Regulation of the Serotonergic System by Kainate in the Avian Retina.

Serotonin (5-HT) has been recognized as a neurotransmitter in the vertebrate retina, restricted mainly to amacrine and bipolar cells. It is involved with synaptic processing and possibly as a mitogenic factor. We confirm that chick retina amacrine and bipolar cells are, respectively, heavily and faintly immunolabeled for 5-HT. Amacrine serotonergic cells also co-express tyrosine hydroxylase (TH), a marker of dopaminergic cells in the retina. Previous reports demonstrated that serotonin transport can be modulated by neurotransmitter receptor activation. As 5-HT is diffusely released as a neuromodulator and co-localized with other transmitters, we evaluated if 5-HT uptake or release is modulated by several mediators in the avian retina. The role of different glutamate receptors on serotonin transport and release in vitro and in vivo was also studied. We show that L-glutamate induces an inhibitory effect on [3H]5-HT uptake and this effect was specific to kainate receptor activation. Kainate-induced decrease in [3H]5-HT uptake was blocked by CNQX, an AMPA/kainate receptor antagonist, but not by MK-801, a NMDA receptor antagonist. [3H]5-HT uptake was not observed in the presence of AMPA, thus suggesting that the decrease in serotonin uptake is mediated by kainate. 5-HT (10-50 µM) had no intrinsic activity in raising intracellular Ca2+, but addition of 10 µM 5-HT decreased Ca2+ shifts induced by KCl in retinal neurons. Moreover, kainate decreased the number of bipolar and amacrine cells labeled to serotonin in chick retina. In conclusion, our data suggest a highly selective effect of kainate receptors in the regulation of serotonin functions in the retinal cells.

Cerebral malaria induces electrophysiological and neurochemical impairment in mice retinal tissue: possible effect on glutathione and glutamatergic system.

BACKGROUND: Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has usually been associated with cognitive, behavioural and motor dysfunctions, being the retinopathy the most serious consequence resulting from the disease. The pathophysiological mechanisms underlying this complication remain incompletely understood. Several experimental models of CM have already been developed in order to clarify those mechanisms related to this syndrome. In this context, the present work has been performed to investigate which possible electrophysiological and neurochemistry alterations could be involved in the CM pathology. METHODS: Experimental CM was induced in Plasmodium berghei-infected male and female C57Bl/6 mice. The survival and neurological symptoms of CM were registered. Brains and retina were assayed for TNF levels and NOS2 expression. Electroretinography measurements were recorded to assessed a- and b-wave amplitudes and neurochemicals changes were evaluated by determination of glutamate and glutathione levels by HPLC. RESULTS: Susceptible C57Bl/6 mice infected with ≈ 106 parasitized red blood cells (P. berghei ANKA strain), showed a low parasitaemia, with evident clinical signs as: respiratory failure, ataxia, hemiplegia, and coma followed by animal death. In parallel to the clinical characterization of CM, the retinal electrophysiological analysis showed an intense decrease of a- and-b-wave amplitude associated to cone photoreceptor response only at the 7 days post-infection. Neurochemical results demonstrated that the disease led to a decrease in the glutathione levels with 2 days post inoculation. It was also demonstrated that the increase in the glutathione levels during the infection was followed by the increase in the 3H-glutamate uptake rate (4 and 7 days post-infection), suggesting that CM condition causes an up-regulation of the transporters systems. Furthermore, these findings also highlighted that the electrophysiological and neurochemical alterations occurs in a manner independent on the establishment of an inflammatory response, once tumour necrosis factor levels and inducible nitric oxide synthase expression were altered only in the cerebral tissue but not in the retina. CONCLUSIONS: In summary, these findings indicate for the first time that CM induces neurochemical and electrophysiological impairment in the mice retinal tissue, in a TNF-independent manner.

Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation.

