The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein.

Conformational diseases, such as Alzheimer, Parkinson and Huntington diseases, are part of a common class of neurological disorders characterized by the aggregation and progressive accumulation of proteins bearing aberrant conformations. Huntington disease (HD) has autosomal dominant inheritance and is caused by mutations leading to an abnormal expansion in the polyglutamine (polyQ) tract of the huntingtin (HTT) protein, leading to the formation of HTT inclusion bodies in neurons of affected patients. Interestingly, recent experimental evidence is challenging the conventional view by which the disease pathogenesis is solely a consequence of the intracellular accumulation of mutant protein aggregates. These studies reveal that transcellular transfer of mutated huntingtin protein is able to seed oligomers involving even the wild-type (WT) forms of the protein. To date, there is still no successful strategy to treat HD. Here, we describe a novel functional role for the HSPB1-p62/SQSTM1 complex, which acts as a cargo loading platform, allowing the unconventional secretion of mutant HTT by extracellular vesicles. HSPB1 interacts preferentially with polyQ-expanded HTT compared with the WT protein and affects its aggregation. Furthermore, HSPB1 levels correlate with the rate of mutant HTT secretion, which is controlled by the activity of the PI3K/AKT/mTOR signalling pathway. Finally, we show that these HTT-containing vesicular structures are biologically active and able to be internalized by recipient cells, therefore providing an additional mechanism to explain the prion-like spreading properties of mutant HTT. These findings might also have implications for the turn-over of other disease-associated, aggregation-prone proteins.

MMP-2, MMP-9, VEGF and CA 15.3 in breast cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of extracellular matrix degrading proteinases. Owing to their matrix-degrading abilities and high expression in advanced tumours, MMPs were originally implicated in cancer progression, invasion and metastasis. PATIENTS AND METHODS: In this study, the correlation was determined between the expression of gelatinases (MMP-2 and MMP-9) in the sera of breast cancer patients from zymographic analysis and serum concentrations of VEGF and CA 15.3, before surgery and after 1 and 6 months; the association of both markers with clinicopathological features including histological type, stage of disease and estrogen (ER) and progesterone (PgR) receptors status were also analysed. In all, 88 breast cancer patients and 20 healthy women were involved in this study. RESULTS: No statistically significant correlation between pro MMP-2, pro MMP-9, VEGF and CA 15.3 serum levels was found (p>0.05). In breast cancer patients, a significant decrease of the pro MMP-2 serum expression 1 month after surgery with respect to serum levels before surgery (p=0.0008) was evident, as well as of CA 15.3 serum levels at baseline and after 1 month (p=0.017). Moreover a strong decrease of pro MMP-9 serum levels was found in 88 breast cancer patients after 1 month (p=0.028) and after 6 months (p =0.009) from surgery. On the other hand, no significant differences in the serum levels of VEGF, CA 15.3, pro MMP-2 or pro MMP-9 between 88 breast cancer patients preoperatively and 20 healthy women as controls were found. Our findings did indicate a significant positive association between higher preoperative levels of CA 15.3 and progression of disease (p=0.03), as well as a longer disease-free survival in patients who exhibited a decrease of serum pro MMP-9 expression compared to other biomarkers. No relationship between these four markers and the main clinical and pathological parameters was found. CONCLUSION: The present study failed to demonstrate any association between serum levels of MMPs, VEGF and CA 15.3 and well-known clinicopathological characteristics of breast carcinoma, while demonstrating the prognostic value of CA 15.3 and pro MMP-9 in the follow-up of breast cancer patients.

Cardiac aging, calcium overload, and arrhythmias.

