Impaired Intracellular Transport Research Articles

Tensile Stress on Microtubules Facilitates Dynein-Driven Cargo Transport.

Mechanical stress significantly affects the physiological functions of cells, including tissue homeostasis, cytoskeletal alterations, and intracellular transport. As a major cytoskeletal component, microtubules respond to mechanical stimulation by altering their alignment and polymerization dynamics. Previously, we reported that microtubules may modulate cargo transport by one of the microtubule-associated motor proteins, dynein, under compressive mechanical stress. Despite the critical role of tensile stress in many biological functions, how tensile stress on microtubules regulates cargo transport is yet to be unveiled. The present study demonstrates that the low-level tensile stress-induced microtubule deformation facilitates dynein-driven transport. We validate our experimental findings using all-atom molecular dynamics simulation. Our study may provide important implications for developing new therapies for diseases that involve impaired intracellular transport.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

A Review of Oral Fixed-Dose Sodium Phenylbutyrate and Ursodoxicoltaurine in People with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is estimated to affect approximately 300,000 individuals worldwide. From symptom onset, the disease has rapid progression, and typically leads to death in approximately 3 years, though there is wide phenotypic variability. ALS pathophysiology is probably driven by several cellular and molecular mechanisms, including endoplasmic reticulum (ER) dysfunction, apoptosis, oxidative stress, impaired intracellular transport, neuroinflammation, and defective RNA metabolism and protein homeostasis. Several agents that target these pathways are in development, and a few are approved in certain regions. A fixed-dose combination of sodium phenylbutyrate and ursodoxicoltaurine (PB and TURSO, also known as AMX0035) was developed to target ER stress and mitochondrial dysfunction. This combination was approved for the treatment of ALS in the USA and Canada in 2022, following findings from the CENTAUR trial. CENTAUR was a Phase II trial comprising a 24-week randomised placebo-controlled phase and an open-label extension (OLE) phase. Treatment with PB and TURSO significantly slowed the rate of functional decline over 24 weeks compared with placebo, meeting the primary endpoint of the study. Over long-term follow-up, median survival duration was about 4.8 months longer in the group originally randomised to PB and TURSO, compared with the group originally randomised to placebo. PHOENIX, a Phase III trial of PB and TURSO planned to be completed in 2024, includes a 48-week randomised controlled phase, followed by an OLE. The PHOENIX trial is expected to provide additional insights regarding the effects of PB and TURSO in ALS.

184 The role of the Laminin 332 in cholesterol transport in dry and Aged skin

The skin provides a barrier function mediated by a combination of cornified epithelial cells, cell-cell adhesions and epidermal lipids. The barrier function is compromised in old age which is exemplified by features such as improper lipid metabolism and flattened dermal-epidermal junction. Laminin 332 is a skin basement membrane glycoprotein which was shown to be decreased in aged skin. We have previously demonstrated that the loss of laminin 332 leads to an upregulation of cholesterol biosynthesis genes and alters the skin’s lipid profile. We identified that this change is due to an impaired intracellular cholesterol transport. To understand the mechanism around cholesterol transport we have identified a panel of proteins of interest for further investigation. We investigated the role that laminin 332, actin and microtubule disruption have on both subcellular location of our proteins of interest and cholesterol transport. We have demonstrated that NPC2, Myo5b and e-cadherin are decreased in laminin α3 knockdown nTERT keratinocytes compared to shC controls. Interestingly, NPC1, a binding partner for NPC2, was shown to be increased in the laminin α3 knockdown nTERT keratinocytes. Normal levels and localisation of each protein can be restored by the addition of recombinant laminin 332. We have also demonstrated that in keratinocytes free cholesterol localises to Rab11a-positive vesicles which is a binding partner for Myo5b. By using actin- and microtubule-disrupting drugs, we have shown that it is the loss of actin that’s driving the observed phenotype. These findings suggest that the disrupted cholesterol transport and impaired barrier function in laminin α3 knockdown is due to lower levels of Myo5b and e-cadherin resulting from a disorganised actin cytoskeleton with a concomitant weakening of cell-cell contacts.

