Triple associated with other neurological deficiencies (e.g. intellectual

            Triple A Syndromeis an inherited autosomal recessive disorder defined by three features: alacrima(absence of tear secretion), achalasia (inability of the loweresophageal sphincter to relax), and adrenal insufficiency, though thislast feature fails to manifest in select patientsi.In addition to these hallmark features, this disease may impact the autonomicnervous system, which controls several diverse and involuntary processes suchas blood pressure and body temperature i. Consequently, this diseaseis 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 motorfunctions.

As the condition is a progressive disorder, many symptoms of triple-Asyndrome may present later in life and worsen over timeii.Currently, there is no cure and available treatments are tailored to manage individualsigns and symptoms of the 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 termed AAASiii.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 tryptophan-asparticacid (WD)-repeat regions.

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This discovery of particular interest to theinvestigators because this repeat motif is known to form b-propeller structures involvedin protein-protein interactions and proper protein foldingiv.Defects in WD-repeat proteins have been implicated in the pathogenesis of severaldiseases such as Cockayne syndrome and dactylaplasiav.While the presence of these repeat regions in the protein sequence provides aclue on how this protein functions in normal cells, it is insufficient to makeconclusions on the precise activity of ALADIN, or how mutations could affectits function, based on this evidence alone.

This is partly due to the diversityof WD-repeat proteins, which are involved in a diverse array of cellularprocesses such as signal transduction, RNA processing, and vesiculartrafficking iv. Thus, after the discovery of the AAAS gene, scientists aimed to determineits pattern of expression to better understand the alterations in the gene areinvolved in triple-A pathogenesis. 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 AAASallele in human tissues using the multiple human tissue northern (MTN) blottechniquevi.Labelled DNA probes consisting of exon 1 or spanning exons 4-16 of the genewere used to detect AAAS mRNA in 16different human tissues, including those unaffected by the disease. Interestingly,MTN blot results showed that the gene was expressed in all tissues tested, butmore highly expressed in the placenta, testis, pancreas, kidneys, cerebellum, gastrointestinaltract, and the adrenal and pituitary glands vi.

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. Western blots showed that the protein was only expressed inpancreas, adrenal and pituitary glands but not in the kidney, skeletal muscle,and placenta vi. Since the triple-A syndrome is associated withdefects in tissues in which ALADIN is expressed, it is highly likely that theprotein performs a crucial function that when absent results (at least somewhat)in the disease 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-ALADINvii.

These cells were then fixed, labelled with fluorophore-conjugated antibodiesagainst Nup358 and Tpr to visualize nuclear pore complexes (NPCs), and observedby deconvoluted microscopy. NPC and ALADIN fluorescence signals co-localized,however the ALADIN signal was shown to overlap more closely with the Nup358signal than that of the Tpr signal. While Tpr is localized to the nuclearbasket, Nup358 (also known as RanBP2) is present on the cytoplasmic face ofNPCs, where it carries out essential functions in nuclear transport.  Therefore, these imaging results implicate ALADINas a nucleoporin and pinpoint its localization to the cytoplasmic face ofnuclear pores.             Next,Cronshaw and Matunis examined the specific domains of the protein essential totarget ALADIN to the NPC.

  Many of thetriple-A mutations result in the C-terminal truncation of ALADIN, so the subcellularlocalization of ALADINR478X, the most severe of these C-terminallytruncated mutants, was analyzed iv, vii. HeLa cells were transfectedwith GFP-tagged ALADINR478X and the NPCs were visualized as before(with antibodies against Nup358 and Tpr). Unlike the wild-type protein, GFP-ALADINR478Xwas found dispersed in the cytoplasm, suggesting that the C-terminus of ALADINis necessary for the targeting of the nucleoporin to NPCs. However, theC-terminus alone is 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 vii. To find other domains necessaryfor targeting ALADIN to NPCs, the authors created a series of N-terminal deletionmutants. When transfected into HeLa cells, a fluorescently tagged ALADIN mutantlacking the first 100 residues was found distributed throughout the cell,including the nucleus, indicating that the N-terminal domain is also needed totarget ALADIN to the NPC. As N-terminally truncated ALADIN was found in thenucleus, this domain may also contain a cytoplasmic retention signal, howeverthere is not strong evidence to support this claim and this result may be dueto experimental design vii. Interestingly, one triple-A linked point mutation in theN-terminus (Q15K) did not affect ALADIN localization iv.

