Abelson Helper Integration Site Research Articles

Abelson Helper Integration Site 1 haplotypes and peripheral blood expression associates with lithium response and immunomodulation in bipolar patients

RationaleIn bipolar disorder (BD), immunological factors play a role in the pathogenesis and treatment of the illness. Studies showed the potential link between Abelson Helper Integration Site 1 (AHI1) protein, behavioural changes and innate immunity regulation. An immunomodulatory effect was suggested for lithium, a mood stabilizer used in BD treatment.ObjectivesWe hypothesized that AHI1 may be an important mediator of lithium treatment response. Our study aimed to investigate whether the AHI1 haplotypes and expression associates with lithium treatment response in BD patients. We also examined whether AHI1 expression and lithium treatment correlate with innate inflammatory response genes.ResultsWe genotyped seven AHI1 single nucleotide polymorphisms in 97 euthymic BD patients and found that TG haplotype (rs7739635, rs9494332) was significantly associated with lithium response. We also showed significantly increased AHI1 expression in the blood of lithium responders compared to non-responders and BD patients compared to healthy controls (HC). We analyzed the expression of genes involved in the innate immune response and inflammatory response regulation (TLR4, CASP4, CASP5, NLRP3, IL1A, IL1B, IL6, IL10, IL18) in 21 lithium-treated BD patients, 20 BD patients treated with other mood stabilizer and 19 HC. We found significantly altered expression between BD patients and HC, but not between BD patients treated with different mood stabilizers.ConclusionsOur study suggests the involvement of AHI1 in the lithium mode of action. Moreover, mood-stabilizing treatment associated with the innate immunity-related gene expression in BD patients and only the lithium-treated BD patients showed significantly elevated expression of anti-inflammatory IL10, suggesting lithium’s immunomodulatory potential.

Role of Abelson Helper Integration Site 1, Nebulin, and Paired Box 3 Genes in the Development of Nonsyndromic Strabismus in a Series of Iranian Families: Sequence Analysis and Systematic Review of the Genetics of Nonsyndromic Strabismus.

To look for causative genetic mutations in a series of Iranian families with strabismus. In addition, we systematically reviewed all the published articles regarding the role of genetic variations in primary and nonsyndromic comitant strabismus. Four families with a history of multiple cases of primary and nonsyndromic comitant strabismus were enrolled in this study. Polymerase chain reaction and Sanger sequencing of exons 23, 11, and 3 of the Abelson helper integration site 1 (AHI1), nebulin (NEB), and paired box 3 (PAX3) genes were performed, respectively. One offspring of a consanguineous marriage underwent whole-exome sequencing (WES) to look for possible causative variants. To conduct a systematic review, we thoroughly searched PubMed, Scopus, and ISI Web of Knowledge extracting relevant publications, released by April 2021. We examined four Iranian strabismus pedigrees with multiple affected offspring in different generations. Among these 17 participants, 10 family members had strabismus and 7 were healthy. Sanger sequencing did not reveal a causative mutation. Therefore, to further investigate, one affected offspring was chosen for WES. The WES study demonstrated two possible variants in MYO5B and DHODH genes. These genetic variants showed high allele frequency in our population and are thought to be polymorphisms in our series of Iranian families. We demonstrated that mutations in AHI1, NEB, and PAX3 genes were not common in a series of Iranian patients with familial strabismus. Moreover, by performing WES, we revealed that two variants of uncertain significance as possible causative variants for strabismus are not related to this disease in our population.

The mitochondrial Ahi1/GR participates the regulation on mtDNA copy numbers and brain ATP levels and modulates depressive behaviors in mice

BackgroundPrevious studies have shown that depression is often accompanied by an increase in mtDNA copy number and a decrease in ATP levels; however, the exact regulatory mechanisms remain unclear.MethodsIn the present study, Western blot, cell knockdown, immunofluorescence, immunoprecipitation and ChIP-qPCR assays were used to detect changes in the Ahi1/GR-TFAM-mtDNA pathway in the brains of neuronal Abelson helper integration site-1 (Ahi1) KO mice and dexamethasone (Dex)-induced mice to elucidate the pathogenesis of depression. In addition, a rescue experiment was performed to determine the effects of regular exercise on the Ahi1/GR-TFAM-mtDNA-ATP pathway and depression-like behavior in Dex-induced mice and Ahi1 KO mice under stress.ResultsIn this study, we found that ATP levels decreased and mitochondrial DNA (mtDNA) copy numbers increased in depression-related brain regions in Dex-induced depressive mice and Ahi1 knockout (KO) mice. In addition, Ahi1 and glucocorticoid receptor (GR), two important proteins related to stress and depressive behaviors, were significantly decreased in the mitochondria under stress. Intriguingly, GR can bind to the D-loop control region of mitochondria and regulate mitochondrial replication and transcription. Importantly, regular exercise significantly increased mitochondrial Ahi1/GR levels and ATP levels and thus improved depression-like behaviors in Dex-induced depressive mice but not in Ahi1 KO mice under stress.ConclusionsIn summary, our findings demonstrated that the mitochondrial Ahi1/GR complex and TFAM coordinately regulate mtDNA copy numbers and brain ATP levels by binding to the D-loop region of mtDNA Regular exercise increases the levels of the mitochondrial Ahi1/GR complex and improves depressive behaviors.BE2neeR3CuVf7a9CPyYjGYVideo