Elucidating the mutational impact in causing Niemann–Pick disease type C: an in silico approach

Niemann–Pick disease type C is a rare autosomal recessive of lysosomal storage disorder characterized by impaired intracellular lipid transport and has a tendency to accumulate the fatty acids and glycosphingolipids in a variety of neurovisceral tissues. This work includes computational tools to deciphere the mutational effect in NPC protein. The study initiated with the collection of 471 missense mutations from various databases, which were then analyzed using computational tools. The mutations (G549V, F703S, Q775P and L1244P) were said to be disease associated, altering the biophysical properties, in highly conserved regions and reduces the stability using several in silico methods and were subjected to molecular docking analysis. To analyze the ligand (Itraconazole: a small molecule of antifungal drug class, which is known to inhibit cholesterol export from lysosomes) activity Molecular docking study was performed for all the complex proteins. The average binding affinity was taken and found to be −10.76 kcal/mol (native) and −11.06 kcal/mol (Q775P was located in transmembrane region IV which impacts the sterol-sensing domain of the NPC1 protein and associated with a severe infantile neurological form). Finally, molecular dynamic simulation was performed in duplicate and trajectories were built for the backbone of the RMSD, RMSF, the number of intramolecular hydrogen bonds, the radius of gyration and the SSE percent for both the complex proteins. This work contributes to understand the effectiveness and may provide an insight on the stability of the drug with the complex variant structures. Communicated by Ramaswamy H. Sarma

Consistently High Agreement Between Independent Raters of Niemann-Pick Type C1 Clinical Severity Scale in Phase 2/3 Trial

BackgroundNiemann-Pick disease, type C1 (NPC1) is a rare neurodegenerative genetic disorder characterized by impaired intracellular transport of cholesterol and other lipids. The Niemann-Pick Disease, type C1 Severity Scale (NPC-SS) was developed to quantify neurological progression of NPC; it is used to monitor the natural history of disease progression and assess response to treatment. The objective of the study was to examine the interrater reliability of the NPC-SS in a phase 2/3 trial. MethodsStudy data were from a multicenter, prospective, randomized, double-blind trial of adrabetadex in 56 subjects with NPC1. Clinical data recorded at each study site were distributed to two independent blinded central raters to generate a severity score. A composite four-item score was utilized as the primary clinical study end point, whereas a five-item focused score has been utilized in other NPC1 trials. Interrater reliability was assessed using two-way mixed models for instrument stability, Cohen kappa, weighted kappa, and percent agreement for the four- and five-item scores. ResultsThe frequency distribution and mean (S.D.) of the NPC-SS domain assessments by the raters were almost identical. Evaluation at the patient visit level showed wide variability between visits; however, weighted kappa calculation provided a lower variability between visits. The average kappa coefficients ranged between 0.69 and 0.89, indicating good to very good agreement between raters. ConclusionsThese results support the NPC-SS, including derived four- and five-item composite scores, as reliable measures for use in a clinical trial setting.

