Defects In Intracellular Trafficking Research Articles

Restore the Secretion and Activity of Factor VIII Mutants By Proteostasis Regulator

Quantitative or qualitative deficiency in factor VIII (FVIII) result in hemophilia A. FVIII deficiency is caused by broad spectrum mutations, and missense mutation is the most common type of mutation, which lead to various defects of FVIII from decreased secretion to impaired function. Cellular protein homeostasis is disturbed by FVIII mutants, and thus excessive misfolding and degradation of FVIII mutant result in FVIII deficiency. We hypothesized that folding and/or trafficking of FVIII can be enhanced by proteostasis regulators, leading to increased coagulation factor levels sufficient to restore hemostasis. We performed a cell-based screening using a FDA-approved drug library and a natural compound library looking for compounds that enhance secretion of FVIII with a B domain deletion. We identified SAHA (Suberanilohydroxamic acid) as a compound that increased both wild-type (WT) and a missense mutant FVIII. SAHA is a histone deacetylase inhibitor that was also reported to enhance proteostaisis of mutant CFTR, a-1 antitrypsin, lysosomal glucocerebrosidase and the GABA receptor. Here we show that SAHA can increase the secretion and activity of FVIII mutants by enhancing their folding and trafficking as well transcription. To confirm the ability of SAHA to enhance FVIII mutant secretion, we first used the WT FVIII A2 domain and the M614T mutant A2 domain to detect the proper working time and concentration of SAHA in HEK 293T cells. M614T is a missense mutation that was shown to cause secretion defect of FVIII in our previous study. Western blot showed a dose-dependent increase in levels of both A2-WT and A2-M614T in cell culture media between 0 and 2.5ug/ml of SAHA after 24-hour treatment. Levels of both WT and mutant A2 domain dropped when SAHA concentration exceeded 5 ug/ml due to cellular toxicity. SAHA enhanced the secretion of A2-M614T much more than that of A2-WT. Of note, protein levels of both A2-WT and A2-M614T in cell lysate were also slightly elevated by SAHA. Time course study showed that the maximum effect on secretion was achieved after 24 hours of SAHA treatment. We next studied the effect of SAHA on activity and secretion of several full-length FVIII mutants (A469S, R527W, R531G, I566T, N582D, S584T, R593C, M614T, R1997G, R2150C), which were shown to decrease FVIII secretion due to various defects in intracellular trafficking in our previous studies. Except for N582D, activity levels of all FVIII mutants in cell culture media were elevated 3-14 folds after a 24-hour SAHA treatment (P<0.05), whereas the activity level of FVIII WT only increased by ~2 folds (P<0.05). Antigen levels of FVIII mutants in both cell lysates and cell culture media increased by ~3 folds following SAHA treatment (P<0.05). The increase in activities of several FVIII mutants (I566T, S584T, R593C, R1997G, R2150C) is higher than the increase in antigen levels of the same mutants in cell culture media, which indicates that, in addition to enhancing secretion, SAHA treatment also increased the proportions of functional proteins in secreted FVIII mutants in cell culture media. To investigate the mechanism, we studied the interactions of WT FVIII and several responsive mutant FVIII (I566T, S584T, R593C and R1997C) with endoplasmic reticulum (ER) chaperones BiP and calreticulin by co-Immunoprecipitation. The protein levels of BiP and calreticulin in HEK 293T cells were not affected by SAHA. However, the interactions of BiP with both WT FVIII and FVIII mutants were remarkably enhanced by SAHA, while no significant differences in the interactions of calreticulin with FVIII WT or FVIII mutants were observed. The effect of SAHA on FVIII secretion can be blocked by simultaneously treating cells with a BiP inhibitor SubAB. We next measured mRNA levels of BiP as well as FVIII in cells expressing FVIII WT and FVIII mutants with or without SAHA treatment via real time PCR. Similar to protein levels, BiP mRNA levels were not changed by SAHA. However, FVIII mRNA levels were elevated by 1.2 to 3.1 folds (P<0.05) by SAHA, in keeping with the histone deacetylase inhibitor activity of SAHA. In summary, we have shown that SAHA increases FVIII antigen and activity levels in cell culture media by increasing FVIII mRNA levels and by enhancing its folding and trafficking via specifically promoting its interaction with BiP. SAHA is a potential proteostaisis regulator that can increase the secretion and restore the function of certain FVIII missense mutants. Disclosures No relevant conflicts of interest to declare.

