Tenomodulin expression in the periodontal ligament enhances cellular adhesion.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain

BRICHOS interactions with amyloid proteins and implications for Alzheimer disease

To date, about 30 diseases, in which amyloid fibrils form extracellular deposits, have been identified in humans. It is not known if the fibrils have a function, like storage of misfolded proteins, or if they just reflect failure of the cell to manage misfolded proteins. There is no treatment for the majority of the amyloid diseases and therefore disease modifying therapies are sought for. The work in this thesis is focused on studying the BRICHOS domain, which is expressed as part of proproteins found in several protein families involved in a wide range of functions, and some of them are associated with amyloid disease, e.g. interstitial lung disease (proSP-C) and dementia (Bri2). BRICHOS is suggested to have a role in preventing amyloid aggregation of its proproteins. Alzheimer disease (AD) is the most common form of dementia, and aggregation of the amyloid-β peptide (Aβ) is widely considered as the causative event. Aβ is derived by sequential cleavages of the Aβ precursor protein AβPP. Previous studies have shown that proSP-C BRICHOS reduces Aβ aggregation, and suggested that the monomer is the active form. In Paper I we studied ways to increase the monomer/trimer ratio of proSP-C BRICHOS expressed in E. coli, and how this affects its activity against Aβ fibrillation. We found that treatment with amphipathic agents increased proSP-C BRICHOS monomer/trimer ratio and its activity. We also determined that proSP-C BRICHOS is monomeric in mammalian cells. ProSP-C BRICHOS is only expressed in alveolar type II cells where it facilitates folding of the extremely aggregation prone transmembrane region of proSP-C. In Paper II we studied whether proSP-C BRICHOS could reduce amyloid aggregation of a designed amyloidogenic protein in the secretory pathway of mammalian cells. We found that co-expression of BRICHOS led to reduced amyloid aggregation, and prevented subsequent inhibition of proteasomal degradation. This suggests that BRICHOS has generic anti-amyloid properties. The BRICHOS containing Bri2 and Bri3 proteins are expressed in the central nervous system and have been proposed to be involved in AβPP processing. In Paper III we studied interactions between Bri2 and Bri3 BRICHOS and endogenous neuronal AβPP and Aβ. We found that Bri2 BRICHOS is shed from cells, and interacts with intracellular Aβ and AβPP. Bri3 BRICHOS was not shed into the extracellular space, showed abundant interactions with intracellular Aβ, and exhibited reduced hippocampal and cortical levels in AD. In Paper IV we studied proSP-C and Bri2 BRICHOS effects on Aβ aggregation in vivo in a mouse model overexpressing mutant AβPP and presenilin1 (PS1). Both proSP-C and Bri2 BRICHOS reduced Aβ levels and aggregation without affecting AβPP processing. Mice co- expressing BRICHOS and AβPP/PS1 showed improved memory and reduced neuroinflammation compared to AβPP/PS1 control animals. The results in this thesis show that BRICHOS reduces amyloid aggregation in vitro, in cells and in a mouse AD model, and indicate a potential physiological relationship between BRICHOS and Aβ. These findings together support that BRICHOS and its properties are worth continuing to study in relation to amyloid aggregation and AD

Amyloid and intracellular accumulation of BRI2

Familial British dementia (FBD) and familial Danish dementia (FDD) are caused by mutations in the BRI2 gene. These diseases are characterized clinically by progressive dementia and ataxia and neuropathologically by amyloid deposits and neurofibrillary tangles. Herein, we investigate BRI2 protein accumulation in FBD, FDD, Alzheimer disease and Gerstmann-Sträussler-Scheinker disease. In FBD and FDD, we observed reduced processing of the mutant BRI2 pro-protein, which was found accumulating intracellularly in the Golgi of neurons and glial cells. In addition, we observed an accumulation of a mature form of BRI2 protein in dystrophic neurites, surrounding amyloid cores. Accumulation of BRI2 was also observed in dystrophic neurites of Alzheimer disease and Gerstmann-Sträussler-Scheinker disease cases. Although it remains to be determined whether intracellular accumulation of BRI2 may lead to cell damage in these degenerative diseases, our study provides new insights into the role of mutant BRI2 in the pathogenesis of FBD and FDD and implicates BRI2 as a potential indicator of neuritic damage in diseases characterized by cerebral amyloid deposition

4-Phenylbutyric acid treatment rescues trafficking and processing of a mutant surfactant protein C

