Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Examination of Longitudinal Alterations in Alzheimer's Disease-Related Neurogenesis in an APP/PS1 Transgenic Mouse Model, and the Effects of P33, a Putative Neuroprotective Agent Thereon.

Neurogenesis plays a crucial role in cognitive processes. During aging and in Alzheimer's disease (AD), altered neurogenesis and neuroinflammation are evident both in C57BL/6J, APPSwe/PS1dE9 (Tg) mice and humans. AD pathology may slow down upon drug treatment, for example, in a previous study of our group P33, a putative neuroprotective agent was found to exert advantageous effects on the elevated levels of APP, Aß, and neuroinflammation. In the present study, we aimed to examine longitudinal alterations in neurogenesis, neuroinflammation and AD pathology in a transgenic (Tg) mouse model, and assessed the putative beneficial effects of long-term P33 treatment on AD-specific neurological alterations. Hippocampal cell proliferation and differentiation were significantly reduced between 8 and 12 months of age. Regarding neuroinflammation, significantly elevated astrogliosis and microglial activation were observed in 6- to 7-month-old Tg animals. The amounts of the molecules involved in the amyloidogenic pathway were altered from 4 months of age in Tg animals. P33-treatment led to significantly increased neurogenesis in 9-month-old animals. Our data support the hypothesis that altered neurogenesis may be a consequence of AD pathology. Based on our findings in the transgenic animal model, early pharmacological treatment before the manifestation of AD symptoms might ameliorate neurological decline.

Impact of Two Neuronal Sigma-1 Receptor Modulators, PRE084 and DMT, on Neurogenesis and Neuroinflammation in an Aß1-42-Injected, Wild-Type Mouse Model of AD.

Alzheimer's disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-ß-(Aß-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aß-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aß1-42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury.

BACKGROUND: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. METHODS: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. RESULTS: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. CONCLUSIONS: Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

Post-diaminobenzidine Treatments for Double Stainings: Extension of Sulfide-Silver-Gold Intensification for Light and Fluorescent Microscopy.

Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3'-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions.

Effects of the Pentapeptide P33 on Memory and Synaptic Plasticity in APP/PS1 Transgenic Mice: A Novel Mechanism Presenting the Protein Fe65 as a Target.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer's disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aß) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-ß42 monomer.

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.

Peripheral cyclic ß-amino acids balance the stability and edge-protection of ß-sandwiches.

Engineering water-soluble stand-alone ß-sandwich mimetics is a current challenge because of the difficulties associated with tailoring long-range interactions. In this work, single cis-(1R,2S)-2-aminocyclohexanecarboxylic acid mutations were introduced into the edge strands of the eight-stranded ß-sandwich mimetic structures from the betabellin family. Temperature-dependent NMR and CD measurements, together with thermodynamic analyses, demonstrated that the modified peripheral strands exhibited an irregular and partially disordered structure but were able to exert sufficient shielding on the hydrophobic core to retain the predominantly ß-sandwich structure. Although the frustrated interactions decreased the free energy of unfolding, the temperature of the maximum stabilities increased to or remained at physiologically relevant temperatures. We found that the irregular peripheral strands were able to prevent edge-to-edge association and fibril formation in the aggregation-prone model. These findings establish a ß-sandwich stabilization and aggregation inhibition approach, which does not interfere with the pillars of the peptide bond or change the net charge of the peptide.

Multivalent foldamer-based affinity assay for selective recognition of Aß oligomers.

Mimicking the molecular recognition functionality of antibodies is a great challenge. Foldamers are attractive candidates because of their relatively small size and designable interaction surface. This paper describes a sandwich type enzyme-linked immunoassay with a tetravalent ß-peptide foldamer helix array as capture element and enzyme labeled tracer antibodies. The assay was found to be selective to ß-amyloid oligomeric species with surface features transiently present in ongoing aggregation. In optimized conditions, with special emphasis on the foldamer immobilization, a detection limit of 5 pM was achieved with a linear range of 10-500 pM. These results suggest that protein mimetic foldamers can be useful tools in biosensors and affinity assays.

Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG.

The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1- naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 (o)C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol.

The anxiolytic buspirone shifts coping strategy in novel environmental context of mice with different anxious phenotype.

Patients suffering from anxiety disorders show increased fear when encounter a novel environment. Rodents, placed in new environmental context may respond either with increased novelty seeking (active), or enhanced anxiety (passive coping style), which may depend on the trait anxiety of the animal. Here, the connection between the initial level of anxiety and the behavioral responses in a novel environment was investigated. Two inbred mouse strains having either high- or low-anxiety related behavior (AX and nAX) were exposed to elevated plus maze (EPM), a standard test for assessing anxiety level, for 8 consecutive days. The initial anxiety level was modulated by chronic treatment with buspirone (bus) treatment, a clinically effective anxiolytic, using 2.5mg/kg and 5.0mg/kg doses. Both strains showed a gradual decrease of open-arm exploration, which was not prevented by bus treatment. Another cohort of animals was exposed to EPM for 2 days, and then we changed to blue light illumination and used a different cleaning substance with citrus odor (context change, CC). It was found that upon CC AX mice exhibited increased, while nAX mice showed decreased anxiety. Bus in 2.5mg/kg changed the coping strategy from passive to active exploration after CC in the AX mice; however, the same treatment rendered nAX mice passive upon CC. Bus in 5.0mg/kg failed to alter the overall coping style in the novel environment of both strains. These results suggest that these mouse lines use different coping strategy in novel context, which can be changed with bus treatment.