Intracellular trafficking defects induced by α-synuclein as a pathogenic mechanism for Parkinson's disease

Intracellular trafficking defects induced by α-synuclein as a pathogenic mechanism for Parkinson's disease

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging.

SummaryInability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule‐based minus‐end‐directed motor proteins, the well‐characterized dynein, and the less‐studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti‐aging purposes.

Reduced RON Expression, DM1 resistance and MRP1 Upregulation Contributes to Resistance in Colon Cancer Cells against anti‐RON Antibody‐Drug Conjugate Zt/g4‐DM1

Zt/g4‐DM1 is an antibody‐drug conjugate that specifically delivers the potent cytotoxic payload maytansinoid (DM1) to RON receptor‐positive cells. Despite its impressive preclinical efficacy in the initial stages of treatment in xenograft models, tumors‐mediated by colon and PDAC cells were only partially inhibited by Zt/g4‐DM1. In light of these findings, we wanted to investigate mechanisms involved in Zt/g4‐DM1 resistance. As part of this work, we developed anti‐RON Zt/g4‐SMCC‐DM1 resistant colon cancer HT29, HCT116, SW620 cells derived from tumors by continuous treatment at lower doses followed by incremental dose increases. Approximately 100–200 fold resistance to Zt/g4‐DM1 developed in these cells and cross‐resistance was observed other ADCs containing drugs with similar mechanism of action. Reduced RON expression, DM1 resistance and increased expression of the ABCC1 (MRP1) drug exporter compared to parental cells were primary mediators of resistance. Interestingly we found a small percentage of RON positive population in resistant cells, when isolated these RON+ clones from three cell lines exhibited stable drug refractoriness and altered intracellular trafficking as a unique underlying mechanism of resistance to Zt/g4‐DM1. HT29R, HCT116R, SW620R cell lines showed resistance to unconjugated DM1 but not to other chemotherapeutic agents. Tumors derived from HT29R, HCT116R, SW620R grew in mice and were refractory to Zt/g4‐DM1 compared with parental cells. Hence, acquired resistance to Zt/g4‐DM1 was generated from cells derived from tumors by continuous drug exposure, and either increased ABCC1 protein or reduced RON expression and defective intracellular trafficking in case of RON+ resistant cells were primary mediators of resistance. These resistant cell models retained sensitivity to chemotherapeutics, suggesting that alternative therapies may help in overcoming acquired resistance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OP.5A.08] HCARG/COMMD5 IMPROVES KIDNEY REPAIR BY REGULATING EGF RECEPTOR TRAFFICKING

