Targeting TMEM16A to reverse vasoconstriction and remodelling in idiopathic pulmonary arterial hypertension.

Our systematic analysis of anion channels and transporters in idiopathic pulmonary arterial hypertension (IPAH) showed marked upregulation of the Cl- channel TMEM16A gene. We hypothesised that TMEM16A overexpression might represent a novel vicious circle in the molecular pathways causing pulmonary arterial hypertension (PAH).We investigated healthy donor lungs (n=40) and recipient lungs with IPAH (n=38) for the expression of anion channel and transporter genes in small pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs).In IPAH, TMEM16A was strongly upregulated and patch-clamp recordings confirmed an increased Cl- current in PASMCs (n=9-10). These cells were depolarised and could be repolarised by TMEM16A inhibitors or knock-down experiments (n=6-10). Inhibition/knock-down of TMEM16A reduced the proliferation of IPAH-PASMCs (n=6). Conversely, overexpression of TMEM16A in healthy donor PASMCs produced an IPAH-like phenotype. Chronic application of benzbromarone in two independent animal models significantly decreased right ventricular pressure and reversed remodelling of established pulmonary hypertension.Our findings suggest that increased TMEM16A expression and activity comprise an important pathologic mechanism underlying the vasoconstriction and remodelling of pulmonary arteries in PAH. Inhibition of TMEM16A represents a novel therapeutic approach to reverse remodelling in PAH.

Importance of kynurenine in pulmonary hypertension.

The tryptophan metabolite kynurenine is significantly increased in pulmonary arterial hypertension (PAH) patients, and it is a potent vasodilator of systemic arteries. Our aim was to investigate the role of kynurenine in the pulmonary circulation. Serum tryptophan, kynurenine, and kynurenic acid levels were measured in 20 idiopathic PAH (IPAH) patients, 20 healthy controls, and 20 patients with chronic lung disease or metabolic syndrome without PH. Laser-dissected pulmonary arteries from IPAH and control lungs were tested for the expression of indoleamine-2, 3-dioxygenase (IDO), the rate-limiting enzyme for the conversion from tryptophan to kynurenine. Acute effects of kynurenine were tested in pulmonary vascular preparations, two different models of chronic pulmonary hypertension (PH), and in human pulmonary arterial smooth muscle cells (hPASMCs). In IPAH vs. control serum, kynurenine was significantly elevated (3.6 ± 0.2 vs. 2.6 ± 0.1 µM, P < 0.0001), and strongly associated with PH (area under the curve = 0.86), but kynurenine levels were not elevated in lung disease and metabolic syndrome. Among all investigated tryptophan metabolites, kynurenine displayed the strongest correlation with mean pulmonary arterial pressure (mPAP) (ρ: 0.770, P < 0.0001). Tryptophan was significantly decreased in IPAH lungs; however, IDO expression was not changed. In hPASMCs, kynurenine increased both cAMP and cGMP; in intrapulmonary arteries, it relaxed the preconstriction via NO/cGMP and cAMP pathways, and in two models of established PH, it acutely decreased the mPAP. Our data suggest that kynurenine elevation might be specifically associated with mPAP; kynurenine acts on hPASMCs in synergy with NO and exerts acute pulmonary vasodilatation in chronic PH models. Kynurenine might provide both a new biomarker and a new therapeutic option for PH.

Hypoxic vascular response and ventilation/perfusion matching in end-stage COPD may depend on p22phox.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease in which the amount of emphysema and airway disease may be very different between individuals, even in end-stage disease. Emphysema formation may be linked to the involvement of the small pulmonary vessels. The NAPDH oxidase (Nox) family is emerging as a key disease-related factor in vascular diseases, but currently its role in hypoxia-induced pulmonary remodelling in COPD remains unclear.Here we investigate the role of p22phox, a regulatory subunit of Nox, in COPD lungs, hypoxic pulmonary vasoconstriction (HPV), hypoxia-induced pulmonary vascular remodelling and pulmonary hypertension.In COPD, compared to control lungs, p22phox expression was significantly reduced. The expression was correlated positively with mean pulmonary arterial pressure and oxygenation index and negatively with the diffusing capacity of the lung for carbon monoxide (p<0.02). This suggests a role of p22phox in ventilation/perfusion ratio matching, vascular remodelling and loss of perfused lung area. In p22phox-/- mice, HPV was significantly impaired. In the chronic hypoxic setting, lack of p22phox was associated with improved right ventricular function and decreased pulmonary vascular remodelling.p22phox-dependent Nox plays an important role in the COPD phenotype, by its action on phase II HPV and chronic vascular remodelling.