The study of drug candidates for the treatment of amyloidosis and neurodegenerative diseases frequently involves in vitro measurements of amyloid fibril formation. Macromolecular crowding and off-pathway aggregation (OPA) are, by different reasons, two important phenomena affecting the scalability of amyloid inhibitors and their successful application in vivo. On the one hand, the cellular milieu is crowded with macromolecules that drastically increase the effective (thermodynamic) concentration of the amyloidogenic protein. On the other hand, off-pathway aggregates, rather than amyloid fibrils, are increasingly appointed as causative agents of toxicity. The present contribution reveals that insoluble off-pathway aggregates of hen egg-white lysozyme (HEWL) are a peculiar type of crowding agents that, unlike classical macromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resume after lowering the fraction of insoluble aggregates at a constant soluble HEWL concentration. Protein depletion and thioflavin-T fluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. The crystallization-like model extended to account for OPA and time-dependent activity coefficients is able to fit multiple kinetic results using a single set of three parameters describing amyloid nucleation, autocatalytic growth, and off-pathway nucleation. The list of fitted results notably includes the cases of aggregation rebirth and all types of progress curves measured for different HEWL concentrations. The quantitative challenges posed by macromolecular crowding and OPA find here a unified response with broader implications for the development of on- and off-pathway inhibitors.

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests off-pathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include non-linear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

Enzyme kinetics: the whole picture reveals hidden meanings.

The methodology adopted by Michaelis and Menten in 1913 is still routinely used to characterize the catalytic power and selectivity of enzymes. These kinetic measurements must be performed soon after the purified enzyme is mixed with a large excess of substrate. Other time scales and solution compositions are no less physiologically relevant, but fall outside the range of applicability of the classical formalism. Here we show that the complete picture of an enzyme's mode of function is critically obscured by the limited scope of conventional kinetic analysis, even in the simplest case of a single active site without inhibition. This picture is now unveiled in a mathematically closed form that remains valid over the reaction time for all combinations of enzyme/substrate concentrations and rate constants. Algebraic simplicity is maintained in the new formalism when stationary reaction phases are considered. By achieving this century-old objective, the otherwise hidden role of the reversible binding step is revealed and atypical kinetic profiles are explained. Most singular kinetic behaviors are identified in a critical region of conditions that coincide with typical cell conditions. Because it is not covered by the Michaelis-Menten model, the critical region has been missed until now by low- and high-throughput screenings of new drugs. New possibilities are therefore raised for novel and once-promising inhibitors to therapeutically target enzymes.

Fluorinated beta-sheet breaker peptides.

The aggregation of amyloid-ß peptide (Aß) has been linked to the formation of neuritic plaques, which are pathological hallmarks of Alzheimer's disease. We synthesized peptides containing fluorinated amino acids and studied their effect on the Aß aggregation. The peptides were based on the sequence LVFFD, in which valine was substituted by either 4,4,4-trifluorovaline or 4-fluoroproline, or the phenylalanine at position 3 was replaced by 3,4,5-trifluorophenylalanine. Our results demonstrate that fluorination of the hydrophobic residue valine or phenylalanine is effective in preventing the Aß aggregation. This study opens up the possibility of using new sequences based on fluorinated amino acids to inhibit the amyloid-fibril formation.

A generic crystallization-like model that describes the kinetics of amyloid fibril formation.

Associated with neurodegenerative disorders such as Alzheimer, Parkinson, or prion diseases, the conversion of soluble proteins into amyloid fibrils remains poorly understood. Extensive "in vitro" measurements of protein aggregation kinetics have been reported, but no consensus mechanism has emerged until now. This contribution aims at overcoming this gap by proposing a theoretically consistent crystallization-like model (CLM) that is able to describe the classic types of amyloid fibrillization kinetics identified in our literature survey. Amyloid conversion represented as a function of time is shown to follow different curve shapes, ranging from sigmoidal to hyperbolic, according to the relative importance of the nucleation and growth steps. Using the CLM, apparently unrelated data are deconvoluted into generic mechanistic information integrating the combined influence of seeding, nucleation, growth, and fibril breakage events. It is notable that this complex assembly of interdependent events is ultimately reduced to a mathematically simple model, whose two parameters can be determined by little more than visual inspection. The good fitting results obtained for all cases confirm the CLM as a good approximation to the generalized underlying principle governing amyloid fibrillization. A perspective is presented on possible applications of the CLM during the development of new targets for amyloid disease therapeutics.

Thyroid hormone action is required for normal cone opsin expression during mouse retinal development.

PURPOSE: The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS: The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS: Hypothyroid newborn mice showed an increase in S-opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TRbeta1 and -beta2 were T3-binding defective, the pattern of opsin expression was similar to TRbeta ablation, showing increased S-opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TRbeta were mutated in the helix 12 AF-2 domain. CONCLUSIONS: The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.