Mutations in the SFTPC gene, encoding surfactant protein–C (SP-C), are associated with interstitial lung disease (ILD). Knowledge of the intracellular fate of mutant SP-C is essential in the design of therapies to correct trafficking/processing of the proprotein, and to prevent the formation of cytotoxic aggregates. We assessed the potential of a chemical chaperone to correct the trafficking and processing of three disease-associated mutant SP-C proteins. HEK293 cells were stably transfected with wild-type (SP-C(WT)) or mutant (SP-C(L188Q), SP-C(Δexon4), or SP-C(I73T)) SP-C, and cell lines with a similar expression of SP-C mRNA were identified. The effects of the chemical chaperone 4-phenylbutyric acid (PBA) and lysosomotropic drugs on intracellular trafficking to the endolysosomal pathway and the subsequent conversion of SP-C proprotein to mature peptide were assessed. Despite comparable SP-C mRNA expression, proprotein concentrations varied greatly: SP-C(I73T) was more abundant than SP-C(WT) and was localized to the cell surface, whereas SP-C(Δexon4) was barely detectable. In contrast, SP-C(L188Q) and SP-C(WT) proprotein concentrations were comparable, and a small amount of SP-C(L188Q) was localized to the endolysosomal pathway. PBA treatment restored the trafficking and processing of SP-C(L188Q) to SP-C(WT) concentrations, but did not correct the mistrafficking of SP-C(I73T) or rescue SP-C(Δexon4). PBA treatment also promoted the aggregation of SP-C proproteins, including SP-C(L188Q). This study provides proof of the principle that a chemical chaperone can correct the mistrafficking and processing of a disease-associated mutant SP-C proprotein

Mechanisms of amyloid-beta cytotoxicity in hippocampal network function : rescue strategies in Alzheimer's disease

The origin and nature of cognitive processes are strongly associated with synchronous rhythmic activity in the brain. Gamma oscillations that span the frequency range of 30–80 Hz are particularly important for sensory perception, attention, learning, and memory. These oscillations occur intrinsically in brain regions, such as the hippocampus, that are directly linked to memory and disease. It has been reported that gamma and other rhythms are impaired in brain disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia; however, little is known about how these oscillations are affected. In the studies contained in this thesis, we investigated a possible involvement of toxic Amyloid-beta (Aβ) peptide associated with Alzheimer’s disease in degradation of gamma oscillations and the underlying cellular mechanismsin rodent hippocampi. We also aimed to prevent possible Aβ- induced effects by using specially designed molecular tools known to reduce toxicity associated with Aβ by interfering with its folding and aggregation steps. Using electrophysiological techniques to study thelocal field potentials and cellular properties in the CA3 region of the hippocampus, we found that Aβ in physiological concentrations acutely degrades pharmacologically- induced hippocampal gamma oscillations in vitro in a concentration- and time- dependent manner. The severity of degradation also increased with the amount of fibrillar Aβ present. We report that the underlying cause of degradation of gamma oscillations is Aβ-induced desynchronization of action potentials in pyramidal neurons and a shift in the equilibrium of excitatory-inhibitory synaptic transmission. Using specially designed molecular tools such as Aβ-binding ligands and molecular chaperones, we provide evidence that Aβ-induced effects on gamma oscillations, cellular firing, and synaptic dynamics can be prevented. We also show unpublished data on Aβ effects on parvalbumin-positive baskets cells or fast-spiking interneurons, in which Aβ causes an increase in firing rate during gamma oscillations. This is similar to what is observed in neighboring pyramidal neurons, suggesting a general mechanism behind the effect of Aβ. The studies in this thesis provide a correlative link between Aβ-induced effects on excitatory and inhibitory neurons in the hippocampus and extracellular gamma oscillations, and identify the Aβ aggregation state responsible for its toxicity. We demonstrate that strategies aimed at preventing peptide aggregation are able to prevent the toxic effects of Aβ on neurons and gamma oscillations. The studies have the potential to contribute to the design of future therapeutic interventions that are aimed at preserving neuronal oscillations in the brain to achieve cognitive benefits for patients

Itm2a, a Target Gene of GATA-3, Plays a Minimal Role in Regulating the Development and Function of T Cells

The integral membrane protein 2a (Itm2a) is one of the BRICHOS domain-containing proteins and is structurally related to Itm2b and Itm2c. It is expressed preferentially in the T lineage among hematopoietic cells and is induced by MHC-mediated positive selection. However, its transcriptional regulation and function are poorly understood. Here we showed Itm2a to be a target gene of GATA-3, a T cell-specific transcription factor. Deficiency of Itm2a had little impact on the development and function of polyclonal T cells but resulted in a partial defect in the development of thymocytes bearing a MHC class I-restricted TCR, OT-I. In addition, Itm2a-deficient mice displayed an attenuated T helper cell-dependent immune response in vivo. We further demonstrated that Itm2b but not Itm2c was also expressed in T cells, and was induced upon activation, albeit following a kinetic different from that of Itm2a. Thus, functional redundancy between Itm2a and Itm2b may explain the minimal phenotype of Itm2a deficiency

Biophysical approaches for the study of interactions between molecular chaperones and protein aggregates.