TGF-ß directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury.

TGF-ß is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-ß pathway is described, which rapidly promoted internalization of the αßγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-ß applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which--together with patch-clamp and flow cytometry studies--identified ENaC as the target of TGF-ß. TGF-ß rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of ßENaC, the subunit responsible for cell-surface stability of the αßγENaC complex. ENaC internalization was dependent on oxidation of ßENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove ßENaC internalization, which was inhibited by a TGF-ß neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-ß signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-ß-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

Docosahexaenoic acid causes rapid pulmonary arterial relaxation via KCa channel-mediated hyperpolarisation in pulmonary hypertension.

Cardioprotective benefits of ω-3 fatty acids such as docosahexaenoic acid (DHA) are well established, but the regulatory effect of DHA on vascular tone and pressure in pulmonary hypertension is largely unknown.As DHA is a potent regulator of K+ channels, we hypothesised that DHA modulates the membrane potential of pulmonary artery smooth muscle cells (PASMCs) through K+ channels and thus exerts its effects on pulmonary vascular tone and pressure.We show that DHA caused dose-dependent activation of the calcium-activated K+ (KCa) current in primary human PASMCs and endothelium-dependent relaxation of pulmonary arteries. This vasodilation was significantly diminished in KCa-/- (Kcnma1-/-) mice. In vivo, acute DHA returned the right ventricular systolic pressure in the chronic hypoxia-induced pulmonary hypertension animal model to the level of normoxic animals. Interestingly, in idiopathic pulmonary arterial hypertension the KCa channels and their subunits were upregulated. DHA activated KCa channels in these human PASMCs and hyperpolarised the membrane potential of the idiopathic pulmonary arterial hypertension PASMCs to that of the PASMCs from healthy donors.Our findings indicate that DHA activates PASMC KCa channels leading to vasorelaxation in pulmonary hypertension. This effect might provide a molecular explanation for the previously undescribed role of DHA as an acute vasodilator in pulmonary hypertension.

Bradycardia alters Ca(2+) dynamics enhancing dispersion of repolarization and arrhythmia risk.

Bradycardia prolongs action potential (AP) durations (APD adaptation), enhances dispersion of repolarization (DOR), and promotes tachyarrhythmias. Yet, the mechanisms responsible for enhanced DOR and tachyarrhythmias remain largely unexplored. Ca(2+) transients and APs were measured optically from Langendorff rabbit hearts at high (150 × 150 µm(2)) or low (1.5 × 1.5 cm(2)) magnification while pacing at a physiological (120 beats/min) or a slow heart rate (SHR = 50 beats/min). Western blots and pharmacological interventions were used to elucidate the regional effects of bradycardia. As a result, bradycardia (SHR 50 beats/min) increased APDs gradually (time constant τf→s = 48 ± 9.2 s) and caused a secondary Ca(2+) release (SCR) from the sarcoplasmic reticulum during AP plateaus, occurring at the base on average of 184.4 ± 9.7 ms after the Ca(2+) transient upstroke. In subcellular imaging, SCRs were temporally synchronous and spatially homogeneous within myocytes. In diastole, SHR elicited variable asynchronous sarcoplasmic reticulum Ca(2+) release events leading to subcellular Ca(2+) waves, detectable only at high magnification. SCR was regionally heterogeneous, correlated with APD prolongation (P < 0.01, n = 5), enhanced DOR (r = 0.9277 ± 0.03, n = 7), and was gradually reversed by pacing at 120 beats/min along with APD shortening (P < 0.05, n = 5). A stabilizer of leaky ryanodine receptors (RyR2), 3-(4-benzylcyclohexyl)-1-(7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)propan-1-one (K201; 1 µM), suppressed SCR and reduced APD at the base, thereby reducing DOR (P < 0.02, n = 5). Ventricular ectopy induced by bradycardia (n = 5/15) was suppressed by K201. Western blot analysis revealed spatial differences of voltage-gated L-type Ca(2+) channel protein (Cav1.2α), Na(+)-Ca(2+) exchange (NCX1), voltage-gated Na(+) channel (Nav1.5), and rabbit ether-a-go-go-related (rERG) protein [but not RyR2 or sarcoplasmic reticulum Ca(2+) ATPase 2a] that correlate with the SCR distribution and explain the molecular basis for SCR heterogeneities. In conclusion, acute bradycardia elicits synchronized subcellular SCRs of sufficient magnitude to overcome the source-sink mismatch and to promote afterdepolarizations.

