Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS)

Background

Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts.

Methodology/Principal Findings

Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP.

Conclusions/Significance

Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.     

A Novel Inhibitor of Amyloid β (Aβ) Peptide Aggregation: FROM HIGH THROUGHPUT SCREENING TO EFFICACY IN AN ANIMAL MODEL OF ALZHEIMER DISEASE*

Compelling evidence indicates that aggregation of the amyloid β (Aβ) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aβ42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aβ42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aβ42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aβ42 aggregation and reducing Aβ42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aβ42. The ability of D737 to prevent Aβ42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.     

Trafficking of α4* Nicotinic Receptors Revealed by Superecliptic Phluorin: EFFECTS OF A β4 AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTATION AND CHRONIC EXPOSURE TO NICOTINE*

We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4β4 nAChRs, but many α4β2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the β4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4β4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2–3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4β4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 μm) did not alter the PM insertion frequency or trafficking behavior of α4β4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4β2-containing vesicle fusions at the PM; this stage in α4β2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.     

SUMMER DECLINE OF ULVA LACTUCA (CHLOROPHYTA) IN A EUTROPHIC EMBAYMENT: INTERACTIVE EFFECTS OF TEMPERATURE AND NITROGEN AVAILABILITY?1

Throughout the summer, abundance of Ulva lactuca L. declined while biomass of Cladophora vagabunda (L.) van den Hoek and Gracilaria tikvahiae McLachlan increased in a New England embayment undergoing eu-trophication (Waquoit Bay, Massachusetts). We investigated the physiological basis for the summer dieback, focusing on temporal variations in photosynthetic performance and tissue nitrogen (N). We also compared photosynthetic and N uptake capabilities of U. lactuca with other abundant species in this eutrophic system. Photosynthetic egiciency and capacity of U. lactuca declined markedly at 25°C, compared with a spring (15°C) peak in photosynthetic performance; P_max was 4.6 ± 0.3 and 1.8 ± 0.6 μmol O₂.m^?2.s^?1 during spring and summer, respectively. Notably, summer p_max of other abundant species of the embayment was 1.5–3 × higher than that measured for U. lactuca. Ulva lactuca showed a signifciant photosynthetic response to dissolved inorganic carbon enrichment during summer, when water-column-dissolved CO₂ levels were 20% of spring values. Although ammonium uptake rates of U. lactuca were extremely high at both subsaturating (15μM) and saturating (75 μM) N concentrations, as predicted by the functional-form hypothesis, tissue N fell to 1% by late summer. We suggest that a carbon imbalance, initiated by rising water temperatures and declining water-column N; thermal stress; and biological factors (competition, grazing) all contribute to the recurrent summer decline of U. lactuca in this shallow, eutrophic embayment. Thus, while the morphology of U. lactuca might be considered a successful strategy for disturbed, or “stressed” (sensu Littler and Littler 1980), habitats, its inability to persist and flourish in this environment emphasizes the complexity of factors at work in natural systems.     

RELATIONSHIP BETWEEN FISH AND THE NUMBER OF HORNS IN CERATIUM HIRUNDINELLA (DINOPHYCEAE): A FOOD‐WEB‐MEDIATED EFFECT ON ALGAL MORPHOLOGY?1

In a freshwater mesocosm experiment, we explored the potential for direct and indirect effects of roach (Rutilus rutilus) and Eurasian perch (Perca fluviatilis), two planktivorous fishes with different feeding behaviors, on the morphology of Ceratium hirundinella (O. F. Müll.) Dujard., a large dinoflagellate. Three morphs were detected: one with two hypothecal horns, one with a third rudimentary horn, and one with three well‐developed horns. We observed a strong negative relationship between the presence of fish and the proportion of three‐horned cells. The two fishes had strikingly similar effects on C. hirundinella morphology, despite their different capabilities to retain particles of the size of C. hirundinella. This finding suggests that the morphological variation in C. hirundinella was not related to selection by fish. Morphological variations in C. hirundinella could not be explained by fish‐mediated variations in turbidity (i.e., light climate) or by predation pressure by the fish. In contrast, the proportion of three‐horned cells was directly related to the biomass of filter‐feeding cladocerans. This result was unexpected since cladocerans are not considered to consume C. hirundinella and they did not depress C. hirundinella numbers in our experiment. Without excluding other possible mechanisms, we suggest that the third horn might help these dinoflagellates avoid physical contact with the filtering apparatus of the cladocerans and the consequent potential damage caused by these herbivores, which were more abundant in the absence of planktivorous fish.