Dem-Aging: autophagy-related pathologies and the "two faces of dementia".

Neuronal ceroid lipofuscinosis (NCL) is an umbrella term referring to the most frequent childhood-onset neurodegenerative diseases, which are also the main cause of childhood dementia. Although the molecular mechanisms underlying the NCLs remain elusive, evidence is increasingly pointing to shared disease pathways and common clinical features across the disease forms. The characterization of pathological mechanisms, disease modifiers, and biomarkers might facilitate the development of treatment strategies.The DEM-AGING project aims to define molecular signatures in NCL and expedite biomarker discovery with a view to identifying novel targets for monitoring disease status and progression and accelerating clinical trial readiness in this field. In this study, we fused multiomic assessments in established NCL models with similar data on the more common late-onset neurodegenerative conditions in order to test the hypothesis of shared molecular fingerprints critical to the underlying pathological mechanisms. Our aim, ultimately, is to combine data analysis, cell models, and omic strategies in an effort to trace new routes to therapies that might readily be applied in the most common forms of dementia.

Epileptiform activity in the mouse visual cortex interferes with cortical processing in connected areas.

Epileptiform activity is associated with impairment of brain function even in absence of seizures, as demonstrated by failures in various testing paradigm in presence of hypersynchronous interictal spikes (ISs). Clinical evidence suggests that cognitive deficits might be directly caused by the anomalous activity rather than by its underlying etiology. Indeed, we seek to understand whether ISs interfere with neuronal processing in connected areas not directly participating in the hypersynchronous activity in an acute model of epilepsy. Here we cause focal ISs in the visual cortex of anesthetized mice and we determine that, even if ISs do not invade the opposite hemisphere, the local field potential is subtly disrupted with a modulation of firing probability imposed by the contralateral IS activity. Finally, we find that visual processing is altered depending on the temporal relationship between ISs and stimulus presentation. We conclude that focal ISs interact with normal cortical dynamics far from the epileptic focus, disrupting endogenous oscillatory rhythms and affecting information processing.

Extracellular matrix inhibits structural and functional plasticity of dendritic spines in the adult visual cortex.

Brain cells are immersed in a complex structure forming the extracellular matrix. The composition of the matrix gradually matures during postnatal development, as the brain circuitry reaches its adult form. The fully developed extracellular environment stabilizes neuronal connectivity and decreases cortical plasticity as highlighted by the demonstration that treatments degrading the matrix are able to restore synaptic plasticity in the adult brain. The mechanisms through which the matrix inhibits cortical plasticity are not fully clarified. Here we show that a prominent component of the matrix, chondroitin sulfate proteoglycans (CSPGs), restrains morphological changes of dendritic spines in the visual cortex of adult mice. By means of in vivo and in vitro two-photon imaging and electrophysiology, we find that after enzymatic digestion of CSPGs, cortical spines become more motile and express a larger degree of structural and functional plasticity.

3,5,3'-Triiodothyronine deprivation affects phenotype and intracellular [Ca2+]i of human cardiomyocytes in culture.

OBJECTIVE: A decrease in plasma T3 concentration is a frequent finding in patients with heart failure. However, the role of this 'low T3 syndrome' on disease evolution has never been clarified. As phenotypic and functional cardiomyocyte impairments are alterations that correlate with the failing myocardium, we studied the long-term effects of T3 deprivation on human cardiomyocyte structure and calcium handling. METHODS: Atrial cardiomyocytes and myocardial tissue were cultured with or without 3 nM T3. Microscopical examination of structural features was followed by analysis of alpha-sarcomeric actinin and sarcoplasmic reticulum calcium ATP-ase (SERCA-2) content. Calcium handling was studied by [Ca2+](i) imaging. RESULTS: When stimulated with cyclopiazonic acid, a SERCA-2 inhibitor, T3-deprived cardiomyocytes showed significantly faster (P=0.03) and more transient (P=0.04) increases in [Ca(2+)](i) than T3-supplemented cells. Moreover, in the T3-free cultures a significantly lower number of cells (P=0.003) responded to caffeine, a typical activator of sarcoplasmic reticulum Ca(2+)-release channel. T3-deprived cardiomyocytes also presented altered morphology with larger dimensions than T3-supplemented cells (P < 0.0001). Additionally, in T3-deprived samples alpha-sarcomeric actinin and SERCA-2 protein levels were reduced to 65.6 +/- 3% (P < 0.0001) and 74.1 +/- 4% (P=0.005), respectively, when compared with the T3-supplemented group. CONCLUSIONS: Our data show that human cardiomyocyte calcium handling and phenotype are strongly influenced by T3 suggesting important implications of the 'low T3 syndrome' on the progression of heart failure.