Targeting a Highly Deregulated eIF4F Translation Initiation Complex Sensitises IM-Resistant Cells to Tyrosine Kinase Inhibitors and Effectively Suppresses BCR-ABL1 Protein Expression

Background We previously reported that Abelson helper integration site-1 (AHI-1) is highly deregulated in chronic myeloid leukemia (CML) leukemic stem cells (LSCs) and mediates tyrosine kinase inhibitor (TKI) resistance. AHI-1 physically interacts with BCR-ABL1 and its SH3 domain interacts with BCR-ABL1 substrates, like dynamin-2, which regulate leukemic properties of CML LSCs. However, the molecular and biological roles of AHI-1 and its interacting partners in mediating TKI resistance remain largely unknown. Objective To investigate the molecular functions of AHI-1 and its SH3 domain in regulation of TKI resistance, using advanced antibody microarray analysis. Methods A high content antibody microarray was performed in BCR-ABL1+ cells co-transduced with wild-type (WT) AHI-1 or the deletion of AHI-1 SH3 domain (SH3Δ) with or without imatinib (IM). Changes in antibody signals for protein expression or phosphorylation were determined using limma and pathway enrichment analysis by g:Profiler. eIF4G1 genetic inhibition by lentiviral-mediated shRNA or pharmacological inhibition by SBI-756, was performed in TKI-resistant cells to assay translation initiation activity by proximity ligation assay (PLA), o-propargyl-puromycin (OPP), and polysome profiling. Results The antibody microarray analysis revealed that WT AHI-1 cells have the greatest number of changes in the phospho-proteome and proteome compared to BCR-ABL1+ cells and AHI-1 SH3Δ cells with and without IM. Pathway enrichment analysis identified that the targets with significantly increased differential antibody signal after IM treatment in WT AHI-1 cells were related to the regulation of translation initiation complex (p < 0.0001). Interestingly, our RNA-seq dataset analysis further identified several eukaryotic initiation factor 4F (eIF4F) complex members to be significantly increased in CD34+ CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex (2-fold, p = 0.001). WT AHI-1 cells also showed increased expression and phosphorylation of eIF4G1 (>2-fold) and eIF4B (>2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, and cyclin D3 (a downstream protein of eIF4F translational activity) as compared to BCR-ABL1+ cells, by immunoblotting. These results were similarly demonstrated in IM-resistant cells as compared to IM-sensitive cells (2-5-fold, p < 0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p < 0.0001) and increased TKI sensitivity in IM-resistant cells (p < 0.0021). These cells showed reduced levels of eIF4F complex formation by PLA assay (p = 0.013) and reduced protein expression of cyclin D3 (40%, p = 0.0036) and BCR-ABL1 (70%). Similarly, IM-resistant cells were more sensitive to SBI-756 treatment, an eIF4G1 inhibitor (50% reduction, p = 0.0001) than IM-sensitive cells (30% reduction); these effects were enhanced by a combination of SBI-756 and IM (80%, p < 0.02). Furthermore, SBI-756 treatment reduced PLA foci formation (p = 0.014) and global protein synthesis rates (p < 0.0001). Polysome profiling demonstrated that IM-resistant cells had higher levels of translation activity, which was inhibited by SBI-756 treatment. Most interestingly, SBI-756 treatment reduced protein expression of BCR-ABL1 (70%) and cyclin D3 (40%) in these cells. Conclusion We have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells. eIF4G1 inhibitor treatment sensitises IM-resistant cells to TKI and reduces BCR-ABL1 protein expression, providing potential treatment strategies to overcome TKI resistance.

Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis.

Depression is a serious public-health issue. Recent reports have suggested higher susceptibility to viral infections in depressive patients. However, how depression affects antiviral innate immune signaling remains unknown. Here, we revealed a reduction in expression of Abelson helper integration site 1 (AHI1) in the peripheral blood mononuclear cells (PBMCs) and macrophages from the patients with major depressive disorder (MDD), which leads to attenuated antiviral immune response. We found that depression-related arginine vasopressin (AVP) induces reduction of AHI1 in macrophages. Further studies demonstrated that AHI1 is a critical stabilizer of basal type-I-interferon (IFN-I) signaling. Mechanistically, AHI1 recruits OTUD1 to deubiquitinate and stabilize Tyk2, while AHI1 reduction downregulates Tyk2 and IFN-I signaling activity in macrophages from both MDD patients and depression model mice. Interestingly, we identified a clinical analgesic meptazinol that effectively stimulates AHI1 expression, thus enhancing IFN-I antiviral defense in depression model mice. Our study promotes the understanding of the signaling mechanisms of depression-mediated antiviral immune dysfunction, and reveals meptazinol as an enhancer of antiviral innate immunity in depressive patients.

