Single-molecule Force Spectroscopy Predicts a Misfolded,Domain-swapped Conformation in human γD-Crystallin Protein

Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the β1 and β2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.     

Eurasian otters, Lutra lutra, have a dominant mtDNA haplotype from the Iberian Peninsula to Scandinavia

The Eurasian otter, Lutra lutra, has a Palaearctic distribution and has suffered a severe decline throughout Europe during the last century. Previous studies in this and other mustelids have shown reduced levels of variability in mitochondrial DNA, although otter phylogeographic studies were restricted to central-western Europe. In this work we have sequenced 361 bp of the mtDNA control region in 73 individuals from eight countries and added our results to eight sequences available from GenBank and the literature. The range of distribution has been expanded in relation to previous works north towards Scandinavia, east to Russia and Belarus, and south to the Iberian Peninsula. We found a single dominant haplotype in 91.78% of the samples, and six more haplotypes deviating a maximum of two mutations from the dominant haplotype restricted to a single country. Variability was extremely low in western Europe but higher in eastern countries. This, together with the lack of phylogeographical structuring, supports the postglacial recolonization of Europe from a single refugium. The Eurasian otter mtDNA control region has a 220-bp variable minisatellite in Domain III that we sequenced in 29 otters. We found a total of 19 minisatellite haplotypes, but they showed no phylogenetic information.     

Increased Oxidative Metabolism and Neurotransmitter Cycling in the Brain of Mice Lacking the Thyroid Hormone Transporter Slc16a2 (Mct8)

Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T₄ and T₃) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T₄ and T₃. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-¹³C) glucose and brain extracts prepared and analyzed by ¹³C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T₃ by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.     

Glutamate receptors on dopamine neurons control the persistence of cocaine seeking

Cocaine strengthens excitatory synapses onto midbrain dopamine neurons through the synaptic delivery of GluR1-containing AMPA receptors. This cocaine-evoked plasticity depends on NMDA receptor activation, but its behavioral significance in the context of addiction remains elusive. Here, we generated mice lacking the GluR1, GluR2, or NR1 receptor subunits selectively in dopamine neurons. We report that in midbrain slices of cocaine-treated mice, synaptic transmission was no longer strengthened when GluR1 or NR1 was abolished, while in the respective mice the drug still induced normal conditioned place preference and locomotor sensitization. In contrast, extinction of drug-seeking behavior was absent in mice lacking GluR1, while in the NR1 mutant mice reinstatement was abolished. In conclusion, cocaine-evoked synaptic plasticity does not mediate concurrent short-term behavioral effects of the drug but may initiate adaptive changes eventually leading to the persistence of drug-seeking behavior.     

Assessing the performance of nonparametric estimators of species richness in meadows

To accurately measure the number of species in a biological community, a complete inventory should be performed, which is generally unfeasible; hopefully, estimators of species richness can help. Our main objectives were (i) to assess the performance of nonparametric estimators of plant species richness with real data from a small set of meadows located in the Basque campiña (northern Spain), and (ii) to apply the best estimator to a larger dataset to test the effects on plant species richness caused by environmental conditions and human practices. Two non-asymptotic and seven asymptotic accumulation functions were fitted to a randomized sample-based rarefaction curve computed with data from three well sampled meadows, and information theoretic methods were used to select the best fitting model; this was the Morgan-Mercer-Flodin, and its asymptote was taken as our best guess of true richness. Then, five nonparametric estimators were computed: ICE, Chao 2, Jackknife 1 and 2, and Bootstrap; MMRuns and MMMeans were also assessed. According to the criteria set for our performance assessment (i.e., bias, precision, and accuracy), the best estimator was Jackknife 1. Finally, Jackknife 1 was applied to assess the effects of terrain slope and soil parent material, and also fertilization, grazing, and mowing, on plant species richness from a larger dataset (20 meadows). Results suggested that grass cutting was causing a loss of richness close to 30%, as compared to unmowed meadows. It is concluded that the use of nonparametric estimators of species richness can improve the evaluation of biodiversity responses to human management practices.     

<Emphasis Type="Italic">MICA</Emphasis> response to gliadin in intestinal mucosa from celiac patients

MHC class I chain-related gene A (MICA), a putative independent susceptibility gene in autoimmune diseases, encodes a surface protein present in epithelial cells that binds to NKG2D, an activating receptor of NK, and T cells, and could function as a stress-inducible activator of the innate immune response. There is no evidence of a long-term implication of MICA in the celiac autoimmune process. However, it could be that gliadin activation of MICA occurs only during the initial stages of the disease. In order to determine whether MICA is activated in response to gliadin in patients with celiac disease (CD), small intestinal mucosa biopsy samples from ten long-standing celiac patients on a gluten-free diet and from five non-celiac individuals were incubated with and without gliadin for 4 h. Total RNA was purified and MICA, IFNG and NKG2D mRNA were quantified by fluorescent real-time RT-PCR. Expression levels were calculated relative to GAPDH. MICA expression was detected in both patients and controls, but incubation with gliadin induced a strong increase in samples from the treated CD group compared with the non-CD controls (P=0.028), while no differences were observed for IFNG or NKG2D mRNA levels. The gliadin-provoked over-expression of MICA in normalized tissues from CD patients suggests a role for this stress-induced activator of the immune response in the early stages of organ-specific autoimmune destruction, probably preceding the onset of inflammation.     

Neuronal Hyperactivity Disturbs ATP Microgradients,Impairs Microglial Motility,and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.