Requirement of ERK activation for visual cortical plasticity.

Experience-dependent plasticity in the developing visual cortex depends on electrical activity and molecular signals involved in stabilization or removal of inputs. Extracellular signal-regulated kinase 1,2 (also called p42/44 mitogen-activated protein kinase) activation in the cortex is regulated by both factors. We show that two different inhibitors of the ERK pathway suppress the induction of two forms of long-term potentiation (LTP) in rat cortical slices and that their intracortical administration to monocularly deprived rats prevents the shift in ocular dominance towards the nondeprived eye. These results demonstrate that the ERK pathway is necessary for experience-dependent plasticity and for LTP of synaptic transmission in the developing visual cortex.

Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL-2 expression levels.

Treatment of transplanted patients with cyclosporin A (CSA) may cause adverse effects such as nephrotoxicity and hypertension. As CSA is known to induce oxidative stress in several tissues, it may cause vascular problems by triggering oxidative stress in endothelial cells (EC). However, oxidative stress has been reported for acute exposure to CSA concentrations exceeding its clinical range, whereas immunosuppression requires life-long treatment with therapeutic concentrations. We therefore compared the effects of 21 h pharmacological (200 microM) vs. 8 days clinical (0.5-2.5 microM) doses of CSA on cultured human EC. Pharmacological doses of CSA cause a decrease in cell density via apoptosis and a down-regulation of the antiapoptotic protein Bcl-2. However, these effects are independent of CSA-induced oxidative stress. In contrast, therapeutic concentrations of CSA cause Bcl-2 up-regulation and modification of EC morphology, both effects blocked by antioxidants. Therefore, a low level of oxidants may act in EC as second messengers that up-regulate Bcl-2, thus promoting survival of impaired EC. Our data suggest that the oxidative stress induced by clinical concentrations of CSA may be involved in the adverse effects of the drug on the vascular system of transplanted patients via an adaptive response involving Bcl-2 up-regulation rather than an apoptotic process

Brain-derived neurotrophic factor causes cAMP response element-binding protein phosphorylation in absence of calcium increases in slices and cultured neurons from rat visual cortex.

Neurotrophins play a crucial role in the developmental plasticity of the visual cortex, but very little is known about the cellular mechanisms involved in their action. In many models of synaptic plasticity, increases in cytosolic calcium concentration and activation of the transcription factor cAMP response element-binding protein (CREB) are crucial factors for the induction and maintenance of long-lasting changes of synaptic efficacy. Whether BDNF modulates intracellular calcium levels in visual cortical neurons and the significance of this action for BDNF signal transduction is still controversial. We investigated whether CREB phosphorylation and calcium changes are elicited by acute BDNF presentation in postnatal visual cortical slices and cultures. We found that BDNF did not cause any calcium increase, but it induced robust CREB phosphorylation in neurons from both preparations. We further analyzed signal transduction and its dependency on calcium changes in cultured neurons. CREB phosphorylation required trkB activation because treatment with the trk inhibitor k252a completely blocked CREB phosphorylation. In agreement with the imaging experiments, we verified that calcium changes were not necessary for CREB activation because preincubation with BAPTA-AM did not diminish the level of CREB phosphorylation induced by BDNF stimulation. CREB phosphorylation was accompanied by gene expression, because we observed the upregulation of c-fos expression, which was also not affected by preincubation with BAPTA-AM. Finally, BDNF caused phosphorylation of mitogen-activated protein kinase (MAPK), and because the treatment with the MAPK inhibitor U0126 completely abolished CREB activation and c-fos upregulation, it is likely that both processes depend mainly on the MAP kinase pathway. These results indicate that MAPK and CREB, but not intracellular calcium, are important mediators of neurotrophin actions in the visual cortex.

Protection of retinal ganglion cells and preservation of function after optic nerve lesion in bcl-2 transgenic mice.

Multicellular organisms face the necessity of removing superfluous or injured cells during normal development, tissue turn-over and in response to damaging conditions. These finalised killings occur throughout a process, commonly called programmed cell death (PCD), which is placed under strict cellular control. PCD is regulated by the products of the expression of a number of genes. This fact raises the intriguing possibility of inhibiting such degenerative processes by operating on some of the controlling genes. Central neurons of transgenic mice overexpressing bcl-2, a powerful inhibitor of PCD, are remarkably resistant to degeneration induced by noxious stimuli. We have explored the ate of retinal ganglion cells and of their axons, when such transgenic animals have been challenged by a lesion of the optic nerve. These results have direct bearing on the possibility of attaining functional restoration of the injured pathway.

Activity-regulated cell death contributes to the formation of ON and OFF alpha ganglion cell mosaics.