Oestrogen upregulates L-type Ca²âº channels via oestrogen-receptor- by a regional genomic mechanism in female rabbit hearts.

In type-2 long QT (LQT2), adult women and adolescent boys have a higher risk of lethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbit hearts, similar sex- and age-dependent TdP risks were attributed to higher expression levels of L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger, at the base of the female epicardium. Here, the effects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby I(Ca,L) density is upregulated in adult female rabbit hearts. I(Ca,L) density was measured by the whole-cell patch-clamp technique on days 0-3 in cardiomyocytes isolated from the base and apex of adult female epicardium. Peak I(Ca,L) was 28% higher at the base than apex (P < 0.01) and decreased gradually (days 0-3), becoming similar to apex myocytes, which had stable currents for 3 days. Incubation with oestrogen (E2, 0.1-1.0 nm) increased I(Ca,L) (∼2-fold) in female base but not endo-, apex or male myocytes. Progesterone (0.1-10 µm) had no effect at base myocytes. An agonist of the α- (PPT, 5 nm) but not the ß- (DPN, 5 nm) subtype oestrogen receptor (ERα/ERß) upregulated I(Ca,L) like E2. Western blots detected similar levels of ERα and ERß in male and female hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA (RT-PCR) levels but did not change I(Ca,L) kinetics. I(Ca,L) upregulation by E2 was suppressed by the ER antagonist ICI 182,780 (10 µm) or by inhibition of transcription (actinomycin D, 4 µm) or protein biosynthesis (cycloheximide, 70 µm). Therefore, E2 upregulates I(Ca,L) by a regional genomic mechanism involving ERα which is a known determinant of sex differences in TdP risk in LQT2.

Reverse rate-dependent changes are determined by baseline action potential duration in mammalian and human ventricular preparations.

Class III antiarrhythmic agents exhibit reverse rate-dependent lengthening of the action potential duration (APD). In spite of the several theories developed so far to explain this reverse rate-dependency (RRD), its mechanism has not yet been clarified. The aim of the present work was to further elucidate the mechanisms responsible for RRD in mammalian ventricular myocardium. Action potentials were recorded using conventional sharp microelectrodes from human, canine, rabbit and guinea pig ventricular myocardium in a rate-dependent manner varying the cycle length (CL) between 0.3 and 5 s. Rate-dependent drug effects were studied using agents known to lengthen or shorten action potentials, and these drug-induced changes in APD were correlated with baseline APD values. Both drug-induced lengthening (by dofetilide, sotalol, E-4031, BaCl(2), veratrine, BAY K 8644) and shortening (by mexiletine, tetrodotoxin, lemakalim) of action potentials displayed RRD, i.e., changes in APD were greater at longer than at shorter CLs. In rabbit, where APD is a biphasic function of CL, the drug-induced APD changes were proportional to baseline APD values but not to CL. Similar results were obtained when repolarization was modified by injection of inward or outward current pulses in isolated canine cardiomyocytes. In each case the change in APD was proportional to baseline APD (i.e., that measured before the superfusion of drug or injection of current). Also, the net membrane current (I (net)), determined from the action potential waveform at the middle of the plateau, was inversely proportional to APD and consequently with to CL. The results indicate that RRD is a common characteristic of all the drugs tested regardless of the modified ion current species. Thus, drug-induced RRD can be considered as an intrinsic property of cardiac membranes based on the inverse relationship between I (net) and APD.

Changes in gene expression following cardiac pacing-induced delayed cardioprotection in the canine heart.

The aim of the present study was to identify gene expression changes in the rapid cardiac pacing-induced delayed antiarrhythmic protection in the canine, using cDNA microarrays and quantitative real-time PCR (QRT -PCR) techniques. In all dogs under light pentobarbitone anaesthesia, a pacing electrode was introduced into the right ventricle, and then the animals were divided into three groups: (1) sham-operated and sham-paced group (SP, n = 3) (2) ischaemic control group (IC; n = 3); these were without cardiac pacing and subjected only to a 25 min occlusion of the left anterior descending coronary artery (LAD), and (3) paced group (PC, n = 3); these animals were paced at a rate of 220-240 beats min-1 24 h prior to ischaemia. With cDNA chip 23 genes were found with altered expression in response to rapid cardiac pacing and 10 genes in the IC group when compared to SP dogs. These genes encode transcription factors (MEF2); members of signaling pathways (TGFß2, PDE4D9), hormone related proteins (e.g. vasopressin V1 and V2 receptors). RT-QPCR was used either to confirm the results of the microarray analysis and also to study 46 genes which are already known to have a role in the late phase of PC. By this method 17 genes were up-regulated and 6 genes down-regulated in the IC group; their expression ratios changed either to the opposite or showed no alteration after cardiac pacing. This study would add some new information about those transcriptional changes that are involved in the delayed phase of cardiac protection.