Abelson helper integration site 1 haplotypes and peripheral blood expression associates with lithium response in bipolar patients

Abelson helper integration site 1 haplotypes and peripheral blood expression associates with lithium response in bipolar patients

Identification of a Highly Deregulated eIF4F Translation Initiation Complex in Drug-Resistant BCR-ABL + Cells By a Phospho-Proteomic Antibody Microarray

Protein-tyrosine kinase inhibitors (TKIs) have been effective for treatment of early stages of chronic myeloid leukemia (CML). However, BCR-ABL-dependent resistance mechanisms and TKI-unresponsive quiescent leukemic stem cells (LSCs) can result in drug resistance and disease relapse. We have demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs. It interacts with BCR-ABL through the AHI-1 WD40 domain and with other proteins, like dynamin-2 (DNM2), through its SH3 domain, to enhance leukemic-initiating activities and TKI resistance. To uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance in CML stem/progenitor cells, an advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL + cells co-transduced with wild-type Ahi-1 or Ahi-1 SH3 Δ mutant in the presence or absence of imatinib (IM). The microarray simultaneously quantified the differences in expression and phosphorylation sites of proteins in multiple signaling pathways using 1325 antibodies in duplicate measurements, and each microarray analysis was performed in duplicate. Significant changes in antibody signals for protein expression or phosphorylation were determined using limma. This analysis revealed that the overexpression of wild-type Ahi-1 (WT Ahi-1) has a profound differential effect, compared to the SH3 domain deleted Ahi-1 (Ahi-1 SH3 Δ), on the expression and phosphorylation status of proteins in BCR-ABL + cells with and without IM treatment. BCR-ABL + cells co-expressing WT Ahi-1 had a greater number of significantly differential antibody signals (7 increases, 42 decreases) when compared to BCR-ABL + cells, while Ahi-1 SH3 Δ expressing cells resulted in fewer significantly differential antibody signals (12 increases, 2 decreases) compared to BCR-ABL + cells. IM treatment resulted in WT Ahi-1 expressing cells having the greatest number of significantly differential antibody signals (7 increases, 56 decreases) compared to BCR-ABL + cells (5 decreases) and those co-expressing Ahi-1 SH3 Δ (2 increases, 9 decreases). Pathway enrichment analysis, using gProfiler, identified that the targets with significantly increased differential antibody signal after IM treatment in WT Ahi-1 cells were related to the regulation of translation initiation complex (p>0.0001). Interestingly, our RNA-seq dataset analysis further identified several members of the eukaryotic initiation factor 4F (eIF4F) complex to be significantly upregulated in CD34 + CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex involved in translation initiation (2-fold, p=0.001), as well as mTOR, a key regulator that controls the assembly of the eIF4F complex. This finding prompted us to further explore the regulation of translation initiation and the members of the eIF4F complex in BCR-ABL + cells. Immunoblotting demonstrated that BCR-ABL + cells co-transduced with WT Ahi-1 showed increased expression of eIF4G1 (3-fold) and eIF4B (2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, compared to BCR-ABL + cells. Additionally, Cyclin D3, a gene reported to be sensitive to eIF4F translational activity, was found to have slightly increased expression (1.4-fold) in WT Ahi-1 cells compared to BCR-ABL + cells. Most interestingly, these results were also demonstrated in IM-resistant CML cells as compared to IM-sensitive cells, with an increase in eIF4G1 expression (2-fold), phosphorylation of eIF4B (5-fold, p=0.003), and Cyclin D3 expression (4-fold, p<0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p<0.0001) and increases TKI sensitivity in IM-resistant cells (3-fold, p<0.05). A new protein interaction between eIF4G1 and the mRNA cap-binding protein eIF4E was further identified in these cells using a proximity ligation assay. Thus, we have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells, which may contribute to the regulation of TKI resistance in CML. DisclosuresNo relevant conflicts of interest to declare.

Stress modulates Ahi1-dependent nuclear localization of ten-eleven translocation protein 2.

Major depression disorder is one of the most common psychiatric diseases. Recent evidence supports that environmental stress affects gene expression and promotes the pathological process of depression through epigenetic mechanisms. Three ten-eleven translocation (Tet) enzymes are epigenetic regulators of gene expression that promote 5-hydroxymethylcytosine (5hmC) modification of genes. Here, we show that the loss of Tet2 can induce depression-like phenotypes in mice. Paradoxically, using the paradigms of chronic stress, such as chronic mild stress and chronic social defeat stress, we found that depressive behaviors were associated with increased Tet2 expression but decreased global 5hmC level in hippocampus. We examined the genome-wide 5hmC profile in the hippocampus of Tet2 knockout mice and identified 651 dynamically hydroxymethylated regions, some of which overlapped with known depression-associated loci. We further showed that chronic stress could induce the abnormal nuclear translocation of Tet2 protein from cytosol. Through Tet2 immunoprecipitation and mass spectrum analyses, we identified a cellular trafficking protein, Abelson helper integration site-1 (Ahi1), which could interact with Tet2 protein. Ahi1 knockout or knockdown caused the accumulation of Tet2 in cytosol. The reduction of Ahi1 protein under chronic stress explained the abnormal Ahi1-dependent nuclear translocation of Tet2. These findings together provide the evidence for a critical role of modulating Tet2 nuclear translocation in regulating stress response.