Molecular chaperones are key components of the arsenal of cellular defence mechanisms active against protein aggregation. In addition to their established role in assisting protein folding, increasing evidence indicates that molecular chaperones are able to protect against a range of potentially damaging aspects of protein behaviour, including misfolding and aggregation events that can result in the generation of aberrant protein assemblies whose formation is implicated in the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The interactions between molecular chaperones and different amyloidogenic protein species are difficult to study owing to the inherent heterogeneity of the aggregation process as well as the dynamic nature of molecular chaperones under physiological conditions. As a consequence, understanding the detailed microscopic mechanisms underlying the nature and means of inhibition of aggregate formation remains challenging yet is a key objective for protein biophysics. In this review, we discuss recent results from biophysical studies on the interactions between molecular chaperones and protein aggregates. In particular, we focus on the insights gained from current experimental techniques into the dynamics of the oligomerisation process of molecular chaperones, and highlight the opportunities that future biophysical approaches have in advancing our understanding of the great variety of biological functions of this important class of proteins.We acknowledge financial support from the Frances and Augustus Newman Foundation (TPJK), the Biological Sciences Research Council (TPJK), the European Research Council (TPJK and MAW), the Wellcome Trust (CMD, TPJK and MV), and the Marie Curie fellowship scheme (PA).This is the final version of the article. It was first available from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C5CC03689

Epigenetic mechanisms underlying Gastrokine 1 gene silencing in gastric cancer progression

Gastric cancer (GC) is still one of the leading causes of cancer-related deaths worldwide and high mortality rate is mainly due to late-stage diagnosis. New insights show that epigenetic alterations contribute significantly to the development and progression of GC and if nowadays the role of somatic mutations as drivers of carcinogenesis in the alimentary tract is well established, the importance of gene silencing by epigenetic mechanisms is increasingly recognized. Gastrokine1 (GKN1) is a highly expressed stomach protein important for maintaining the physiological function of the gastric mucosa. GKN1 is down-regulated in gastric tumor tissues and derived cell lines so it has recently emerged as a potential biomarker for gastric cancer. It has also been demonstrated that GKN1 expression induces apoptosis in gastric cancer cells thus suggesting a possible role of the protein as tumor suppressor. The mechanism by which GKN1 gene is inactivated in GC remains still unknown, so here I have investigated on the possible causes of GKN1 gene silencing in order to determine if epigenetic mechanisms could also contribute to its down-regulation. To these aim, chromatin immunoprecipitation (ChIP) assays for the repressive trimethylation of histone 3 at lysine 9 (H3K9triMe) and its specific histone-lysine Nmethyltransferase (SUV39H1) were performed on biopsies of normal and tumor human gastric tissues. The results showed that GKN1 downregulation in gastric cancer tissues is associated with high levels of H3K9triMe and with the recruitment of SUV39H1 on GKN1 promoter, suggesting the presence of an epigenetic transcriptional complex that negatively regulates GKN1 expression in gastric tumor. It was also investigated whether underacetylation might contribute to GKN1 transcriptional inhibition using TSA to increase general histone acetylation. The results showed that inhibition of HDACs leads to GKN1 restoration at transcriptional level, but no at traslational level. These findings led to hypothesize that a second regulatory block occurs at translational level, perhaps by mechanisms mediated by microRNAs (miRNAs), resulting in translational repression and gene silencing. So, the possible involvment of miRNAs in this process was investigated. The results demostrated that GKN1 3’UTR was a direct target of hsa-miR-544a and miR-1245b-3p and showed an increase of miR-544a expression in the gastric cancer cell lines after TSA treatment. The up-regulation of miR-544a could be the cause of the GKN1 translational repression, thus suggesting its potential role as biomarker and therapeutic target in GC patients. These findings indicate that epigenetic mechanisms leading to the inactivation of GKN1 play a key role in the multi-step process of gastric 2 carcinogenesis and would provide an essential starting point for the development of new therapeutic strategies based on epigenetic targets for alternatives gene

The proteostasis network and its decline in ageing

Ageing is a major risk factor for the development of many diseases, prominently including neurodegenerative disorders such as Alzheimer disease and Parkinson disease. A hallmark of many age-related diseases is the dysfunction in protein homeostasis (proteostasis), leading to the accumulation of protein aggregates. In healthy cells, a complex proteostasis network, comprising molecular chaperones and proteolytic machineries and their regulators, operates to ensure the maintenance of proteostasis. These factors coordinate protein synthesis with polypeptide folding, the conservation of protein conformation and protein degradation. However, sustaining proteome balance is a challenging task in the face of various external and endogenous stresses that accumulate during ageing. These stresses lead to the decline of proteostasis network capacity and proteome integrity. The resulting accumulation of misfolded and aggregated proteins affects, in particular, postmitotic cell types such as neurons, manifesting in disease. Recent analyses of proteome-wide changes that occur during ageing inform strategies to improve proteostasis. The possibilities of pharmacological augmentation of the capacity of proteostasis networks hold great promise for delaying the onset of age-related pathologies associated with proteome deterioration and for extending healthspan