Gap junctional uncoupling plays a trigger role in the antiarrhythmic effect of ischaemic preconditioning.

OBJECTIVE: The aim of this study was to determine whether uncoupling of gap junctions (GJ) prior to ischaemia would modify the antiarrhythmic effect of ischaemic preconditioning (PC) in a canine model of ischaemia/reperfusion. METHODS: Twenty control dogs, anaesthetised with chloralose and urethane, were thoracotomised and subjected either to a 25 or a 60 min occlusion of the left anterior descending (LAD) coronary artery. This prolonged ischaemia was preceded 20 min earlier by a single 5 min LAD occlusion in preconditioned dogs (PC group; n=14) or by a 20 min intracoronary infusion of 50 microM carbenoxolone (CBX group; n=15), a relatively selective uncoupler of gap junctions. CBX was also infused in PC dogs (CBX+PC group; n=11). The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and of ventricular arrhythmias, such as ventricular premature beats (VPBs), ventricular tachycardiac (VT) episodes and ventricular fibrillation (VF), as well as changes in electrical impedance was assessed throughout the experiments. Connexin 43 (Cx43) phosphorylation and GJ permeability were determined at the end of the occlusion periods. RESULTS: Compared to the controls PC and, interestingly, CBX markedly reduced, e.g. the total number of VPBs (440+/-104 vs 47+/-11 and 60+/-15; P<0.05) during the prolonged occlusion. This protection was, however, attenuated when CBX was infused in PC dogs (VPBs: 203+/-32). Changes in electrical impedance, GJ permeability and Cx43 dephosphorylation were significantly less in the PC and CBX groups than in the controls but these were again increased in the CBX+PC group. CONCLUSIONS: Uncoupling of GJs prior to ischaemia either by PC or CBX preserves the electrical coupling of cells and results in an antiarrhythmic effect during a subsequent ischaemic insult, indicating that a partial closure of gap junctions may play a trigger role in the protection. In contrast, when CBX is administered in PC dogs the protection both against GJ uncoupling and arrhythmias is markedly attenuated, suggesting that the antiarrhythmic protection, at least in part, is mediated through GJs.

Genomic upregulation of cardiac Cav1.2α and NCX1 by estrogen in women.

BACKGROUND: Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts. METHODS: Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs. RESULTS: In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1-2 days) increased ICa,L (31%, P < 0.05) and INCX (7.5-fold, - 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter ICa,L and INCX. CONCLUSIONS: The results show that estrogen upregulates cardiac ICa,L and INCX in women through genomic mechanisms that account for sex differences in Ca2+ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.

TASK-1 Regulates Apoptosis and Proliferation in a Subset of Non-Small Cell Lung Cancers.

Lung cancer is the leading cause of cancer deaths worldwide; survival times are poor despite therapy. The role of the two-pore domain K+ (K2P) channel TASK-1 (KCNK3) in lung cancer is at present unknown. We found that TASK-1 is expressed in non-small cell lung cancer (NSCLC) cell lines at variable levels. In a highly TASK-1 expressing NSCLC cell line, A549, a characteristic pH- and hypoxia-sensitive non-inactivating K+ current was measured, indicating the presence of functional TASK-1 channels. Inhibition of TASK-1 led to significant depolarization in these cells. Knockdown of TASK-1 by siRNA significantly enhanced apoptosis and reduced proliferation in A549 cells, but not in weakly TASK-1 expressing NCI-H358 cells. Na+-coupled nutrient transport across the cell membrane is functionally coupled to the efflux of K+ via K+ channels, thus TASK-1 may potentially influence Na+-coupled nutrient transport. In contrast to TASK-1, which was not differentially expressed in lung cancer vs. normal lung tissue, we found the Na+-coupled nutrient transporters, SLC5A3, SLC5A6, and SLC38A1, transporters for myo-inositol, biotin and glutamine, respectively, to be significantly overexpressed in lung adenocarcinomas. In summary, we show for the first time that the TASK-1 channel regulates apoptosis and proliferation in a subset of NSCLC.

Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells.

Reliable in vitro assays are essential for study of the effects of neurotoxic compounds such as beta-amyloid peptides (Abeta). The MTT assay has been used in cultures of different cells, e.g. SH-SY5Y neuroblastoma cells, for the quantitative measurement of Abeta toxicity. In our laboratory differentiated SH-SY5Y cells were used in the MTT assay. Cell differentiation with 10 microM all-trans-retinoic acid resulted in a constant cell number. The cells possess highly developed neurites and exhibit high sensitivity against Abeta. Owing to the constant cell number in differentiated SH-SY5Y cultures the decrease of the redox activity is directly proportional to the neurotoxicity of the substances, no correction is needed. The results of the MTT assay of Abeta peptides on differentiated SH-SY5Y cells displayed a good correlation also with the in vivo results. The present experiments reveal an effective assay for the study of potentially neurotoxic compounds.

Ion channels and transporters as therapeutic targets in the pulmonary circulation.

Pulmonary circulation is a low pressure, low resistance, high flow system. The low resting vascular tone is maintained by the concerted action of ion channels, exchangers and pumps. Under physiological as well as pathophysiological conditions, they are targets of locally secreted or circulating vasodilators and/or vasoconstrictors, leading to changes in expression or to posttranslational modifications. Both structural changes in the pulmonary arteries and a sustained increase in pulmonary vascular tone result in pulmonary vascular remodeling contributing to morbidity and mortality in pediatric and adult patients. There is increasing evidence demonstrating the pivotal role of ion channels such as K(+) and Cl(-) or transient receptor potential channels in different cell types which are thought to play a key role in vasoconstrictive remodeling. This review focuses on ion channels, exchangers and pumps in the pulmonary circulation and summarizes their putative pathophysiological as well as therapeutic role in pulmonary vascular remodeling. A better understanding of the mechanisms of their actions may allow for the development of new options for attenuating acute and chronic pulmonary vasoconstriction and remodeling treating the devastating disease pulmonary hypertension.

Modulation of gap junctions by nitric oxide contributes to the anti-arrhythmic effect of sodium nitroprusside?

BACKGROUND AND PURPOSE: Nitric oxide (NO) donors provide a preconditioning-like anti-arrhythmic protection in the anaesthetized dog. As NO may modulate gap junction (GJ) function, the present study investigated whether this anti-arrhythmic effect is due to a modification of GJs by NO, derived from the NO donor sodium nitroprusside (SNP). EXPERIMENTAL APPROACH: In chloralose-urethane-anaesthetized, open-chest dogs, either saline (controls; n= 11) or SNP (0.2 microg x kg(-1) x min(-1); n= 10) was infused at a rate of 0.5 mL x min(-1) by the intracoronary route. The infusions were started 20 min prior to and maintained throughout the entire 60 min occlusion period of the left anterior descending coronary artery. The severity of ischaemia and of arrhythmias, tissue electrical impedance and permeability, as well as the phosphorylation of connexin43, were assessed. KEY RESULTS: Compared with the controls, SNP infusion markedly suppressed the total number of ventricular premature beats (666 +/- 202 vs. 49 +/- 18; P < 0.05), and the number of ventricular tachycardiac episodes (8.1 +/- 2.3 vs. 0.2 +/- 0.1; P < 0.05) without significantly modifying the incidence of ventricular tachycardia or ventricular fibrillation. The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and tissue electrical impedance changes were significantly less in the SNP-treated dogs. SNP improved GJ permeability and preserved the phosphorylated form of connexin43. CONCLUSION AND IMPLICATIONS: The anti-arrhythmic protection resulting from SNP infusion in the anaesthethized dog may, in part, be associated with the modulation of gap junctional function by NO.

In vitro model of neurotoxicity of Abeta 1-42 and neuroprotection by a pentapeptide: irreversible events during the first hour.

The cell biology of Alzheimer's disease (AD) is characterized mainly by the neurodegeneration caused by the beta-amyloid (Abeta) peptides and by the formation of neurofibrillary tangles. The initial events of neurodegeneration in the brain tissue include synaptic dysfunction and axonopathy. Abeta-induced axonopathy and neurite degeneration were studied in vitro on differentiated human-derived neurotypic SH-SY5Y cells. Different methods were used to investigate the mechanism of action of aggregated Abeta on neuroblastoma cells. Abeta 1-42 aggregated for 1 h induced irreversible changes in the neurite morphology. Change of tau hyperphosphorylation and cell viability (cytoplasmic redox state and active membrane uptake) was irreversible during the first hour after the addition of Abeta 1-42 to the cells. These rapid events indicate that Abeta might induce neurodegeneration even at an early stage of Abeta-cell contact. A novel pentapeptide LPYFD-amide, an analog of Soto's LPFFD, significantly decreased neurite degeneration, tau aggregation, and cell viability reduction induced by Abeta 1-42.