Loss of Ahi-1 Impairs the Self-Renewal Activity of Normal and BCR-ABL+ Stem Cells in Vitro and in Vivo

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have identified AHI-1 (Abelson helper integration site-1) oncogene as a potential therapeutic target in CML. It encodes a scaffold protein with a SH3 domain, multiple proline rich motifs and several WD40 repeats. Its expression is highly increased in BCR-ABL+ LSCs. Overexpression of Ahi-1 in primitive hematopoietic cells enhances cell proliferation in vitro and induces leukemia in vivo, and these effects can be enhanced by BCR-ABL. We have also demonstrated that AHI-1 interacts with multiple kinases, including BCR-ABL, β-catenin and JAK2 to mediate LSC activities and TKI resistance in vitro and in vivo . Interestingly, mouse Ahi-1 is highly expressed in hematopoietic stem cells (HSCs, CD45+EPCR+CD48-CD150+ (E-SLAM)) and progenitors (lin-Sca-1+c-kit+ (LSK)), but expressed at low levels in differentiated cells. In addition, Ahi-1 transcripts are found to be expressed in human embryonic stem cells (ES) and at all stages of mouse embryo development, with increasing expression just prior to birth, suggesting that Ahi-1 expression is developmentally regulated. To investigate the precise role of Ahi-1 in regulating normal and LSC self-renewal and differentiation, we have developed a new conditional Ahi-1 knockout mouse model. Ahi-1 deficient mice (AKO) were viable and fertile, but displayed around 30% reduction of bone marrow (BM) and thymus cellularity and altered lineage differentiation as compared to control wild-type mice (AWT). Interestingly, cell cycle analysis showed that loss of Ahi-1 reduced quiescent G0 cells by about 17% in HSC (lin-Sca-1+c-kit+CD48-) as compared to AWT cells. Ahi-1 deficient LSK cells did not exhibit differences in output of colony forming cells (CFCs) but did have dramatically reduced CFC secondary replating efficiency as compared to control cells. In vivo limited dilution assays (LDA), measured by bone marrow transplantation, demonstrated that Ahi-1 loss did not result in significant changes in the frequency of HSC numbers in primary transplanted mice (AWT vs AKO: 1/85000 vs 1/74000), but greatly reduced the reconstitution ability of these cells in serial transplantation assays, since no detectable donor cells were found in tertiary transplanted mice with AKO cells, while engrafted donor cells were still detected in tertiary transplanted mice with AWT cells. These results suggest that Ahi-1 plays a key role in regulating long-term repopulation activity of normal HSCs.The effects of Ahi-1 on LSC growth and maintenance were more profound when BCR-ABL was expressed in Ahi-1-deficient LSCs. Primitive Ahi-1-deficient BM cells transduced with BCR-ABL retroviruses (AKOBA) displayed reduced cell proliferation in the presence of growth factors, especially 14 days after in vtiro culture, compared to BCR-ABL-expressing BM cells from AWT mice (AWTBA). The FACS analysis for these cells after 7 days in culture showed reduced LSK numbers (30%) in AKOBA cells compared to AWTBA control cells. CFC assays revealed a 40% reduction in colonies in AKOBA as compared to AWTBA cells, but the effect was strikingly enhanced in the second and third replating assays (~70% reduction) in AKOBA cells. Most interestingly, we demonstrated that loss of Ahi-1 significantly enhanced survival of leukemic mice generated by crossing of Ahi-1 conditional KO mice with BCR-ABL-transgenic mice as compared to Ahi-1 wild-type/BCR-ABL control mice (65 days vs. 14 days, P=0.008). These results demonstrate that Ahi-1 plays a critical role in regulating HSC functionality and that its deletion perturbs self-renewal and leukemogenesis of BCR-ABL+ LSCs. DisclosuresNo relevant conflicts of interest to declare.

The Transition Zone Protein AHI1 Regulates Neuronal Ciliary Trafficking of MCHR1 and Its Downstream Signaling Pathway.