The association of the expression of calcium handling proteins and their polymorphic variants of genes with the structural and functional state of the heart of patients with atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): The Russian Science Foundation, the Cardiology Research Institute, Tomsk National Research Medical Center of the Russian Academy of Sciences. Background. The cellular mechanisms of the genesis of atrial fibrillation are associated with impaired intracellular transport of calcium ions, which is determined by the activity of calcium-transporting proteins of the sarcoplasmic reticulum (SR). The aim of the study to investigate the expression of calcium handling proteins of the sarcoplasmic reticulum and their polymorphic variants of genes with the structural and functional state of the heart of patients with permanent atrial fibrillation. Methods. The study included 45 patients aged 29 to 65 years with atrial fibrillation. A complete echocardiographic study was performed according to standard views (parasternal and apical views) by an En Visor CHD Philips sonograph (Netherlands). All patients underwent catheter ablation, during which a biopsy of myocardial tissue from the top of the left ventricle (1-3 mg) was taken. The protein content of SERCA2a and calsequestrin (CASQ2) was determined by immunoblotting in patient’s myocardium. We identified polymorphic variants rs1860561 of the SERCA2a and rs6684209 and rs7521023 of the CASQ2 gene by real-time polymerase chain reaction. Results. The expression of both SERCA2a and CASQ2 proteins correlated with the size of the left atrium (LA). Thus, a higher level of SERCA2a expression in the myocardium corresponded to a larger LA size in patients. Despite the fact that the LV ejection fraction did not correlate with the expression level of the studied proteins, a direct correlation was found between the SERCA2a level and the values of LV end-diastolic and systolic volumes. In addition, the hemodynamic parameters of the heart, characterizing the diastolic function of the heart of patients, such as the rates of early (peak E) and late diastolic filling (peak A) LV were lower in patients with a higher SERCA2a level, although the ratio of these parameters (peak E/peak A) had no significant differences. In the homozygous GG genotype of the ATP2A2 gene, the expression level of the SERCA2a protein was (p = 0.039) higher than in patients with the heterozygous genotype (GA). Analysis of the parameters of the ECHO-KG study showed that the presence of the A allele of the rs1860561 variant of the ATP2A2 gene is associated with an increase in the LV sphericity index. The expression of the CASQ2 protein in patients with CC genotype of the rs6684209 variant of the CASQ2 gene was 2.5 times higher than in patients with CT genotype. The genotypes of the rs7521023 variant of the CASQ2 gene were not associated with the level of expression of the corresponding protein in the myocardium of the studied patients. Conclusions. The different genotypes of the rs1860561 variant of the ATP2A2 gene and rs6684209 of the CASQ2 gene can modulate the expression level of the SERCA2a protein and the CASQ2 protein. Moreover, the expression of calcium handling proteins to a greater extent affects the functional and structural characteristics of the heart of patients with permanent AF.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

Deletion of Mea6 in Cerebellar Granule Cells Impairs Synaptic Development and Motor Performance.

The cerebellum is conceptualized as a processor of complex movements. Many diseases with gene-targeted mutations, including Fahr’s disease associated with the loss-of-function mutation of meningioma expressed antigen 6 (Mea6), exhibit cerebellar malformations, and abnormal motor behaviors. We previously reported that the defects in cerebellar development and motor performance of Nestin-Cre;Mea6F/F mice are severer than those of Purkinje cell-targeted pCP2-Cre;Mea6F/F mice, suggesting that Mea6 acts on other types of cerebellar cells. Hence, we investigated the function of Mea6 in cerebellar granule cells. We found that mutant mice with the specific deletion of Mea6 in granule cells displayed abnormal posture, balance, and motor learning, as indicated in footprint, head inclination, balanced beam, and rotarod tests. We further showed that Math1-Cre;Mea6F/F mice exhibited disrupted migration of granule cell progenitors and damaged parallel fiber-Purkinje cell synapses, which may be related to impaired intracellular transport of vesicular glutamate transporter 1 and brain-derived neurotrophic factor. The present findings extend our previous work and may help to better understand the pathogenesis of Fahr’s disease.

MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion.

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterized by compromised function of the neuromuscular junction, manifesting with fatigable muscle weakness. Mutations in MYO9A were previously identified as causative for CMS but the precise pathomechanism remained to be characterized. On the basis of the role of MYO9A as an actin-based molecular motor and as a negative regulator of RhoA, we hypothesized that loss of MYO9A may affect the neuronal cytoskeleton, leading to impaired intracellular transport. To investigate this, we used MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells), revealing altered expression of a number of cytoskeletal proteins important for neuron structure and intracellular transport. On the basis of these findings, the effect on protein transport was determined using a vesicular recycling assay which revealed impaired recycling of a neuronal growth factor receptor. In addition, an unbiased approach utilizing proteomic profiling of the secretome revealed a key role for defective intracellular transport affecting proper protein secretion in the pathophysiology of MYO9A-related CMS. This also led to the identification of agrin as being affected by the defective transport. Zebrafish with reduced MYO9A orthologue expression were treated with an artificial agrin compound, ameliorating defects in neurite extension and improving motility. In summary, loss of MYO9A affects the neuronal cytoskeleton and leads to impaired transport of proteins, including agrin, which may provide a new and unexpected treatment option.