The effect of aging was tested on experimental ventricular arrhythmias in isolated heart preparations from normal Wistar rats (NWR), Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Delayed afterdepolarizations and triggered activity induced by high-calcium perfusion (16 mM) in isolated papillary muscles were more frequent in the 24-month-old than in 6-month-old NWR. Reperfusion-VA were more severe in 14-month-old SHR than in WKY. The authors have previously shown that: (1) reperfusion- and reoxygenation-induced VA, in the isolated Langendorff perfused heart, were significantly more severe and frequent in 24-month-old than in 6-month-old NWR; (2) no age-related difference in the incidence of programmed electrical stimulation (PES, train of stimuli + 1 or 2 extrastimuli)-induced VA was observed in isolated NWR hearts during control perfusion, after coronary artery ligation or during hypoxia; (3) on the contrary, the incidence of PES-induced VA was significantly higher in isolated hearts from 14-month-old SHR than from 3-month-old SHR, and 3-month-old and 14-month-old WKY. It was concluded that "physiological" aging is associated with a higher propensity to calcium-related VA, while "pathological" aging characterized by hypertension of long duration increases the incidence of PES-induced VA, probably caused by myocardial fibrosis, which could facilitate reentry.

Caffeine actions on currents induced by calcium-overload in Purkinje fibers.

The ionic events underlying the changes induced by caffeine in calcium-overloaded Purkinje fibers were studied by means of a voltage-clamp technique. The following results were obtained. In fibers exposed to strophanthidin (5 X 10(-7) M), a depolarizing clamp of suitable magnitude or duration is followed by an oscillatory current (Ios) often superimposed on a decaying inward tail current (the "tail current"). Caffeine (9 mM) abolishes Ios and increases the tail current. Caffeine has similar actions when calcium overload is induced by increasing [Ca]0 or decreasing [Na]0. The magnitude of the tail current is reduced by decreasing [Ca]0. The tail current appears with repolarizations to -40 mV or more negative values as Ios appears in the absence of caffeine. As with Ios the tail current can be triggered twice (during and after a test clamp of suitable characteristics), becomes more inward with repeated clamps and becomes larger with larger or longer conditioning clamps. During the recovery from caffeine exposure, the tail current decreases gradually as Ios returns progressively. It is concluded that both Ios and tail current are caused by calcium overload but are affected in opposite direction by caffeine, apparently because caffeine decreases the calcium overload in the sarcoplasmic reticulum (abolition of Ios) and increases the calcium to be extruded from the sarcoplasm (increase in the tail current).

Summer camps for diabetic children: an experience in Campania, Italy.

To evaluate the effectiveness of summer camps with objective parameters, the authors examined data relative to nine summer camps organized by the Young Diabetics Association in Campania, Italy. The mean duration of camps was 10 days (range, 8-15) and a total of 256 diabetic children with an average age of 10 (range 8-16) participated in them. The medical personnel consisted of three pediatric endocrinologists, one psychologist, two male nurses and two parents who were directors of the Association. A significant improvement in knowledge and self-management of the disease was noted at the end of the camps. A beneficial effect on mean HbA1c level was also observed in the diabetic children who attended the monthly meetings and follow-up checks with their parents after the camp. On the other hand, a worsening of these values was noted in diabetic children who did not participate in them. No increased incidence of hypoglycaemia or ketoacidosis was found during or after camps, in contrast with previous studies. From a psychological viewpoint, the results suggest that summer camps have an important bearing on achieving acceptance of the disease. Sharing personal experiences with actively involved parents who participated in self-management training together with their children, has favourably influenced the results of this experience in Campania.

Tall right precordial R waves in members of a family with hypertrophic cardiomyopathy.

We recently observed a family with tall R waves from V1 to V3 in different relatives, regardless of the actual presence and localization of idiopathic left ventricular hypertrophy.

Increased propensity of the aged myocardium to develop calcium-related electrophysiological alterations.

The electrico-mechanical effects of calcium overload have been studied in cardiac papillary muscles of young adult and senescent Wistar rats driven at different rates. Action potential duration and contraction duration were found to be more prolonged in senescent than in younger animals during control conditions. Oscillatory afterpotentials and aftercontractions were more frequent in senescent than in younger rats during high calcium perfusion. Triggered activity was induced in senescent fibers but not in younger ones. Resting tension increased more in senescent fibers whereas the peak tension was not different in the two groups. It is concluded that aging is associated with a reduced capacity of the cardiac cells to handle an increased calcium load.

Calcium antagonists in the therapy of acute myocardial infarction.