The Abelson-helper integration site 1 (AHI1) gene encodes for a ciliary transition zone localizing protein that when mutated causes the human ciliopathy, Joubert syndrome. We prepared and examined neuronal cultures derived from male and female embryonic Ahi1+/+ and Ahi1-/- mice (littermates) and found that the distribution of ciliary melanin-concentrating hormone receptor-1 (MchR1) was significantly reduced in Ahi1-/- neurons; however, the total and surface expression of MchR1 on Ahi1-/- neurons was similar to controls (Ahi1+/+). This indicates that a pathway for MchR1 trafficking to the surface plasma membrane is intact, but the process of targeting MchR1 into cilia is impaired in Ahi1-deficient mouse neurons, indicating a role for Ahi1 in localizing MchR1 to the cilium. Mouse Ahi1-/- neurons that fail to accumulate MchR1 in the ciliary membrane have significant decreases in two downstream MchR1 signaling pathways [cAMP and extracellular signal-regulated kinase (Erk)] on MCH stimulation. These results suggest that the ciliary localization of MchR1 is necessary and critical for MchR1 signaling, with Ahi1 participating in regulating MchR1 localization to cilia, and further supporting cilia as critical signaling centers in neurons.SIGNIFICANCE STATEMENT Our work here demonstrates that neuronal primary cilia are powerful and focused signaling centers for the G-protein-coupled receptor (GPCR), melanin-concentrating hormone receptor-1 (MCHR1), with a role for the ciliary transition zone protein, Abelson-helper integration site 1 (AHI1), in mediating ciliary trafficking of MCHR1. Moreover, our manuscript further expands the repertoire of cilia functions on neurons, a cell type that has not received significant attention in the cilia field. Lastly, our work demonstrates the significant influence of ciliary GPCR signaling in the overall signaling of neurons.

Autism spectrum disorder-like behavior caused by reduced excitatory synaptic transmission in pyramidal neurons of mouse prefrontal cortex

Autism spectrum disorder (ASD) is thought to result from deviation from normal development of neural circuits and synaptic function. Many genes with mutation in ASD patients have been identified. Here we report that two molecules associated with ASD susceptibility, contactin associated protein-like 2 (CNTNAP2) and Abelson helper integration site-1 (AHI1), are required for synaptic function and ASD-related behavior in mice. Knockdown of CNTNAP2 or AHI1 in layer 2/3 pyramidal neurons of the developing mouse prefrontal cortex (PFC) reduced excitatory synaptic transmission, impaired social interaction and induced mild vocalization abnormality. Although the causes of reduced excitatory transmission were different, pharmacological enhancement of AMPA receptor function effectively restored impaired social behavior in both CNTNAP2- and AHI1-knockdown mice. We conclude that reduced excitatory synaptic transmission in layer 2/3 pyramidal neurons of the PFC leads to impaired social interaction and mild vocalization abnormality in mice.

Alzheimer's Disease and Diabetes: Insulin Signaling as the Bridge Linking Two Pathologies.

Alzheimer's (or Alzheimer) disease (AD) is the most prevalent subset of dementia, affecting elderly populations worldwide. The cumulative costs of the AD care are rapidly accelerating as the average lifespan increases. Onset and risk factors for AD and AD-like dementias have been largely unknown until recently. Studies show that chronic type II diabetes mellitus (DM) is closely associated with neurodegeneration, especially AD. Type II DM is characterized by the cells' inability to take up insulin, as well as chronic hyperglycemia. In the central nervous system, insulin has crucial regulatory roles, while chronic hyperglycemia leads to formation and accumulation of advanced glycation end products (AGEs). AGEs are the major contributor to insulin resistance in diabetic cells, due to their regulatory role on sirtuin expression. Insulin activity in the central nervous system is known to interact with key proteins affected in neurodegenerative conditions, such as amyloid-β precursor protein (AβPP or APP), huntingtin-associated protein-1 (HAP1), Abelson helper integration site-1 (AHI1 or Jouberin), kinesin, and tau. Sirtuins have been theorized to be the mechanism for insulin resistance, and have been found to be affected in neurodegenerative conditions as well. There are hints that all these neuronal proteins may be closely related, although the mechanisms remain unclear. This review will gather existing research on these proteins and highlight the link between neurodegenerative conditions and diabetes mellitus.

Reduction of AHI1 in the serum of Taiwanese with probable Alzheimer’s disease

ObjectiveThe development of blood-based biomarkers for early diagnosis and treatment of Alzheimer’s disease (AD) is desirable. In AD model mouse brain and neuronal cells, Abelson helper integration site-1 (AHI1) protein is reduced. AHI1 facilitates intracellular amyloid precursor protein (APP) translocation to inhibit amyloidogenic pathology of AD, and thus may be an AD biomarker. MethodsThis study was conducted among 32 AD patients and 54 healthy control (HC) subjects. AHI1-related protein levels from initially collected serum samples in each group were screened using Western blotting. The protein concentrations of AHI1 and amyloid-β (Aβ), peptide(s) derived from APP, from all serum samples were analyzed using ELISA. ResultsIn AD serum, AHI1 and a large truncated C-terminal APP fragment were significantly reduced. The average concentrations of serum AHI1 and Aβ in AD were significantly lower than those in HC. Notably, AHI1 concentration in HC serum was decreased in an age-dependent manner, while it was consistently low in AD serum and had no correlation with Aβ or mini-mental state examination score. The receiver operating characteristic analysis on all subjects demonstrated an area under curve (AUC) value of 0.7 for AHI1 on AD diagnosis, while the AUC increased to 0.82 on the subjects younger than 77 years old, suggesting a good diagnostic performance of serum AHI1 for AD especially at relatively young age. ConclusionAn early event of AHI1 reduction in the body of AD patients was observed. Serum AHI1 may be valuable for early diagnosis of AD.