This residuemay be involved in interactions with other proteins or factors essential forALADIN function, such as transport cargo or structural proteins. Analysis ofmutations in the WD-repeats of ALADIN yielded similar results. While some WD-mutationsdo disrupt protein folding leading to ALADIN mislocalization, some WD-ALADINmutants do localize to NPCs and (like Q15K) may disrupt the ability of ALADINto interact with proteins or exist within a critical protein complex iv.In conclusion, 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 correct function. These types of mutations could cause defects in NPCstructure and/or nucleocytoplasmic transport.             Nucleoporins like ALADIN play roles essential to thestructure and/or function of NPCs.

Cronshaw and Matunis attempted to refine therole of ALADIN in generalnucleocytoplasmic transport or NPC structure and assembly in patient fibroblastcells with non-functional ALADIN (due to an AAASsplice-site mutation). First, the structure of the NE and NPCs in patient-derivedfibroblasts were examined via electron microscopy. Compared to controlfibroblasts, the nuclei, NEs, and NPCs of these cells displayed a normalmorphology vii.

These results were confirmed throughimmunofluorescence microscopy using nucleoporin specific antibodies. To detectif these ALADIN mutants affected the selectivity barrier of NPCs, cells werealso immunostained with antibodies against importin b andtransportin vii. Localization of these proteins were unchangedcompared to control cells suggesting that the selectivity barrier isunaffected. Based on these results, ALADIN mutations must cause 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 i,ii.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 demonstrated that this protein interacted with ALADIN viii.This interaction was first discovered through co-immunoprecipitation assays in 293Tcells transfected with FLAG-NDC1.

When lysates were immunoprecipitated with ananti-FLAG antibody, ALADIN was one of the proteins identified by LC-basedtandem mass spectrometry (MS/MS).  Thisinteraction was confirmed to occur in living cells as when HeLacells were transfected with FLAG-NDC1 and GFP-ALADIN, the two fusion proteinswere 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 andALADIN 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 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 viii. Theseresults strongly imply that NDC1 is important in ALADIN localization to theNPCs and suggests a mechanism by which it acts to tether the protein at thecytoplasmic face of the NPC through interactions with WD-repeats and Q15 ofALADIN. These results also suggest that the genetic cause of triple-A syndromein patients without mutations in AAASmay be the disruption 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 of SIV40 large T antigen and XRCC1 in NDC1knockdown cells viii. HeLa cells were co-transfected with either Dronpa-taggedNLSSV40 or Dronpa-XRCC1 and visualized via confocal imaging. Dronpa-NLSSV40mislocalized 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 NPCsis essential for the nuclear import of essential proteins whose absence in thenucleus contribute to the triple-A phenotype.            The work of Storr et. al. elaborated on this conclusion by attempting to find proteincargos whose transport is mediated by ALADIN. Through bacterial two-hybridscreens, in which constructs containing the full-length ALADIN coding sequence wereused as “bait” for “prey” cDNA libraries constructed from a HeLa cell line or humancerebellar tissue, ALADIN was found to interact with ferritin heavy-chainprotein (FTH1) ix.

Thisinteraction was independently confirmed through co-immunoprecipitation and FRETtechniques. FTH1 is a well-known nuclear protein, so it was thought that ALADINcould 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 ormutant) and imaged through immunofluorescence microscopy. FTH1-V5-HIS localized to the nucleus in cells co-transfected with wild-typeAAAS constructs, but was aberrantlylocalized to the cytoplasm when co-transfected with the EGFP-mutant AAAS constructsix. This result shows that ALADIN is needed at NPCs to mediate the importof 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 ix. Thus, the inability of this proteinto localize to the nucleus may lead to increased levels of oxidative stress, whichin turn could result in increased cell death and contribute heavily to thetriple-A phenotype.

To test this hypothesis, 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 reflectsthe activity of the antioxidant enzymes glutathione reductase and glutathioneperoxidase. A decreased GSH/GSSG ratio implies that a greater amount ofglutathione is present in its oxidized/GSSG form and therefore indicates increasedoxidative stress x. WhenAAAS was knocked down via shRNA inH295R, the GSH/GSSG ratio was significantly decreased compared to that of cellstransfected with control shRNA. This increased oxidative stress was shown toinduce apoptosis, evidenced by heightened levels of cleaved PARP, and reducethe viability of H295R adrenal cells, evidenced by reduced propidium iodide(PI) staining x.