The theoretical background and experimental evidence of the use of calcium antagonists in the treatment of acute myocardial infarction are summarised. The main clinical trials that have studied the effects of the three different groups of these drugs in patients with myocardial necrosis are then reviewed. Finally, the conclusion is drawn that there is no proof of the efficacy of calcium blockers in the therapy of patients with acute myocardial infarction but that, in some subgroups of these subjects (Q-wave myocardial infarction with good left ventricular function, non Q-wave myocardial infarction), non-dihydropyridinic compounds should be effective, in particular in patients with contraindications to beta-adrenergic blockade.

Relationship between caffeine effects and calcium in canine cardiac Purkinje fibers.

The actions of caffeine (1-9 mM) on electrical and mechanical events were studied under conditions known to change the intracellular calcium in canine cardiac Purkinje fibers perfused in vitro. It was found that caffeine in a dose-dependent manner 1) shifts the early repolarization to more positive values, 2) shifts the plateau to more negative values, 3) prolongs the terminal phase 3 (induction of a "tail"), 4) transiently increases and then decreases the force of contraction with respect to control values, 5) causes a smaller tail in the presence of agents (local anesthetics, tetrodotoxin, high potassium) that have been shown to diminish the force of contraction, presumably by decreasing cellular calcium, 6) causes the oscillatory potentials in high calcium to peak sooner after the action potential and eventually to disappear as the tail develops, 7) induces a tail at lower concentrations when cellular calcium is enhanced by lowering the external sodium concentration or in the presence of veratridine, and 8) fails to induce a marked tail in the presence of metabolic inhibitors. It is concluded that in Purkinje fibers caffeine induces a marked delay in the final repolarization through a process modulated by calcium. This process may be related to an electrogenic sodium-calcium exchange.

Microsomal iron-dependent NADPH oxidation: evidence for the involvement of membrane-bound nonheme iron in NADPH oxidation by rat heart microsomes.

Rat heart microsomes were found to contain nonheme iron and two lines of evidence suggested that this iron was involved in NADPH oxidation. As first evidence, pretreatment of rats with iron gluconate increased microsomal iron content and NADPH oxidation. As second evidence, treatment of microsomes with nonionic detergent Triton N-101 decreased membrane iron content and NADPH oxidation. Triton N-101-solubilized nonheme iron was nondialyzable and ammonium sulfate-precipitable, indicative of association with protein(s). This protein-bound iron per se did not oxidize NADPH but its addition to detergent-treated microsomes restored very high rates of NADPH oxidation, that were abolished by inhibiting NADPH-cytochrome P450 reductase with p-hydroxymercuribenzoate. Since heart microsomes did not contain cytochrome P450, these results suggested that stimulation of NADPH oxidation was mediated by direct electron transfer from reductase to iron. Purified rat heart ferritin and hemosiderin did not stimulate NADPH oxidation and the stimulation observed with detergent-solubilized microsomal iron was much higher than that observed with EDTA-Fe3+, a very effective electron acceptor for the reductase. This suggested that (i) microsomal iron was different from other intracellular iron-storage proteins, and (ii) microsomal iron was unusually permissive to one-electron transfer from reductase.

Role of calcium on the actions of caffeine in ventricular muscle fibers.

The effects of different concentrations of caffeine on the electrical and mechanical events of ventricular myocardial fibers of the dog were studied in vitro under conditions that alter cellular calcium. Caffeine was found to: (a) increase the contractile force quickly and then slowly in a dose-dependent manner, causing contracture only at high concentrations; (b) shift to more positive values and shorten the plateau, slow the final phase III repolarization, and cause a slow return of potential to the resting value during diastole at high concentrations; (c) increase the magnitude but retard the relaxation of the twitch; (d) cause these effects independently of alpha- or beta-adrenergic mechanisms; (e) shift the plateau to more negative values in the absence of calcium; (f) shift the plateau of the slow response action potentials to more positive values and increase the contractile force without prolonging the late phase III in high K; (g) induce less pronounced effects in the presence of local anesthetics or tetrodotoxin; (h) still increase force in the presence of manganese, verapamil, and ryanodine; (i) induce more pronounced effects in the presence of strophantidin; and (j) retard the fall in contractile force on exposure to zero calcium. It is concluded that caffeine, in addition to and as a consequence of other actions, enhances cellular calcium, which in turn is responsible for some of the electrical and mechanical effects of caffeine.