At maturity, ON and OFF alpha ganglion cells in the cat retina are arrayed in regular mosaics, with adjacent cells commonly forming ON-OFF pairs. In the present study, we investigated the role of activity-mediated ganglion cell death in the formation of such cellular patterns. Because direct measures of ganglion cell mosaics are problematic in the developing retina, we examined the distributions of ON and OFF alpha cells in the postnatal cat retina by assessing the degree to which cells in closest proximity were of opposite sign (i.e., ON-OFF pairs). Computer simulations demonstrated that superimposition of two regular distributions results in a high incidence (approximately 90%) of opposite sign pairs. This is also the case for ON and OFF alpha cells in the mature retina, reflecting the high degree of regularity exhibited by this cell class. In contrast, during the first postnatal month, alpha cells displayed a much lower incidence of opposite sign pairs (approximately 60%), comparable to the superimposition of two simulated random distributions. We also show that there is a 20% loss of alpha cells in the central retina during postnatal development and that this magnitude of loss is sufficient to form regular distributions of ON and OFF cells. To assess the influence of sodium voltage-gated activity on this developmental process, intraocular injections of tetrodotoxin (TTX) were made during the postnatal period of alpha cell loss. When the TTX-treated animals reached maturity, there was a dose-related decrease in the incidence of opposite sign pairs, without any appreciable change in cell density. Moreover, the regularity index of ON and OFF cells was significantly lower than normal in the TTX-treated retinas. These findings demonstrate that a spatially selective pattern of ganglion cell loss contributes to the formation of regular ON and OFF ganglion cell distributions and that such cell loss is regulated by retinal activity.

Long-term survival of retinal ganglion cells following optic nerve section in adult bcl-2 transgenic mice.

The bcl-2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl-2 in adult transgenic mice can protect the axotomized ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice approximately 65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, < 10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl-2 protein in central neurons is a very effective strategy to ensure long-term survival in axotomized cells.

Effect of blocking the Na+/K+ ATPase on Ca2+ extrusion and light adaptation in mammalian retinal rods.

Membrane current and light response were recorded from rods of monkey and guinea pig by means of suction electrodes. The correlation between adaptation and the Na+/K+ pump was investigated by measuring light-dependent changes in sensitivity with and without inhibition of Na+/K+ ATPase by strophanthidin. Strophanthidin was found to reduce the dark current, to slow the time course of the photoresponse, and to increase light sensitivity. At concentrations between 20 and 500 nM, the pump inhibitor suppressed in a reversible way the current re-activation occurring during prolonged illumination and modified the light-dependent decrease in sensitivity, which in control conditions approximates to a Weber-Fechner function. The effects of the pump inhibitor on the adaptive properties of rods are associated with an increased time constant of the membrane current attributed to the operation of the Na+:Ca2+,K+ exchanger. The effects of rapid application of the pump inhibitor on the current re-activation are consistent with the idea that significant changes in the internal sodium occur in rods of mammals during background illumination and that they play an important role in the process of light adaptation.

Interplay between the dendritic trees of alpha and beta ganglion cells during the development of the cat retina.

We have analyzed the effect of a small lesion to the retina of a two-day-old kitten and observed that after degeneration of ganglion cells whose axons were severed, a restricted region of the retina remained depleted of cells. Cells located near the borders of the depleted zone showed an abnormal elongation of dendrites towards the bare area. By means of a computer-aided system, we analyzed the whole population of cells at the two borders, and in agreement with previous data found that the effect was most prominent at the border and progressively decreased to eventually disappear at a distance of approximately 500 microns. The distance from the border, however, is not the only factor to influence the degree of asymmetry; with comparable distances, the vicinity of an alpha-cell reduces the projection of the beta-cell dendrites toward the empty area. We suggest that the organization of the adult retinal pattern is also influenced by interactions occurring between dendrites of different classes of ganglion cells.

Temperature dependence of the light response in rat rods.

1. The effects of temperature on the light responses of rat rods have been investigated over the range 17-40 degrees C. 2. The amplitude of the light-sensitive current increased with temperature with a mean temperature coefficient (Q10) of 2.47. 3. The amplitude of the Na(+)-Ca2+, K+ exchange current decreased with temperature when expressed as a fraction of the light-sensitive current, showing that the light-sensitive channel becomes less permeable to calcium as the temperature is raised. The time constant of relaxation of the exchange current was little affected by temperature. 4. The flash intensity required to give a half-saturating response increased with temperature with a mean Q10 of 1.68. 5. The responses to single photoisomerizations were determined from amplitude histograms of the responses to dim-flash trains. The amplitude of the response to a single photoisomerization decreased with temperature when expressed as a fraction of the light-sensitive current, but the change was not sufficient to account for the overall decrease in sensitivity. 6. The fraction of dim flashes that produced a photoisomerization decreased with temperature. This decrease in photon capture efficiency together with the decrease in the relative size of the single photon event fully accounts for the observed change in sensitivity. 7. The speed of the falling phase of the dim-flash response was accelerated more by warming than the rising phase, and it was therefore not possible to superimpose light responses at different temperatures by a simple change in time scale.