Toxic effects of zinc ions on kinesin – Potential molecular cause of impaired intracellular transport

In healthy organisms the metabolism of the trace element zinc is well balanced. If this balance becomes destroyed the free zinc level might increase and cause toxic effects. The present study demonstrates that under definite conditions zinc ions are able to inhibit the ATPase activity of neuron-specific KIF5A (kinesin-1). Correspondingly, the motility activity of KIF5A also decreased. The inhibition rates have been found to depend on the magnesium ion concentration. Lowering the magnesium concentration weakens the inhibition. In addition, also decreases of temperature or increasing the ATP concentration result in reduced inhibition. Zinc ion-mediated inhibition of KIF5A activity might be one molecular cause contributing to impaired transport processes within brain and other organs in cases of zinc dyshomeostasis.

Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells

High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

Role of Exchange Protein Activated by cAMP 1 in Regulating Rates of Microtubule Formation in Cystic Fibrosis Epithelial Cells.

The regulation of microtubule dynamics in cystic fibrosis (CF) epithelial cells and the consequences of reduced rates of microtubule polymerization on downstream CF cellular events, such as cholesterol accumulation, a marker of impaired intracellular transport, are explored here. It is identified that microtubules in both CF cell models and in primary CF nasal epithelial cells repolymerize at a slower rate compared with respective controls. Previous studies suggest a role for cAMP in modulating organelle transport in CF cells, implicating a role for exchange protein activated by cAMP (EPAC) 1, a regulator of microtubule elongation, as a potential mechanism. EPAC1 activity is reduced in CF cell models and in Cftr(-/-) mouse lung compared with respective non-CF controls. Stimulation of EPAC1 activity with the selective EPAC1 agonist, 8-cpt-2-O-Me-cAMP, stimulates microtubule repolymerization to wild-type rates in CF cells. EPAC1 activation also alleviates cholesterol accumulation in CF cells, suggesting a direct link between microtubule regulation and intracellular transport. To verify the relationship between transport and microtubule regulation, expression of the protein, tubulin polymerization-promoting protein, was knocked down in non-CF human tracheal (9/HTEo(-)) cells to mimic the microtubule dysregulation in CF cells. Transduced cells with short hairpin RNA targeting tubulin polymerization-promoting protein exhibit CF-like perinuclear cholesterol accumulation and other cellular manifestations of CF cells, thus supporting a role for microtubule regulation as a mechanism linking CFTR function to downstream cellular manifestation.

Niemann-Pick Type C-2 Disease: Identification by Analysis of Plasma Cholestane-3β,5α,6β-Triol and Further Insight into the Clinical Phenotype.

Niemann-Pick type C disease is a rare disorder caused by impaired intracellular lipid transport due to mutations in either the NPC1 or the NPC2 gene. Ninety-five % of NPC patients show mutations in the NPC1 gene. A much smaller number of patients suffer from NPC2 disease and present respiratory failure as one of the most frequent symptoms. Several plasma oxysterols are highly elevated in NPC1 and can be used as a biomarker in the diagnosis of NPC1. Plasma cholestane-3β,5α,6β-triol was evaluated as biomarker for NPC2 by GC/MS and LC-MS/MS analysis. The diagnosis was confirmed by Sanger sequencing and filipin staining. We report three NPC2 patients with typical respiratory problems and a detailed description of the nature of the lung disease in one of them. All patients had elevated levels of plasma cholestane-3β,5α,6β-triol. In two of these patients, the positive oxysterol result led to a rapid diagnosis of NPC2 by genetic analysis. The phenotype of the third patient has been described previously. In this patient a cholestane-3β,5α,6β-triol concentration markedly above the reference range was found. Measurement of plasma cholestane-3β,5α,6β-triol enables to discriminate between controls and NPC1 and NPC2 patients, making it a valuable biomarker for the rapid diagnosis not only for NPC1 but also for NPC2 disease.The measurement of oxysterols should be well kept in mind in the differential diagnosis of lysosomal diseases, as the elevation of oxysterols in plasma may speed up the diagnosis of NPC1 and NPC2.

