ofALADIN to interact with proteins or exist within a critical protein complex. Inconclusion, these sets of experiments by Cronshaw and Matunis show that triple-Asyndrome-linked AAAS mutations eitherresult in mislocalization of ALADIN to the cytoplasm by affecting proteinstructure (i.e.  C-terminal truncation)or interfere with the ability of ALADIN to interact with factors essential forits proper function. These types of mutations could cause defects in NPCstructure and/or nucleocytoplasmic transport.

            Nucleoporins like ALADIN play roles essential to thestructure and function of NPCs. Theauthors investigated if failure of ALADIN to localize to the NPC disruptsgeneral nucleocytoplasmic transport or NPC structure and assembly. First, thestructure of the NE and NPCs in fibroblasts derived from a patient possessing non-functionalALADIN (due to an AAAS splice-sitemutation) was examined via electron microscopy. Compared with a normalfibroblast cell line, the nuclei, NEs, and NPCs displayed a normal morphology.

These results were confirmed through immunofluorescence microscopy usingnucleoporin specific antibodies. To detect if these ALADIN mutants affected theselectivity barrier of NPCs, cells were also immunostained with antibodiesagainst importin b and transportin. Localization of theseproteins were unchanged compared to control cells suggesting that theselectivity barrier is unaffected. Thus, ALADIN mutations result in functionalrather than structural defects. This makes sense in the context of the disease,as disruption of normal NPC structure and general nucleocytoplasmic transport wouldalmost certainly be lethal while triple-A syndrome itself is not lethal andmost tissues are unaffected.As the inquiry into triple-A syndromeprogressed, studies began to reveal some rare cases of triple-A syndrome thatare 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 aminoacid or WD-repeat domains of ALADIN interrupt interactions between ALADIN andessential 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 interactionwas first discovered through co-immunoprecipitation assays in 293T cells transfectedwith 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 confirmedto occur in living cells as when HeLa cells were transfected with FLAG-NDC1and GFP-ALADIN, the two fusion proteins were observed to co-localize at thenuclear rim via confocal microscopy. These sets of experiments were importantto 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 thefunction of ALADIN.

 Since failure of ALADIN to localize to theNPC is known to at least partially cause the triple-A phenotype, the authorsinvestigated the role of NDC1 in this process. HeLa cells were transfected withGFP-ALADIN and shRNA against NDC1 to knock down expression of NDC1 andsubjected to fluorescence microscopy. Confocal imaging revealed that while GFP-ALADINlocalized to the NPCs in control co-transfected cells, the fusion protein wasfound dispersed in the cytoplasm in NDC1 knockdown cells. These results stronglyimply that NDC1 is important in ALADIN localization to the NPCs and suggests amechanism by which it acts to tether the protein at the cytoplasmic face of theNPC through interactions with WD-repeats and Q15 of ALADIN.

These results alsosuggest that the genetic cause of triple-A syndrome in patients withoutmutations in AAAS may be thedisruption of NDC1. If impairment of NDC1 is responsible for themanifestation of triple-A syndrome in some patients, then examining how loss ofNDC1 affects nuclear transport may shed light on the disease-causing mechanismof mutated ALADIN. Yamazumi et. al. examined the nuclear import of the NLS ofSIV40 large T antigen and XRCC1 in NDC1 knockdown cells. HeLa cells wereco-transfected with either Dronpa-tagged NLSSV40 or Dronpa-XRCC1 andvisualized via confocal imaging. Dronpa-NLSSV40 mislocalized to thecytoplasm while Dronpa-XRCC1 still localized to the nucleus, which shows that NDC1is required for selective nuclear import of NLSSV40. Importantly,this may indicate that NDC1-mediated anchoring of ALADIN to NPCs is essentialfor the nuclear import of essential proteins whose absence in the nucleuscontribute to the triple-A phenotype.

            The work of Storr et. al. elaborated on thisconclusion by attempting to find protein cargos whose transport is mediated byALADIN. Through bacterial two-hybrid screens, in which constructs containing thefull-length ALADIN coding sequence were used as “bait” for “prey” cDNA librariesconstructed from a HeLa cell line or human cerebellar tissue, ALADIN was foundto interact with ferritin heavy-chain protein (FTH1). This interaction was independentlyconfirmed through co-immunoprecipitation and FRET techniques. FTH1 is a well-knownnuclear protein, so it was thought that ALADIN was necessary for its nuclearimport. To test this, SK-N-SH cells wereco-transfected with FTH1-V5-HIS andEGFP-AAAS constructs (eitherwild-type or mutant) and imaged through immunofluorescence microscopy. FTH1-V5-HIS localized to the nucleus incells co-transfected with wild-type AAASconstructs, but was aberrantly localized to the cytoplasm when co-transfectedwith the EGFP-mutant AAAS constructs.

This result shows that ALADIN is neededat NPCs to mediate the import of FTH1 into the nucleus.            FTH1has an antioxidant activity in the nucleus, where it helps to prevent DNAdamage. In the presence of FTH1, the ability of free iron present in thenucleus to convert reactive oxygen species into free radicals and to induce DNAdamage is markedly reduced (cite).

Thus, the inability of this protein tolocalize to the nucleus when ALADIN is absent from NPCs may lead to increasedlevels of oxidative stress, resulting in increased cell death and contributingheavily to the triple-A phenotype. To test if increased oxidative stress couldbe involved in triple-A pathogenesis, Prasad et. al. assayed the effect of AAASknockdown on redox homeostasis in the adrenocortical cell line H295R bymeasuring the levels of glutathione and glutathione disulfide (also known asoxidized glutathione). The GSH/GSSG ratio represents the redox level and theactivity of the antioxidant enzymes glutathione reductase and glutathioneperoxidase and is commonly used indicator of the intensity of oxidative stress.A lower GSH/GSSG ratio compared to control implies that a greater amount ofglutathione is present in its oxidized (GSSG) form, which is indicative ofoxidative stress.

When AAAS wasknocked down via shRNA in H295R, the GSH/GSSG ratio was significantly decreasedcompared to that of cells transfected with control shRNA. This increasedoxidative stress was shown to induce apoptosis, evidenced by heightened levelsof cleaved PARP, and reduce the viability of H295R adrenal cells, evidenced byreduced propidium iodide staining. These events were confirmed to be caused byoxidative stress as treating these cells with the antioxidant N-acetylcysteine(NAC) returned cell viability levels back to that of controls. This datasupports the conclusion that the absence of ALADIN at the NPCs results in anincrease in oxidative stress and cell death in adrenal cells, most likely dueto the failure to import FTH1 into the nucleus. It is unclear if this effect isspecific to adrenal cells or if other cells have protective or redundantmechanisms since conflicting results were found in other cell types.       Conclusion