Development of the light response in neonatal mammalian rods.

The sensitivity to light is low in many neonatal mammals when compared with that in the adult. In human infants at one month of age, for example, the dark-adapted sensitivity for detection of large stimuli is 50 times lower than in the adult, and in rats the overall sensitivity of the neonatal retina is also low compared with the adult. This low sensitivity in the neonate has been attributed to a number of factors, but the possibility that the photoreceptors themselves might be an important limitation on the overall visual sensitivity has not so far been clearly established. Here we record the light response of single neonatal rat rods and find that the sensitivity is considerably lower than in the adult. The response to a single photoisomerization is normal in the neonate, and the sensitivity deficit can therefore be attributed to a low level of functional rhodopsin. Opsin, the protein component of rhodopsin, must be present in normal amounts, as the sensitivity can be restored to adult levels by treating the retina with 9-cis retinal, an active homologue of the native chromophore 11-cis retinal. The low sensitivity of photoreceptors in the neonate can therefore be attributed mainly to a low concentration of 11-cis retinal in the developing retina.

Two systems of branching axons in monkey's retina.

Several monkey retinae were stained, by using the reduced silver technique, in order to analyse long-distance intraretinal connections. Long, bifurcating processes covering very large areas were identified. Morphological investigation of these processes suggest that they are members of two different systems of branching axons. The first population of these processes originates as axon collaterals from cell in the ganglion cell layer. These cells have a relatively large, elongated soma and straight, sparsely branching dendrites, stratified in the vitreal half of the inner plexiform layer. The main axon (0.6 microns average diameter) passes along the optic fibre bundles, disappearing into the optic disk, whilst its collaterals run mainly in the inner plexiform layer. A cell showing similar morphology has also been found in the ganglion cell layer of a cat retina. The second population of processes consists of very thick fibres (2.1 microns average diameter) apparently originating from the optic disk. The main branches run in the space between the optic fibre layer and the ganglion cell layer, with short, secondary processes crossing the ganglion cel layer orthogonally. Many higher-order processes originate from the second-order branches; these run almost horizontally in the inner plexiform layer. The ganglion cells generating axon collaterals may constitute an intraretinal firing synchronization system, or they may be a residual feature of retinal development. The centrifugal fibres may be related to the sensitivity control during retinal dark adaptation.

Computer aided tracing and encoding of axonal arborizations.

This paper reports a method of acquiring and codifying branching profiles of axonal arborizations. Using a microcomputer interfaced with a motor driven microscope scanning stage, an acquisition strategy was developed. This strategy allows large profiles (covering up to 1250 mm2) to be measured with an accuracy of a few microns. Each histological section is associated with a specific coordinate system. This system was found to locate the profiles with high precision and reliability in the x-y microscope stage. The overall tracing accuracy of the system is fully discussed.

The concentration of cytosolic free calcium in vertebrate rod outer segments measured with fura-2.

The use of fluorescent indicators such as fura-2 (Grynkiewicz et al., 1985) to measure the cytosolic free calcium activity in retinal rods is complicated by the rods' sensitivity both to the fluorescence and to the light that excites it. By stimulating fluorescence from large numbers of rods in whole, loaded retinas and averaging repeated measurements, however, we have been able to monitor changes in free [Ca2+]i during exposure to nonsaturating lights under physiological conditions. Retinas, isolated from the bullfrog Rana catesbeiana, were loaded with fura-2 by incubation and mounted, receptor-side up, in a perfusion chamber placed on the stage of a specially designed apparatus. A step of light delivered from above, whose wavelength alternated between 340 and 380 nm every 110 msec, excited fluorescence from 24 mm2 of retina and evoked a light response (the aspartate-isolated pIII component of the electroretinogram--ERG). By comparing the fluorescence intensities excited by the 2 wavelengths (corrected for background and dark-noise), the free [Ca2+]i of the rod outer segment was determined. In darkness, the [Ca2+]i of the outer segment was found to be approximately 220 nM. A bright light caused it to fall exponentially to approximately 140 nM, with a time constant of approximately 1.6 sec. The value of [Ca2+]i at the onset of illumination was independent of stimulus intensity over a 2 log-unit range, and in all cases the fall was monotonic. After terminating the illumination, [Ca2+]i rose again to its time-zero value.(ABSTRACT TRUNCATED AT 250 WORDS)