The AHI-1-BCR-ABL-DNM2 Complex Mediates Mitochondrial Dynamics in Drug-Resistant BCR-ABL+ Cells

Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 oncoprotein with constitutively active protein-tyrosine kinase activity, perturbing multiple signaling pathways. Although therapies with tyrosine kinase inhibitors (TKIs) can effectively treat early phase CML, relapses and emergence of TKI resistance are problematic, due to BCR-ABL kinase domain mutations and TKI unresponsive quiescent leukemic stem cells (LSCs). These observations point towards a need for alternate treatment strategies to prevent the development of resistant LSCs. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs and that its WD40-repeat domain physically interacts with BCR-ABL, enhancing leukemia-initiating activity. AHI-1 also contains an SH3 domain, which mediates TKI resistance in LSCs. This domain interacts with dynamin-2 (DNM2) and forms a complex with BCR-ABL, to enhance the phosphorylation and activity of DNM2. The AHI-1-BCR-ABL-DNM2 complex is shown to regulate leukemic properties in patient LSCs, including increased ROS production, endocytosis and autophagy. Interestingly, deletion of the Ahi-1 SH3 domain (Ahi-1 SH3Δ) results in a defect in Ahi-1 localization, with most being present in the nucleus. To test whether Ahi-1 SH3 domain activity directly affects cytoplasmic anchoring and localization, we have generated two Ahi-1 mutants, using site-directed mutagenesis: a mutation in the key tryptophan residue (W939A) involved in SH3 domain binding and in a non-conserved surface residue (M906A), as a negative control, based on the crystal structure of the AHI-1 SH3 domain. Interestingly, the cytoplasm-to-nucleus signal ratio of Ahi-1 W939A was significantly reduced compared to the negative control or wildtype Ahi-1, as assessed by immunofluorescence and confocal microscopy (70% reduction, p&amp;lt;0.0001), indicating that changes in localization of Ahi-1 SH3Δ may result in disruption of the complex and allow for new interactions with nuclear proteins. Investigating changes in the proteome may help uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance. Advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL+ cells co-transduced with wildtype Ahi-1 or Ahi-1 SH3Δ. This system quantifies the differences in expression and phosphorylation states of key signaling proteins simultaneously, using 878 antibodies in duplicate. Twenty leads were identified by the following criteria: a large signal difference of at least 1.5-fold change, high signal strength for high expression, and low error between duplicates. These leads were validated by Western blot analysis and several of them were confirmed. Particularly, phosphorylation of cyclin-dependent kinase 1 (CDK1), a key player in cell cycle control and mitochondrial dynamics, was greatly reduced in cells expressing wildtype Ahi-1 compared to Ahi-1 SH3Δ, indicating that AHI-1-mediated phosphorylation changes in CDK1 may contribute to regulation of mitochondrial functions. Indeed, BCR-ABL-transduced cells co-expressing wildtype Ahi-1 showed increased mitochondria potential in response to TKI treatment or serum starvation, in MitoTracker analysis (p&amp;lt;0.05). However, this was not observed in BCR-ABL-transduced cells co-expressing the Ahi-1 SH3Δ mutant. A similar trend was also observed in immunofluorescence confocal microscopy analysis of the mitochondrial importer receptor, TOM20. To further study the role of DNM2 in mediating mitochondrial dynamics associated with AHI-1 and BCR-ABL, CRISPR-Cas9 mediated DNM2 knockdown was performed in TKI-resistant cells, using two different DNM2-targeting guide RNAs; these resulted in significant reduction in DNM2 (78% &amp; 75%) in Western blot analysis. The knockdown cells showed a reduction in viability (60% reduction) and increased sensitivity to TKI treatment compared to the control (90% vs. 30% reduction) after 48 hours and changes in mitochondrial activity were also observed in these cells. These results support a role for mitochondrial dynamics in the AHI-1-BCR-ABL-DNM2 complex-mediated TKI response and that targeting key biological processes regulated by the AHI-1-BCR-ABL-DNM2 complex and its pathways may lead to new therapeutic strategies to overcome TKI resistance in CML. Disclosures Pelech: Kinexus Bioinformatics Corporation: Equity Ownership.

Expression of AHI1 Rescues Amyloidogenic Pathology in Alzheimer's Disease Model Cells.

A hallmark of Alzheimer's disease (AD) pathogenesis is the accumulation of extracellular plaques mainly composed of amyloid-β (Aβ) derived from amyloid precursor protein (APP) cleavage. Recent reports suggest that transport of APP in vesicles with huntingtin-associated protein-1 (HAP1) negatively regulates Aβ production. In neurons, HAP1 forms a stable complex with Abelson helper integration site-1 (AHI1), in which mutations cause neurodevelopmental and psychiatric disorders. HAP1 and AHI1 interact with tropomyosin receptor kinases (Trks), which are also associated with APP and mediate neurotrophic signaling. In this study, we hypothesize that AHI1 participates in APP trafficking and processing to rescue AD pathology. Indeed, AHI1 was significantly reduced in mouse neuroblastoma N2a cells expressing human Swedish and Indiana APP (designed as AD model cells) and in 3xTg-AD mouse brain. The AD model cells as well as Ahi1-knockdown cells expressing wild-type APP-695 exhibited a significant reduction in viability. In addition, the AD model cells were reduced in neurite outgrowth. APP C-terminal fragment-β (CTFβ) and Aβ42 were increased in the AD cell lysates and the culture media, respectively. To investigate the mechanism how AHI1 alters APP activities, we overexpressed human AHI1 in the AD model cells. The results showed that AHI1 interacted with APP physically in mouse brain and transfected N2a cells despite APP genotypes. AHI1 expression facilitated intracellular translocation of APP and inhibited APP amyloidogenic process to reduce the level of APP-CTFβ in the total lysates of AD model cells as well as Aβ in the culture media. Consequently, AHI1-APP interactions enhanced neurotrophic signaling through Erk activation and led to restored cell survival and differentiation.

Mutant Ahi1 Affects Retinal Axon Projection in Zebrafish via Toxic Gain of Function.

Joubert syndrome (JBTS) is an inherited autosomal recessive disorder associated with cerebellum and brainstem malformation and can be caused by mutations in the Abelson helper integration site-1 (AHI1) gene. Although AHI1 mutations in humans cause abnormal cerebellar development and impaired axonal decussation in JBTS, these phenotypes are not robust or are absent in various mouse models with Ahi1 mutations. AHI1 contains an N-terminal coiled-coil domain, multiple WD40 repeats, and a C-terminal Src homology 3 (SH3) domain, suggesting that AHI1 functions as a signaling or scaffolding protein. Since most AHI1 mutations in humans can result in truncated AHI1 proteins lacking WD40 repeats and the SH3 domain, it remains unclear whether mutant AHI1 elicits toxicity via a gain-of-function mechanism by the truncated AHI1. Because Ahi1 in zebrafish and humans share a similar N-terminal region with a coiled-coil domain that is absent in mouse Ahi1, we used zebrafish as a model to investigate whether Ahi1 mutations could affect axonal decussation. Using in situ hybridization, we found that ahi1 is highly expressed in zebrafish ocular tissues, especially in retina, allowing us to examine its effect on retinal ganglion cell (RGC) projection and eye morphology. We injected a morpholino to zebrafish embryos, which can generate mutant Ahi1 lacking the intact WD40 repeats, and found RGC axon misprojection and ocular dysplasia in 4 dpf (days post-fertilization) larvae after the injection. However, ahi1 null zebrafish showed normal RGC axon projection and ocular morphology. We then used CRISPR/Cas9 to generate truncated ahi1 and also found similar defects in the RGC axon projection as seen in those injected with ahi1 morpholino. Thus, the aberrant retinal axon projection in zebrafish is caused by the presence of mutant ahi1 rather than the loss of ahi1, suggesting that mutant Ahi1 may affect axonal decussation via toxic gain of function.

Loss of the neurodevelopmental Joubert syndrome causing protein, Ahi1, causes motor and muscle development delays independent of central nervous system involvement

Joubert syndrome (JBTS) is a predominantly autosomal recessive neurodevelopmental disorder that presents with characteristic malformations of the cerebellar vermis, superior cerebellar peduncles and midbrain in humans. Accompanying these malformations are a heterogeneous set of clinical symptoms, which frequently include deficits in motor and muscle function, such as hypotonia (low muscle tone) and ataxia (clumsiness). These symptoms are attributed to improper development of the hindbrain, but no direct evidence has been reported linking these in JBTS. Here, we describe muscle developmental defects in a mouse with a targeted deletion of the Abelson helper integration site 1 gene, Ahi1, one of the genes known to cause JBTS in humans. While FVB/NJ Ahi1-/- mice display no gross malformations of the cerebellum, deficits are observed in several measures of motor function, strength, and body development. Specifically, Ahi1-/- mice show delayed physical development, delays in surface reflex righting as neonates, and reductions in grip strength and spontaneous locomotor activity as adults. Additionally, Ahi1-/- mice showed evidence of muscle-specific contributions to this phenotype, such as reductions in 1) myoblast differentiation potential in vitro, 2) muscle desmin expression, and 3) overall muscle mass, myonuclear domain, and muscle fiber cross-sectional area. Together, these data suggest that loss of Ahi1 may cause abnormalities in the differentiation of myoblasts to mature muscle cells. Moreover, Ahi1 loss impacts muscle development directly, outside of any indirect impact of cerebellar malformations, revealing a novel myogenic cause for hypotonia in JBTS.

Ahi1 promotes Arl13b ciliary recruitment, regulates Arl13b stability and is required for normal cell migration.

