Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q < 0.05; enrichment range 1.40–9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans?nosology pathways in diabetes and its co-morbidities.     

Specificity of binding of the plectin actin-binding domain to beta4 integrin

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin beta4 subunit. Previously, a binding site for beta4 was mapped on the actin-binding domain (ABD) of plectin and binding of beta4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to beta4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to beta4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The beta4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of beta4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to beta4 themselves, modulate the binding activity of the ABD for beta4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and beta4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.     

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.     

Ginkgo biloba extract EGb 761 protects against mitochondrial aging in the brain and in the liver.

According to the free radical theory of aging, oxygen-derived free radicals causes the age-associated impairment at the cellular and tissue levels. The mitochondrial theory of aging points to mitochondria, and specially mitochondrial DNA, as the major targets of free radical attack upon aging. Thus, oxidative damage to mtDNA accumulate with age in human and rodent tissues and also is inversely related to maximum life span of mammals. Mitochondrial deficits, such as a decrease in mitochondrial membrane potential, occur upon aging due to oxidative damage. The age-related mitochondrial oxidative stress may be prevented by late onset administration of certain antioxidants, such as Ginkgo biloba extract EGb 761. These antioxidants may also delay the physiological impairment associated with aging.     

Intravesicular Factors Controlling Exocytosis in Chromaffin Cells

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many secretory cell types including neurons. LDCV accumulate solutes at high concentrations (catecholamines, 0.5–1 M; ATP, 120–300 mM; or Ca²⁺, 40 mM (Bulenda and Gratzl Biochemistry 24:7760–7765, 1985). Solutes seem to aggregate to a condensed matrix to elude osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delayed release of catecholamines during exocytosis. The aggregation of solutes occurs due to a specific H⁺ pump denominated V-ATPase that maintains an inner acidic media (pH ≈5.5). This pH gradient against cytosol is also responsible for the vesicular accumulation of amines and Ca²⁺. When this gradient is reduced by modulation of the V-ATPase activity, catecholamines and Ca²⁺ are moved toward the cytosol. In addition, some drugs largely accumulate inside LDCV and not only impair the accumulation of natural solutes, but also act as false neurotransmitters when they are co-released with catecholamines. There is much experimental evidence to conclude that the physiological modulation of vesicle pH and the manipulation of intravesicular media with drugs affect the LDCV cargo and change the kinetics of exocytosis. Here, we will present some experimental data demonstrating the participation of drugs in the kinetics of exocytosis through changes in the composition of vesicular media. We also offer a model to explain the regulation of exocytosis by the intravesicular media that conciliate the experimentally obtained data.     

Circulating TNF-alpha and its soluble receptors during experimental acute pancreatitis

Clinical and experimental studies have shown increased concentrations of TNF-alpha and its soluble receptors in serum of patients with acute pancreatitis. In this work, we have investigated the time-course of TNF-alpha and its soluble receptors during taurocholate-induced acute pancreatitis. In addition, since TNF-alpha itself could mediate the shedding of its receptors, we have assessed the effect of inhibiting TNF-alpha production on the release of soluble TNF-alpha receptors in experimental acute pancreatitis. Our results indicate that soluble receptors are released in the early stages of the disease and this increase is concomitant with the release of TNF-alpha, which is mainly bound to specific proteins. The increased concentrations of its receptors strongly suggest that they could be these binding proteins. Inhibition of TNF-alpha generation with pentoxifylline abrogated the shedding of sTNF-alphaR1, but had no effect on sTNF-alphaR2. This finding suggests that the shedding of sTNF-alphaR1 is induced by TNF-alpha itself, but in the case of sTNF-alphaR2, the shedding appears to be induced by another mechanism.     

Crystal structure of a tandem pair of fibronectin type III domains from the cytoplasmic tail of integrin alpha6beta4.

The integrin alpha6beta4 is an essential component of hemidesmosomes but it also plays a dynamic role in invasive carcinoma cells. The cytoplasmic tail of the beta4 subunit is uniquely large among integrins and includes two pairs of fibronectin type III domains separated by a connecting segment. Here we describe the crystal structure of the first tandem domain pair, a module that is critical for alpha6beta4 function. The structure reveals a novel interdomain interface and candidate protein-binding sites, including a large acidic cleft formed from the surfaces of both domains and a prominent loop that is reminiscent of the RGD integrin-binding loop of fibronectin. This is the first crystal structure of either a hemidesmosome component or an integrin cytoplasmic domain, and it will enable the intracellular functions of alpha6beta4 to be dissected at the atomic level.     

Optimising long‐term monitoring projects for species distribution modelling: how atlas data may help

Long‐term biodiversity monitoring data are mainly used to estimate changes in species occupancy or abundance over time, but they may also be incorporated into predictive models to document species distributions in space. Although changes in occupancy or abundance may be estimated from a relatively limited number of sampling units, small sample size may lead to inaccurate spatial models and maps of predicted species distributions. We provide a methodological approach to estimate the minimum sample size needed in monitoring projects to produce accurate species distribution models and maps. The method assumes that monitoring data are not yet available when sampling strategies are to be designed and is based on external distribution data from atlas projects. Atlas data are typically collected in a large number of sampling units during a restricted timeframe and are often similar in nature to the information gathered from long‐term monitoring projects. The large number of sampling units in atlas projects makes it possible to simulate a broad gradient of sample sizes in monitoring data and to examine how the number of sampling units influences the accuracy of the models. We apply the method to several bird species using data from a regional breeding bird atlas. We explore the effect of prevalence, range size and habitat specialization of the species on the sample size needed to generate accurate models. Model accuracy is sensitive to particularly small sample sizes and levels off beyond a sufficiently large number of sampling units that varies among species depending mainly on their prevalence. The integration of spatial modelling techniques into monitoring projects is a cost‐effective approach as it offers the possibility to estimate the dynamics of species distributions in space and over time. We believe our innovative method will help in the sampling design of future monitoring projects aiming to achieve such integration.     

Pretreatment with Resveratrol Prevents Neuronal Injury and Cognitive Deficits Induced by Perinatal Hypoxia-Ischemia in Rats

Despite advances in neonatal care, hypoxic-ischemic brain injury is still a serious clinical problem, which is responsible for many cases of perinatal mortality, cerebral palsy, motor impairment and cognitive deficits. Resveratrol, a natural polyphenol with important anti-oxidant and anti-inflammatory properties, is present in grapevines, peanuts and pomegranates. The aim of the present work was to evaluate the possible neuroprotective effect of resveratrol when administered before or immediately after a hypoxic-ischemic brain event in neonatal rats by analyzing brain damage, the mitochondrial status and long-term cognitive impairment. Our results indicate that pretreatment with resveratrol protects against brain damage, reducing infarct volume, preserving myelination and minimizing the astroglial reactive response. Moreover its neuroprotective effect was found to be long lasting, as behavioral outcomes were significantly improved at adulthood. We speculate that one of the mechanisms for this neuroprotection may be related to the maintenance of the mitochondrial inner membrane integrity and potential, and to the reduction of reactive oxygen species. Curiously, none of these protective features was observed when resveratrol was administered immediately after hypoxia-ischemia.     

Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.