Objective: Epidermal Growth Factor Receptor (EGFR) is crucially involved in nephrogenesis. Although, constitutive EGFR signaling is needed for normal kidney development and can enhance recovery of renal function and structure following acute kidney injury, its overexpression may leads to excess renal tissue fibrosis and functional deterioration in diabetic and hypertensive nephropathy. Consequently, targeting EGFR in renal diseases is complex. Our group has identified HCaRG (Hypertension-related, Calcium-Regulated Gene), currently named COMMD5, whose overexpression in an in vivo model of renal carcinoma inhibited tumor growth and angiogenesis by inhibiting EGFR activation and downstream signaling. Overexpression of COMMD5 in renal proximal tubules of transgenic mice increased mice survival and recovery of renal tissue integrity and function after acute kidney injury. Our goal is to demonstrate that COMMD5 accelerated kidney repair by controlling EGFR activity, endocytosis and trafficking. Design and method: COMMD5 was silenced by siRNA and EGFR expression and activation were evaluated in renal cell lines. Intracellular localization of COMMD5, EGFR and endosome associated protein, such as Rab, were analyzed by confocal microscopy and live cell imaging. Co-immunoprecipitations were used to investigate interactions between COMMD5 and proteins known to be involved in vesicle trafficking. Results: We demonstrated that COMMD5 is an adaptor protein forming a bridge between endosomes and cytoskeleton by binding Rab protein and microtubules/actin. This tethering is essential because silencing of COMMD5 delayed the intracellular transport dynamic and consequently cargo protein such as EGFR. Defect in intracellular trafficking led to loss of cell polarity and oriented migration which are crucial for tissue repair. Conclusions: Our results demonstrated that COMMD5 plays a crucial role in controlling EGFR signaling/trafficking and suggest that COMMD5 could be used as a novel target for EGFR-targeted therapy in kidney diseases.

An iTRAQ-based proteomic analysis reveals dysregulation of neocortical synaptopodin in Lewy body dementias

Lewy body dementias are the second most common cause of neurodegenerative dementia in the elderly after Alzheimer’s disease (AD). The two clinical subgroups of Lewy body dementias, namely, dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD), are differentiated by the chronology of cognitive symptoms relative to parkinsonism. At present, there remains a debate on whether DLB and PDD are separate disease entities, or fall within the same spectrum of Lewy body dementias. In this study, we compared the detergent-soluble proteome via an 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) analysis of pooled lysates from the prefrontal cortex (BA9) of DLB (n = 19) and PDD (n = 21) patients matched a priori for amyloid (total Aβ42) burden, semi-quantitative scores for Lewy bodies and neurofibrillary tangles together with age-matched control (n = 21) subjects. A total of 1914 proteins were confidently identified by iTRAQ (false discovery rate = 0%). None of the proteins showed a significant yet opposite regulation in between DLB and PDD when compared to aged controls in the proteomic data set as well as following immunoblot analysis of the pooled and individual lysates involving all 61 subjects. The postsynaptic protein, synaptopodin (SYNPO) was significantly down-regulated in both DLB and PDD subgroups, suggesting a defective synaptic transmission in the demented patients. In conclusion, the largely similar proteome of DLB and PDD matched for amyloid burden suggests that variations in concomitant AD-related pathology, abnormal post-translational modifications or protein-protein interactions, defective intracellular trafficking or misfolding of proteins could play a part in driving the clinically observed differences between these two subgroups of Lewy body dementias. This further indicates that amyloid-targeting therapeutic strategies may show different efficacies in DLB versus PDD.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival.

In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6−/−) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6−/− mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6−/− mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.

Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, includingMycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Developmental delay in motor skill acquisition in Niemann-Pick C1 mice reveals abnormal cerebellar morphogenesis.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1−/− mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1nmf164 for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1nmf164/ Npc1nmf164 pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1nmf164 homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-β-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs.These findings indicate that in Npc1nmf164 homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0370-z) contains supplementary material, which is available to authorized users.

Abstract 133: Identification of a key region in the HER2 subdomain III required for transformation capability