On the mechanism by which caffeine abolishes the fast rhythms induced by cardiotonic steroids.

The abolition by caffeine of the arrhythmias induced by cardiotonic steroids was studied in canine Purkinje and ventricular muscle fibers and in guinea pig ventricular muscle perfused in vitro. Both the electrical and mechanical activity were recorded. The following results were obtained: (1) Caffeine (9 mM) quickly abolished the arrhythmias induced by strophanthidin (10(-6)M) in Purkinje fibers by inducing quiescence at a depolarized level; (2) A lower concentration of caffeine (3 mM) hastened the appearance of the arrhythmias by favoring the onset of spontaneous activity at a depolarized level but abolished the oscillatory potential at lower strophanthidin concentrations (2-5 X 10(-7)M); (3) Caffeine (9-10 mM) abolished the oscillatory potentials and after-contractions induced by cardiotonic steroids in ventricular muscle fibers; (4) Caffeine prevented the induction of oscillatory potentials and of arrhythmias by cardiotonic steroids; (5) In the presence of cardiotonic steroids and norepinephrine, the oscillatory potentials can induce fast discharge in myocardial fibers which is eliminated by caffeine; (6) High calcium and norepinephrine (singly or together) induce oscillatory potentials and after-contractions which are abolished by caffeine; (7) The elimination of the oscillatory potentials by caffeine in myocardial fibers is associated with the development of contracture. It is concluded that caffeine eliminates cardiac steroid-induced arrhythmias by quickly depolarizing Purkinje fibers and by eliminating the oscillatory potentials in myocardial fibers and (at suitable concentrations) also in Purkinje fibers.

Induction of overdrive excitation in the sinoatrial node in the presence of caffeine.

The induction of spontaneous repetitive activity by a drive ('overdrive excitation') was studied in the sinoatrial node of the guinea pig by recording electrical and mechanical activity in sinoatrial preparations made quiescent by exposure to 20 mM caffeine of caffeine plus high [Ca]0. Electrical drive induced action potentials that were followed by oscillatory potentials and simultaneous aftercontractions. The oscillatory potentials (and the associated aftercontractions) increased in magnitude with successive action potentials, thereby initiating spontaneous beats during slow drives or after fast drives. The repetitive activity eventually slowed and ceased abruptly as the oscillatory potential failed to attain the threshold. Overdrive induced oscillatory potential and aftercontractions even when it did not initiate spontaneous activity. These results suggest that in the presence of caffeine calcium overload can occur in the sinoatrial node and result in oscillatory potentials and abnormal automaticity.

Abolition of digitalis tachyarrhythmias by caffeine.

Digitalis-induced ventricular tachyarrhythmias (VTAs) are believed to be due to oscillatory afterpotentials (OAPs) generated by an oscillatory release of calcium from the sarcoplasmic reticulum (SR). Caffeine blocks the calcium uptake into the SR and then may influence VTAs by depleting the SR stores of calcium. We studied the action of digitalis and caffeine, singly and in combination, in the isolated guinea pig heart perfused by means of a modified Langendorff apparatus. Digitalis (beta-methyldigoxin 1.27 X 10(-6) M) caused VTAs and ventricular fibrillation (VF) in all the hearts. Caffeine alone decreased heart rate but never caused VTAs. With the administration of digitalis and caffeine (1 mM), VTAs rarely developed and VF never occurs. With digitalis and higher concentration of caffeine (10 mM), neither VTAs nor VF were observed. In hearts with complete atrioventricular block, digitalis increased the ventricular rate from 143 +/- 10 to 270 +/- 13 beats/min (n = 8) in 12 +/- 1.9 min and provoked the appearance of multiple ventricular pacemakers. The addition of 10 mM caffeine to the digitalis-containing solution reduced the ventricular rate to 171 +/- 12 beats/min (P less than 0.001 vs. digitalis alone, not significant vs. control, n = 8) and abolished the digitalis-induced multiple pacemakers. Ventricular asystole was occasionally observed during the perfusion with digitalis + 10 mM caffeine. Caffeine alone did not modify the diastolic pressure, whereas caffeine and digitalis rapidly increase it. These results represent indirect evidence to support that SR plays an important role in the origin of the digitalis-induced VTAs.