Mutations in the Abelson-helper integration site 1 (AHI1) gene are associated with neurological/neuropsychiatric disorders, and cause the neurodevelopmental ciliopathy Joubert syndrome (JBTS). Here, we show that deletion of the transition zone (TZ) protein Ahi1 in mouse embryonic fibroblasts (MEFs) has a small effect on cilia formation. However, Ahi1 loss in these cells results in: (1) reduced localization of the JBTS-associated protein Arl13b to the ciliary membrane, (2) decreased sonic hedgehog signaling, (3) and an abnormally elongated ciliary axoneme accompanied by an increase in ciliary IFT88 concentrations. While no changes in Arl13b levels are detected in crude cell membrane extracts, loss of Ahi1 significantly reduced the level of non-membrane-associated Arl13b and its stability via the proteasome pathway. Exogenous expression of Ahi1-GFP in Ahi1-/- MEFs restored ciliary length, increased ciliary recruitment of Arl13b and augmented Arl13b stability. Finally, Ahi1-/- MEFs displayed defects in cell motility and Pdgfr-α-dependent migration. Overall, our findings support molecular mechanisms underlying JBTS etiology that involve: (1) disruptions at the TZ resulting in defects of membrane- and non-membrane-associated proteins to localize to primary cilia, and (2) defective cell migration.This article has an associated First Person interview with the first author of the paper.

Gene Screening of Astrocytoma Grade III Relative to Grade II via Network Analysis: A New Molecular Insight

Background: Astrocytomas (sub-group of gliomas) are central neuron system malignant diseases, which are originated from astrocytes. There are several documents about molecular mechanism of astrocytomas and many related genes are introduced. Here, gene expression profile alteration for grade III relative to grade II of astrocytoma is analyzed via protein-protein interaction (PPI) network to screen and introduce critical differentially expressed genes (DEGs). Methods: The significant DEGs were extracted from gene expression omnibus (GEO) database and included in a PPI network by Cytoscape software. The common significant DEGs and central nodes of the network were selected and enriched by ClueGO to find related biological terms. Results: Twenty critical genes including zinc finger protein 765 (ZNF765), zinc finger protein 540 (ZNF540), Zeste white 10 (ZW10) ZW10 interacting kinetochore protein (ZWINT), collagen type XVIII alpha 1 chain (COL18A1), protamine 2 (RRM2), kinesin family member 23 (KIF23), minichromosome maintenance 10 (MCM10), anaphase promoting complex subunit 7 (ANAPC7), NADPH oxidase activator 1 (NOXA1), ryanodine receptor 2 (RYR2), myozenin 3 (MYOZ3), myosin binding protein C (MYBPC2), fast type, keratin 17 (KRT17), zinc phosphodiesterase ELAC protein 2 (ELAC2), Abelson helper integration site 1 (AHI1), latent transforming growth factor beta binding protein 1 (LTBP1), kaptin (actin binding protein) (KPTN), BEN domain containing 3 (BEND3), cysteine and histidine rich 1 (CYHR1), and hyaluronoglucosaminidase 2 (HYAL2) were identified. Eight class of biological terms related to the critical genes were determined and discussed. Conclusions: A wide range of the introduced significant genes indicates that there are several possible therapeutic ways to challenge astrocytoma.

Effect of chronic unpredictable stress on mice with developmental under-expression of the Ahi1 gene: behavioral manifestations and neurobiological correlates

The Abelson helper integration site 1 (Ahi1) gene plays a pivotal role in brain development and is associated with genetic susceptibility to schizophrenia, and other neuropsychiatric disorders. Translational research in genetically modified mice may reveal the neurobiological mechanisms of such associations. Previous studies of mice heterozygous for Ahi1 knockout (Ahi1+/−) revealed an attenuated anxiety response on various relevant paradigms, in the context of a normal glucocorticoid response to caffeine and pentylenetetrazole. Resting-state fMRI showed decreased amygdalar connectivity with various limbic brain regions and altered network topology. However, it was not clear from previous studies whether stress-hyporesponsiveness reflected resilience or, conversely, a cognitive-emotional deficit. The present studies were designed to investigate the response of Ahi1+/− mice to chronic unpredictable stress (CUS) applied over 9 weeks. Wild type (Ahi1+/+) mice were significantly affected by CUS, manifesting decreased sucrose preference (p < 0.05); reduced anxiety on the elevated plus maze and light dark box and decreased thigmotaxis in the open field (p < 0.01 0.05); decreased hyperthermic response to acute stress (p < 0.05); attenuated contextual fear conditioning (p < 0.01) and increased neurogenesis (p < 0.05). In contrast, Ahi1+/− mice were indifferent to the effects of CUS assessed with the same parameters. Our findings suggest that Ahi1 under-expression during neurodevelopment, as manifested by Ahi1+/− mice, renders these mice stress hyporesponsive. Ahi1 deficiency during development may attenuate the perception and/or integration of environmental stressors as a result of impaired corticolimbic connectivity or aberrant functional wiring. These neural mechanisms may provide initial clues as to the role Ahi1 in schizophrenia and other neuropsychiatric disorders.