Abstract About 25% of breast carcinomas are characterized by over-expression and/or amplification of the HER2 receptor, one of four members of the HER family of receptor tyrosine kinases. The strong correlation between HER2 levels/activity with more aggressive breast cancer disease, poor prognosis as well as resistance to chemo- and endocrine therapies made it a preferred target for anti-cancer therapy. However, current treatment strategies are only partially effective and the majority of breast cancer patients who initially respond favorably rapidly develop resistance to treatments for still not fully understood reasons. Thus, novel strategies/agents are in need. Structural analyses revealed that sub-domain III in the extracellular domain (ECD) of the HER family members is responsible for dimerization. We hypothesized that disrupting the dimerization loop in the HER2-ECD sub-domain III would ultimately convert HER2 to a non-functional receptor with diminished transforming capacity. To this end, we generated a series of HER2-ECD deletion mutants located in sub-domain III and assessed their oncogenic potential compared to the HER2-wild type (WT) after their expression in MCF10A cells. Importantly, a small deletion (16 amino acids) of the HER2 extracellular domain (named HER2Δ6) abolished its homo- and hetero-dimerization and profoundly affected HER2-catalyzed activation of the HER network. Moreover, this mutant was not able to transform MCF10 cells as it failed to promote anchorage-independent growth and interfered with the activation/Tyr phosphorylation of HER1, HER2 and HER3. Compared to HER2-WT-expressing cells, the HER2α6 mutant proved to be even more effective in inhibiting the oncogenic properties of the receptor than the current drugs of choice such as Trastuzumab and Pertuzumab alone or in combination. In addition, while expression of HER2-WT conferred resistance of MCF10A cells to Paclitaxel, the HER2Δ6 variant failed to do so. To determine the molecular mechanisms underlying this behavior, we assessed the mutant and WT expressing cells lines morphologically and biochemically and demonstrated that the HER2Δ6 is absent from the plasma membrane (PM) in MCF10A cells. Interestingly, we found that the mutant receptor displayed an intracellular trafficking defect and was trapped in the endoplasmic reticulum (ER). Our results reveal that the HER2-ECD bears an essential “activating” region that is indispensable for HER2-mediated oncogenic transformation. Targeting and eliminating this “activating” element in HER2 seems to provide a strong innovative approach for developing a valuable novel anti-HER2 therapeutic drug that would specifically benefit patients with HER2 positive tumors resistant to current therapies. Citation Format: Barbara Schroeder, Ghiara Lugo, Javier Menendez, Ingrid Espinoza, Ruth Lupu. Identification of a key region in the HER2 subdomain III required for transformation capability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 133. doi:10.1158/1538-7445.AM2015-133

Abstract P6-07-08: Blocking a key region in the HER2 subdomain III inhibits the HER2-network in patients with resistance to HER2-targeted therapy

Abstract The HER2 receptor is over-expressed in about 25% of breast carcinomas and correlates with more aggressive breast cancer disease, poor prognosis as well as resistance to chemo- and endocrine therapies. The strong correlation between HER2 levels/activity and the malignancy of the disease made it a preferred target for anti-cancer therapy. For example, Trastuzumab, which targets HER2 homo-dimerization and Pertuzumab, which interferes with HER2 hetero-dimerization, are in clinical use. Unfortunately, Trastuzumab monotherapy is only partially effective and the majority of breast cancer patients who initially respond to Trastuzumab rapidly develop resistance to the drug. Thus, novel strategies/agents are in need, especially drugs that prevent hetero-dimerization with other HER family members. Structural analyses revealed that sub-domain III in the extracellular domain (ECD) of the HER family members is responsible for dimerization. Therefore, we hypothesized that disabling the dimerization loop in the HER2-ECD sub-domain III would ultimately convert HER2 to a non-functional receptor. Therefore, we introduced a series of deletions in the HER2-ECD sub-domain III and determined their oncogenic properties compared to HER2wt expressing cells. Importantly, a small deletion (16 amino acids) of the HER2 extracellular domain (named HER2Δ6) abolished its homo- and hetero-dimerization and profoundly affected HER2-catalyzed activation of the HER network, both in the context of HER2 over-expression and ligand-induced trans-activation of HER2. Expression of the HER2Δ6 variant failed to promote anchorage-independent growth and interfered with the activation/Tyr phosphorylation of HER1, HER2 and HER3. Moreover, this mutant failed to promote resistance to Paclitaxel treatment in HER2-overexpressing breast cancer cells. To determine the molecular mechanisms underlying this behavior, we assessed the mutant and wt expressing cells lines morphologically and biochemically and demonstrated that the HER2Δ6 is absent from the plasma membrane (PM), most likely due to a intracellular trafficking defect that mistargets the receptor to or traps it in an endomembrane compartment. Compared to HER2wt-expressing cells, the HER2Δ6 mutant proves to be even more effective in inhibiting the oncogenic properties of the receptor than the current drugs of choice such as Trastuzumab, Pertuzumab and Cetuximab alone or in combination as measured by anchorage-independent growth. These findings reveal that the HER2-ECD bears an essential "activating" region that is indispensable for HER2 homo-and heterodimerization. Elimination of this "activating" element in HER2 seems to recapitulate and greatly improve the combined actions of Trastuzumab and Pertuzumab. These findings offer a strong rationale for developing this peptide sequence into a valuable anti-HER2 therapeutic drug. Citation Format: Barbara Schroeder, Menendez Javier, Espinoza Ingrid, Lupu Ruth. Blocking a key region in the HER2 subdomain III inhibits the HER2-network in patients with resistance to HER2-targeted therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-08.