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 neuroblastoma cells and othercell types.

            Thestudy of triple-A syndrome has yielded significant results as, for the firsttime, a nucleoporin has been implicated as the cause of a hereditary disease inhumans. Mutations in the AAAS gene preventthe localization of ALADIN to the NPCs and / or its interaction with essentialfactors, including transport machinery and/or cargo. This prevents the nuclearimport of FTH1 in adrenal cells and severely increases oxidative stress ix.

Among the features of triple-A syndrome is adrenal insufficiency which, basedon these results, may be caused by a massive wave of oxidative stress-induced celldeath. Indeed, ATCH-resistant adrenal insufficiency can when more than 90% of adrenalglands are destroyed xi.However, this oxidative stress mechanism does not explain the triple-A phenotypein other tissues. Inconsistent results in other cell lines suggest may hint thatadrenal cells are more susceptible to oxidative stress. Conversely, cells of othertissues may possess compensatory FTH1 transport mechanisms,sparing them from oxidative stress. Puzzlingly, no studies have addressed themechanism by which ALADIN mediates the nuclear import of FTH1. This is a salientquestion as ALADIN has not been shown to contain a NLS and there is noevidence that it can cross the NE ix.

 One popular hypothesis is that ALADIN is neededfor the assembly of an essential transport complex at the cytoplasmic face ofthe NPC that is necessary for the nuclear import of FTH1, but further researchis needed to support this idea. Finally, due to the extreme variation in thedisease phenotype, it is very likely that other ALADIN associated proteins ortriple-A associated genes that are compromised in this disease. Theseadditional factors may be involved in the manifestation of alacrima andachalasia in those affected by triple-A syndrome.

Referencesi Triple A syndrome. Geneticand Rare Diseases Information Center.

gov/diseases/457/triple-a-syndrome.Published September 24, 2015. Accessed December 18, 2017. ii Prpic, I.,Huebner, A., Persic, M.

, Handschug, K. and Pavletic, M. (2003), Triple Asyndrome: genotype–phenotype assessment. Clinical Genetics, 63: 415–417.doi:10.


00070.x iii Huebner A, Yoon SJ, OzkinayF, et al. (Nov 2000).

Triple A syndrome–clinical aspects and moleculargenetics. Endocr. Res. 26 (4): 751–759. doi:10.

3109/07435800009048596 iv Huebner A,Kaindl AM, Knobeloch KP, Petzold H, Mann P, Koehler K. The triple Asyndrome is due to mutations in Aladin, a novel member of the nuclear porecomplex. Endocrine Research 2004.

 30 891–899. doi: 10.1081/ERC-200044138 v Handschug K, Sperling S,Yoon SK, Hennig S, Clark AJL, Huebner A. Triple A syndrome is caused bymutations in AAAS, a new WD-repeat protein gene. Hum Mol Genet 2001;10:283–290. vi Cho A-R, Yang K-J,Bae Y, et al. Tissue-specific expression and subcellular localization ofALADIN, the absence of which causes human triple A syndrome. Experimental& Molecular Medicine.


6.043. vii Cronshaw JM, MatunisMJ. The nuclear pore complex protein ALADIN is mislocalized in triple Asyndrome.

 Proceedings of the National Academy of Sciences of the UnitedStates of America. 2003;100(10):5823-5827. doi:10.

1073/pnas.1031047100. viii YamazumiY, Kamiya A, Nishida A, Nishihara A, Iemura S, Natsume T, Akiyama T.

Thetransmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclearpore complexes. Biochem Biophys Res Commun. 2009;389:100–104 ix Storr HL, Kind B,Parfitt DA, et al. Deficiency of Ferritin Heavy-Chain Nuclear Import in TripleA Syndrome Implies Nuclear Oxidative Damage as the Primary DiseaseMechanism.

 Molecular Endocrinology. 2009;23(12):2086-2094.doi:10.1210/me.2009-0056. x Prasad R, MetherellLA, Clark AJ, Storr HL.

Deficiency of ALADIN Impairs Redox Homeostasis in HumanAdrenal Cells and Inhibits Steroidogenesis. Endocrinology.2013;154(9):3209-3218. doi:10.1210/en.2013-1241.

 xi ACTH resistance.CheckOrphan. AccessedDecember 18, 2017.