Recent advances in the diagnosis and treatment of niemann-pick disease type C in children: a guide to early diagnosis for the general pediatrician.

Niemann-Pick disease (NP-C) is a lysosomal storage disease in which impaired intracellular lipid transport leads to accumulation of cholesterol and glycosphingolipids in various neurovisceral tissues. It is an autosomal recessive disorder, caused by mutations in the NPC1 or NPC2 genes. The clinical spectrum is grouped by the age of onset and onset of neurological manifestation: pre/perinatal; early infantile; late infantile; and juvenile periods. The NP-C Suspicion Index (SI) screening tool was developed to identify suspected patients with this disease. It is especially good at recognizing the disease in patients older than four years of age. Biochemical tests involving genetic markers and Filipin staining of skin fibroblast are being employed to assist diagnosis. Therapy is mostly supportive and since 2009, the first specific therapy approved for use was Miglustat (Zavesca) aimed at stabilizing the rate of progression of neurological manifestation. The prognosis correlates with age at onset of neurological signs; patients with early onset form progress faster. The NP-C disease has heterogeneous neurovisceral manifestations. A SI is a screening tool that helps in diagnostic process. Filipin staining test is a specific biomarker diagnostic test. Miglustat is the first disease-specific therapy.

Loss of lysophosphatidic acid receptor LPA1 alters oligodendrocyte differentiation and myelination in the mouse cerebral cortex.

Lysophosphatidic acid (LPA) is an intercellular signaling lipid that regulates multiple cellular functions, acting through specific G-protein coupled receptors (LPA(1-6)). Our previous studies using viable Malaga variant maLPA1-null mice demonstrated the requirement of the LPA1 receptor for normal proliferation, differentiation, and survival of the neuronal precursors. In the cerebral cortex LPA1 is expressed extensively in differentiating oligodendrocytes, in parallel with myelination. Although exogenous LPA-induced effects have been investigated in myelinating cells, the in vivo contribution of LPA1 to normal myelination remains to be demonstrated. This study identified a relevant in vivo role for LPA1 as a regulator of cortical myelination. Immunochemical analysis in adult maLPA1-null mice demonstrated a reduction in the steady-state levels of the myelin proteins MBP, PLP/DM20, and CNPase in the cerebral cortex. The myelin defects were confirmed using magnetic resonance spectroscopy and electron microscopy. Stereological analysis limited the defects to adult differentiating oligodendrocytes, without variation in the NG2+ precursor cells. Finally, a possible mechanism involving oligodendrocyte survival was demonstrated by the impaired intracellular transport of the PLP/DM20 myelin protein which was accompanied by cellular loss, suggesting stress-induced apoptosis. These findings describe a previously uncharacterized in vivo functional role for LPA1 in the regulation of oligodendrocyte differentiation and myelination in the CNS, underlining the importance of the maLPA1-null mouse as a model for the study of demyelinating diseases.

P 29. BDNF val66met polymorphism influences the time course of changes in corticospinal excitability induced by paired associative and transcranial direct current stimulation

It is widely appreciated that brain-derived neurotrophic factor (BDNF) is released in an activity dependent manner within the brain, and that it plays a key role in regulating synaptic plasticity and long-term potentiation (LTP). A single nucleotide polymorphism (SNP) arises at nucleotide 196 of the eponymous gene in humans. This gives rise to a valine to methionine substitution at codon 66 of the precursor protein. When assessed in animal models, the presence of this substitution is associated with impaired intracellular transport and reduced capacity for LTP. Differences between met carriers and val homozygotes have been reported in studies investigating the plasticity of human motor cortex in response to various forms of brain stimulation. Those who are homozygous for the val allele show elevations in corticospinal excitability following paired associative stimulation (PAS) ( Cirillo et al., 2012 ) and “theta burst” repetitive transcranial magnetic stimulation (rTMS) ( Cheeran et al., 2008 ), that are larger than those exhibited by individuals with a met allele. In contrast, Antal et al. (2010) reported that met carriers show an accentuated response to transcranial direct current stimulation (tDCS). Given the clinical potential of brain stimulation techniques, there is a pressing need to establish the basis of these contrarieties, and the generality of effects attributable to BDNF genotype. In the present study, we focused on corticospinal projections to muscles of the forearm that are of particular significance in rehabilitation following stroke. Consideration was given to two forms of brain stimulation-PAS and tDCS that putatively have utility in this context. A total of 56 participants (29 Female) aged 19–32 (mean ± SD: 22.31 ± 3.42 years) underwent PAS. There were 53 participants (30 Female) aged between 18 and 32 (mean ± SD: 22.35 ± 3.44) who took part in the tDCS condition. PAS consisted of a train of electrical stimulation (1 ms pulse width, 10 Hz, 500 ms duration) delivered to the right Flexor Carpi Radialis (FCR) motor point, followed 25 ms later by a TMS pulse delivered to M1. This was repeated every 10 s for 30 min (total 180 pairs). Anodal 1 mA tDCS was delivered to M1 for 30 min. Motor evoked potentials (MEPs) in response to TMS of the target area were obtained on prior to the intervention, and at 0, 10, 20 and 30 min following the cessation of stimulation. With respect to FCR, val homozygotes exhibited increased corticospinal excitability following tDCS that emerged after 10 min and remained elevated thereafter. In met carriers there was a transient increase in MEP amplitude that peaked 10 min post-intervention. The val homozygotes exhibited a qualitatively similar pattern of response to PAS, whereby increases in corticospinal excitability were delayed. Although weaker overall, the elevations of MEP amplitude exhibited by met carriers were larger immediately following the cessation of stimulation than subsequently. In relation to ECR, the profile of response to tDCS was similar to that manifested in FCR. The val homozygotes exhibited an immediate and sustained elevation in ECR MEP amplitude following PAS. No such changes were present for met carriers. In summary, these findings suggest that met carriers respond transiently, albeit weakly, to brain stimulation. Responses to tDCS are more prominent than those to PAS. Val homozygotes exhibit responses to both forms of stimulation that tend to increase in magnitude over 30 min post-intervention.

Tissue-Specific Transgene Expression in Induced Pluripotent Stem (iPS) Cell-Derived Megakaryocytes: Correction of Glanzmann Thrombasthenia (GT)

Abstract 387Megakaryocyte-specific transgene expression in patient-derived iPS cells offers a new approach to study and potentially treat disorders that affect megakaryocytes and platelets. The feasibility of this method was demonstrated in GT, a disorder resulting from an absence of functional platelet integrin alphaIIb/beta3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. While rare, GT can serve as a model of an inherited platelet disorder that would benefit from corrective cell therapy. For this study, iPS cells were generated from the peripheral blood of healthy controls and two patients with GT. Patient 1 is homozygous for a novel alphaIIb exon/intron 4 splice site mutation, leading to an alternatively spliced transcript and premature stop codon. Patient 2 is homozygous for a previously reported alphaIIb missense mutation in exon 8 (p.G273D), leading to impaired intracellular transport and surface expression of the heterodimer. Both patients express <5% alphaIIb/beta3 on the surface of their platelets, which was confirmed in IPS cell-derived megakaryocytes. To achieve megakaryocyte-specific expression, transgene constructs were created using the murine proximal GPIbalpha promoter driving either an eGFP reporter or wild type alphaIIb cDNA. These constructs were inserted into the AAVS1 safe harbor locus using zinc finger nuclease-mediated homologous recombination. When targeted to a single allele of the AAVS1 locus in human ES cells, the GPIbalpha promoter driving eGFP showed a high-level of expression in ES cell-derived megakaryocytes. Inserting the GPIbalpha promoter driving alphaIIb into a single allele of the AAVS1 locus in GT iPS cells restored ∼60% surface expression of integrin alphaIIb/beta3 when compared to control iPS cell-derived megakaryocytes. Transgenic alphaIIb mRNA levels were similar to endogenous alphaIIb mRNA levels in differentiated megakaryocytes. When stimulated with thrombin, corrected GT iPS cell-derived megakaryocytes demonstrated both PAC-1 antibody binding and fibrinogen binding. This study shows the GPIbalpha promoter construct targeted to the AAVS1 locus drives a high-level of expression in iPS cell-derived megakaryocytes, which offers a novel tool to study megakaryocyte and platelet biology in samples obtained from affected individuals. For GT, this study demonstrates a higher level of correction than seen with a lentiviral approach. To our knowledge, this is the first report of the generation and correction of iPS cell lines from patients with a disease affecting platelet function. Disclosures:No relevant conflicts of interest to declare.

Congenital Sucrase‐Isomaltase Deficiency

Congenital Sucrase‐Isomaltase Deficiency

Mo1804 Contribution of Intestinal Bacteria and Epithelial Mesenchymal Transition to Intestinal Inflammation and Fibrosis in Enterocyte-Specific Pipk III Deficient Mice

BACKGROUND AND AIM: Genome-wide association studies have provided MTMR3 as a Crohn's-disease-associated gene. Pipk III, which cooperatively functions in phosphatidyl inositol 3,5-bisphosphates [PI(3,5)P2] metabolism with MTMR3, is a kinase that produces PI(3,5)P2 from phophatidyl inositol 3-bisphosphate. We generated enterocyte-specific Pipk III deficient (KO) mice which displayed diarrhea, body weight loss, and pathological findings of epithelial vacuoles, transmural inflammatory cell infiltration, increased fibrosis, and goblet cell depletion, sharing the clinical and histological findings with Crohn's diseae. In this context, we tried to elucidate the mechanisms of intestinal inflammation and fibrosis in Crohn's disease by analyzing those in our KOmice. METHODS:We examined the localization of proteins functioning at apical and basolateral membranes of enterocytes using immunofluorescence staining, and the expression of these membranous proteins in intestinal mucosa of both KO and the control mice using western blot analysis. We compared the expression of inflammationand fibrosis-related genes in intestinal mucosa using RT-PCR. In addition, we monitored the survival of Pipk III KO mice with or without antibiotics to study the relevance of intestinal bacteria in intestinal inflammation of KO mice. Furthermore, we injected fluorescent dextran per anus and observed its mucosal distribution using immunofluorescence staining to evaluate how easily intestinal microbiota can invade to mucosa of KO mice. RESULTS: Although western blot analysis did not show any differences in expression of apical and basolateral membranous proteins between KO and the control mice, immunofluorescence staining revealed mislocalization of these proteins in KO enterocytes, suggesting impaired intracellular vesicle transport and cellular polarity. We found the increased genes expression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IL-6. Meanwhile, the expression of fibrosis-promoting genes such as TGF-beta1 and CTGF, and Snail1, which is a transcription factor that promotes epithelial mesenchymal transition (EMT), was increased in KO mice compared to the control mice. Consistent with this result, immunofluorescence staining revealed that KO enterocytes expressed Vimentin protein more intensely than the control. Furthermore, the survival of KO mice treated with antibiotics was prolonged and fluorescent dextran injected per anus was distributed more scattered in KO mice with antibiotics than those without antibiotics. CONCLUSION: Facilitating entry of intestinal bacteria to mucoca based on impaired intracellular vesicle transport and cellular polarity in enterocytes seems to bring about intestinal inflammation in KO mice. Meanwhile, there is a possibility that EMT in KO enterocytes induces intestinal fibrosis.