Traffic problems: inherited disease and intracellular trafficking defect

Traffic problems: inherited disease and intracellular trafficking defect

Impaired trafficking of the very low density lipoprotein receptor caused by missense mutations associated with dysequilibrium syndrome

Dysequilibrium syndrome (DES, OMIM 224050) is a genetically heterogeneous condition that combines autosomal recessive non-progressive cerebellar ataxia with mental retardation. The subclass dysequilibrium syndrome type 1 (CAMRQ1) has been attributed to mutations in the VLDLR gene encoding the very low density lipoprotein receptor (VLDLR). This receptor is involved in the Reelin signaling pathway that guides neuronal migration in the cerebral cortex and cerebellum. Three missense mutations (c.1459G>T; p.D487Y, c.1561G>C; p.D521H and c.2117G>T; p.C706F) have been previously identified in VLDLR gene in patients with DES. However, the functional implications of those mutations are not known and therefore we undertook detailed functional analysis to elucidate the cellular mechanisms underlying their pathogenicity. The mutations have been generated by site-directed mutagenesis and then expressed in cultured cell lines. Confocal microscopy and biochemical analysis have been employed to examine the subcellular localization and functional activities of the mutated proteins relative to wild type. Our results indicate that the three missense mutations lead to defective intracellular trafficking and ER retention of the mutant VLDLR protein. This trafficking impairment prevents the mutants from reaching the plasma membrane and binding exogenous Reelin, the initiating event in Reelin signaling. Collectively, our results provide evidence that ER quality control is involved in the functional inactivation and underlying pathogenicity of these DES-associated mutations in the VLDLR.

A membrane proximal helix in the cytosolic domain of the human APP interacting protein LR11/SorLA deforms liposomes

Over the last decade, compelling evidence has linked the development of Alzheimer's disease (AD) to defective intracellular trafficking of the amyloid precursor protein (APP). Faulty APP trafficking results in an overproduction of Aβ peptides, which is generally agreed to be the primary cause of AD-related pathogenesis. LR11 (SorLA), a type I transmembrane sorting receptor, has emerged as a key regulator of APP trafficking and processing. It directly interacts with APP and diverts it away from amyloidogenic processing. The 54-residue cytosolic domain of LR11 is essential for its proper intracellular localization and trafficking which, in turn, determines the fate of APP. Here, we have found a surprising membrane-proximal amphipathic helix in the cytosolic domain of LR11. Moreover, a peptide corresponding to this region folds into an α-helical structure in the presence of liposomes and transforms liposomes to small vesicles and tubule-like particles. We postulate that this amphipathic helix may contribute to the dynamic remodeling of membrane structure and facilitate LR11 intracellular transport. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P.Weliky.

Chapter 3 - Mutant GABAA receptor subunits in genetic (idiopathic) epilepsy

The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies.