Search

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. The miRNA miR-106b-5p has been linked to epilepsy, but its specific role and mechanism of action remain unclear. This was investigated in the present study using a mouse model of pilocarpine-induced status epilepticus and an in vitro system of HT22 hippocampal cells treated with Mg2+-free solution and cocultured with BV2 microglia cells. We found that inhibiting miR-106b-5p expression promoted microglia M2 polarization, reduced the inflammatory response, and alleviated neuronal injury. These effects involved modulation of the repulsive guidance molecule A (RGMa)–Rac1–c-Jun N-terminal kinase (JNK)/p38–mitogen-activated protein kinase (MAPK) signaling axis. Our results suggest that therapeutic strategies targeting miR-106b-5p or downstream factors can be effective in preventing epileptogenesis or treating epilepsy. [ABSTRACT FROM AUTHOR]

Objective: In the present study, we intend to assess the function of Sema3A in osteointegration of titanium implants both in vivo and in vitro. Material and methods: Briefly, Sema3A was transfected in HBMSCs cells to detect its effect on osteogenesis. Subsequently, an in vivo rabbit model was established. Eighteen female rabbits were randomly assigned into three groups (n=6), and rabbits in the two treatment groups (OVX groups) were subjected to bilateral ovariectomy, while those in the control group were treated with sham operation. Twelve weeks later, we first examined expression levels of Sema3A in rabbits of the three groups. Titanium implants were implanted in rabbit proximal tibia. Specifically, rabbits in sham group were implanted with Matrigel, while the remaining in the OVX experimental group (OVX+Sema3A group) and OVX group were implanted with Matrigel containing Sema3A adeno-associated virus or empty vector, respectively. Results: Histomorphometry results uncovered that rabbits in the OVX+Sema3A group had a significantly higher BIC compared with those of the OVX group on the 12th week of post-implantation. And compared with the OVX group, the maximum push-out force increased by 89.4%, and the stiffness increased by 39.4%, the toughness increased by 63.8% in the OVX+Sema3A group at 12 weeks. Conclusion: Sema3A has a positive effect on promoting early osseointegration of titanium implants in osteoporotic rabbits. Clinical relevance: Our research found that Sema3A can improve the osteogenic ability of bone marrow stem cells and promotes osseointegration during osteoporosis. [ABSTRACT FROM AUTHOR]

Sympathetic nerves innervate most organs and regulate organ blood flow. Specifically, in the uterus, estradiol (E2) elicits rapid degeneration of sympathetic axons and stimulates the growth of blood vessels. Both physiological remodeling processes, critical for reproduction, have been extensively studied but as independent events and are still not fully understood. Here, we examine the neuropilin-1 (NRP1), a shared receptor for axon guidance and angiogenic factors. Systemic estradiol or vehicle were chronically injected to prepubertal rats and uterine and sympathetic chain sections immunostained for NRP1. Uterine semaphorin-3A mRNA was evaluated by in situ hybridization. Control sympathetic uterine-projecting neurons (1-month-old) expressed NRP1 in their somas but not in their intrauterine terminal axons. Estradiol did not affect NRP1 in the distal ganglia. However, at the entrance of the organ, some sympathetic NRP1-positive nerves were recognized. Vascular NRP1 was confined to intrauterine small-diameter vessels in both hormonal conditions. Although the overall pattern of NRP1-IR was not affected by E2 treatment, a subpopulation of infiltrated eosinophil leukocytes showed immunoreactivity for NRP1. Sema3A transcripts were detected in this cellular type as well. No NRP1-immunoreactive axons nor infiltrated eosinophils were visualized in other estrogenized pelvic organs. Together, these data suggest the involvement of NRP1/Sema3A signaling in the selective E2-induced uterine neurovascular remodeling. Our data support a model whereby NRP1 could coordinate E2-induced uterine neurovascular remodeling, acting as a positive regulator of growth when expressed in vessels and as a negative regulator of growth when expressed on axons. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common cause of dementia worldwide with 10–30% prevalence in aging population and a high socioeconomic impact. Because AD definitive diagnostic requires post-mortem verification, new approaches to study the disease are necessary. Here, we analyze the humoral immune response in AD to survey whether APP+1 or UBB+1 frameshift proteins, produced as a consequence of the "molecular misreading" alteration in AD occurring in the APP (amyloid precursor protein) and UBB (ubiquitin-B protein) proteins' mRNA, elicit the production of autoantibodies specific of AD. To this end, APP+1 and UBB+1 peptides were expressed in bacteria as 6xHisHalo fusion proteins and after purification to homogeneity their seroreactivity was analyzed using 81 individual sera from AD patients and 43 individual sera from healthy individuals by luminescence beads immunoassay. We found that as a result of the molecular misreading, APP+1 and UBB+1 frameshift peptides produced a humoral immune response in AD patients, whose autoantibody levels are significantly higher in comparison with healthy controls. Their combination with a previously reported panel of four autoantigens specific of AD (ANTXR1, OR8J1, PYGB, and NUPR1) increased their diagnostic ability assessed by receiver operating characteristic (ROC) curves up to an area under the curve (AUC) of 73.5%. Collectively, our results demonstrate that APP+1 and UBB+1 frameshift proteins, non-previously described as AD-specific autoantigens, elicit the production of autoantibodies which might be useful as blood-based biomarkers to aid in the detection of the disease. [ABSTRACT FROM AUTHOR]

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity. [ABSTRACT FROM AUTHOR]

The most common neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, frontotemporal lobar degeneration, and the motor neuron diseases, with AD affecting approximately 6% of people aged 65 years and older, and PD affecting approximately 1% of people aged over 60 years. Specific proteins are associated with these neurodegenerative diseases, as determined by both immunohistochemical studies on post-mortem tissue and genetic screening, where protein misfolding and aggregation are key hallmarks. Many of these proteins are shown to misfold and aggregate into soluble non-native oligomers and large insoluble protein deposits (fibrils and plaques), both of which may exert a toxic gain of function. Proteotoxicity has been examined intensively in cell culture and in in vivo models, and clinical trials of methods to attenuate proteotoxicity are relatively new. Therapies to enhance cellular protein quality control mechanisms such as upregulation of chaperones and clearance/degradation pathways, as well as immunotherapies against toxic protein conformations, are being actively pursued. In this article, we summarize the common pathophysiology of neurodegenerative disease, and review therapies in early-phase clinical trials that target the proteotoxic component of several neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Determining protein thermal stability is integral in biomedical research. Here, with the use of two thermal stability assays, we show the melting temperature of amyloid precursor protein, an Alzheimer's disease related protein. The average melting temperature for amyloid precursor protein of 55.9 °C was derived from differential scanning fluorometry (55.1 ± 0.3 °C) and cellular thermal melt (56.7 ± 0.7 °C). These experimental methods have significant application for Alzheimer's disease research including their use for amyloid precursor protein stability profiling and for the identification of additional binding partners to further elucidate novel protein functions. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is the most common neurodegenerative disease. Pathological proteins of AD mainly contain amyloid-beta (Aβ) and tau. Their deposition will lead to neuron damage by a series of pathways, and then induce memory and cognitive impairment. Thus, it is pivotal to understand the clearance pathways of Aβ and tau in order to delay or even halt AD. Aβ clearance mechanisms include ubiquitin-proteasome system, autophagy-lysosome, proteases, microglial phagocytosis, and transport from the brain to the blood via the blood-brain barrier (BBB), arachnoid villi and blood-CSF barrier, which can be named blood circulatory clearance. Recently, lymphatic clearance has been demonstrated to play a key role in transport of Aβ into cervical lymph nodes. The discovery of meningeal lymphatic vessels is another direct evidence for lymphatic clearance in the brain. Furthermore, periphery clearance also contributes to Aβ clearance. Tau clearance is almost the same as Aβ clearance. In this review, we will mainly introduce the clearance mechanisms of Aβ and tau proteins, and summarize corresponding targeted drug therapies for AD. [ABSTRACT FROM AUTHOR]

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in DNA damage response. Ectopic expression of PCNA fused at either terminus with ubiquitin (Ub) lacking two C-terminal glycine residues induces translesion DNA synthesis which resembles synthesis mediated by PCNA monoubiquitination. PCNA fused with Ub containing the C-terminal Gly residues at the C-terminus can be further polyubiquitinated in a Gly-dependent manner, which inhibits cell proliferation and induces ATR-dependent replication checkpoint. In this study, we surprisingly found that PCNA fused to a head-to-tail linear Ub chain induces apoptosis in a Ub chain length-dependent manner. Further investigation revealed that the apoptotic effect is actually induced by the linear Ub chain independently from PCNA, as the Ub chain fused to GFP or an epitope tag still efficiently induces apoptosis. It is revealed that the artificial linear Ub chain differs from endogenously encoded linear Ub chains in that its Ubs contain a Ub-G76S substitution, making the Ub chain resistant to cleavage by deubiquitination enzymes. We demonstrated in this study that ectopic expression of the artificial Ub chain alone in cultured human cancer cells is sufficient to inhibit tumor growth in a xenograft mouse model, making the linear Ub chain a putative anti-cancer agent. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B (UBB), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB-expressing transgenic mice display widespread labeling for UBB in brain and exhibit behavioral deficits. Here, we show that UBB is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB-expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties. [ABSTRACT FROM AUTHOR]

An important mechanism of neuronal plasticity is neurogenesis, which occurs during the embryonic period, forming the brain and its structure, and in the postnatal period, providing repair processes and participating in the mechanisms of memory consolidation. Adult neurogenesis in mammals, including humans, is limited in two specific brain areas, the lateral walls of the lateral ventricles (subventricular zone) and the granular layer of the dentate gyrus of the hippocampus (subgranular zone). Neural stem cells (NSC), self-renewing, multipotent progenitor cells, are formed in these zones. Neural stem cells are capable of differentiating into the basic cell types of the nervous system. In addition, NSC may have neurogenic features and non-specific non-neurogenic functions aimed at maintaining the homeostasis of the brain. The microenvironment formed in neurogenic niches has importance maintaining populations of NSC and regulating differentiation into neural or glial cells via cell-to-cell interactions and microenvironmental signals. The vascular microenvironment in neurogenic niches are integrated by signaling molecules secreted from endothelial cells in the blood vessels of the brain or by direct contact with these cells. Accumulation of astrocytes in neurogenic niches if also of importance and leads to activation of neurogenesis. Dysregulation of neurogenesis contributes to the formation of neurological deficits observed in neurodegenerative diseases. Targeting regulation of neurogenesis could be the basis of new protocols of neuroregeneration. [ABSTRACT FROM AUTHOR]

Several integrated proteolytic systems contribute to the maintenance of cellular homeostasis through the continuous removal of misfolded, aggregated or oxidized proteins and damaged organelles. Among these systems, the proteasome and autophagy play the major role in protein quality control, which is a fundamental issue in non-proliferative cells such as neurons. Disturbances in the functionality of these two pathways are frequently observed in neurodegenerative diseases, like Alzheimer's disease, and reflect the accumulation of protease-resistant, deleterious protein aggregates. In this review, we explored the sophisticated crosstalk between the ubiquitin-proteasome system and autophagy in the removal of the harmful structures that characterize Alzheimer's disease neurons. We also dissected the role of the numerous shuttle factors and chaperones that, directly or indirectly interacting with ubiquitin and LC3, are used for cargo selection and delivery to one pathway or the other. [ABSTRACT FROM AUTHOR]

Macroautophagy and chaperone-mediated autophagy (CMA) are two important subtypes of autophagy that play a critical role in cellular quality control under physiological and pathological conditions. Despite the marked differences between these two autophagic pathways, macroautophagy and CMA are intimately connected with each other during the autophagy-lysosomal degradation process, in particular, in the setting of neurological illness. Macroautophagy serves as a backup mechanism to removal of malfunctioning proteins (i.e., aberrant α-synuclein) from the cytoplasm when CMA is compromised, and vice versa. The molecular mechanisms underlying the conversation between macroautophagy and CMA are being clarified. Herein, we survey current overviews concentrating on the complex interactions between macroautophagy and CMA, and present therapeutic potentials through utilization and manipulation of macroautophagy-CMA crosstalk in the treatment of neurological diseases. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) plays a fundamental role in protein degradation in neurons, and there is strong evidence that it fulfills a key role in synaptic transmission. The aim of the present work was to study the implication of one component of the UPS, the HERC1 E3 Ubiquitin Ligase, in motor function and neuromuscular transmission. The tambaleante ( tbl) mutant mouse carries a spontaneous mutation in HERC1 E3 Ubiquitin Ligase, provoking an ataxic phenotype that develops in the second month of life. Our results show that motor performance in mutant mice is altered at postnatal day 30, before the cerebellar neurodegeneration takes place. This defect is associated with by: (a) a reduction of the motor end-plate area, (b) less efficient neuromuscular activity in vivo, and (c) an impaired evoked neurotransmitter release. Together, these data suggest that the HERC1 E3 Ubiquitin Ligase is fundamental for normal muscle function and that it is essential for neurotransmitter release at the mouse neuromuscular junction. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is one key medical challenge of the aging society and despite a great amount of effort and a huge collection of acquired data on molecular mechanisms that are associated with the onset and progression of this devastating disorder, no causal therapy is in sight. The two main hypotheses of AD, the amyloid cascade hypothesis and the Tau hypothesis, are still in the focus of AD research. With aging as the accepted main risk factor of the most important non familial and late onset sporadic forms of AD, it is now mandatory to discuss more intensively aspects of cellular aging and aging biochemistry and its impact on neurodegeneration. Since aging is accompanied by changes in cellular protein homeostasis and an increasing demand for protein degradation, aspects of protein folding, misfolding, refolding and, importantly, protein degradation need to be linked to AD pathogenesis. This is the purpose of this short review. [ABSTRACT FROM AUTHOR]

Aging and neurodegeneration are often accompanied by a functionally impaired ubiquitin-proteasome system (UPS). In tauopathies and polyglutamine diseases, a mutant form of ubiquitin B (UBB) accumulates in disease-specific aggregates. UBB mRNA is generated at low levels in vivo during transcription from the ubiquitin B locus by molecular misreading. The resulting mutant protein has been shown to inhibit proteasome function. To elucidate causative effects and neuropathological consequences of UBB accumulation, we used a UBB expressing transgenic mouse line that models UPS inhibition in neurons and exhibits behavioral phenotypes reminiscent of Alzheimer's disease (AD). In order to reveal affected organs and functions, young and aged UBB transgenic mice were comprehensively phenotyped for more than 240 parameters. This revealed unexpected changes in spontaneous breathing patterns and an altered response to hypoxic conditions. Our findings point to a central dysfunction of respiratory regulation in transgenic mice in comparison to wild-type littermate mice. Accordingly, UBB was strongly expressed in brainstem regions of transgenic mice controlling respiration. These regions included, e.g., the medial part of the nucleus of the tractus solitarius and the lateral subdivisions of the parabrachial nucleus. In addition, UBB was also strongly expressed in these anatomical structures of AD patients (Braak stage #6) and was not expressed in non-demented controls. We conclude that long-term UPS inhibition due to UBB expression causes central breathing dysfunction in a transgenic mouse model of AD. The UBB expression pattern in humans is consistent with the contribution of bronchopneumonia as a cause of death in AD patients. [ABSTRACT FROM AUTHOR]

Oxidative stress has been proposed to be an important factor in the pathogenesis of Alzheimer's disease (AD) and contributed to β-amyloid (A β) generation. Interaction between oxidative stress and neuro-inflammation leads to Aβ generation. AD is associated with an increase in blood-brain barrier (BBB) permeability due to tight junction involvement. Oxidative stress decreases the expression of low-density lipoprotein receptor-related protein 1 and up-regulates receptor for advanced glycation end products in BBB and increases the BBB permeability, which could potentially lead to increased deposition of A β within AD brain. Apoptosis takes place in the pathogenesis of AD, and oxidative stress contributes to apoptosis through both extrinsic pathway and intrinsic pathway. Oxidative stress-induced apoptosis may be a potential factor to A β generation. A β generation requires two sequential cleavages of APP, with the two proteolytic enzymes: β-secretase and γ-secretase. Oxidative damage up-regulates A β via inducing activity of β- and γ-secretases. In this review, we will focus on the mechanism and pathway that oxidative stress contributes to A β generation. [ABSTRACT FROM AUTHOR]

The ubiquitin/proteasome pathway is the major proteolytic quality control system in cells. In this review we discuss the impact of a deregulation of this pathway on neuronal function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in pathological inclusion bodies in all the major chronic neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's diseases as well as amyotrophic lateral sclerosis. We describe the intricate nature of the ubiquitin/proteasome pathway and discuss the paradox of protein aggregation, i.e. its potential toxic/protective effect in neurodegeneration. The relations between some of the dysfunctional components of the pathway and neurodegeneration are presented. We highlight possible ubiquitin/proteasome pathway-targeting therapeutic approaches, such as activating the proteasome, enhancing ubiquitination and promoting SUMOylation that might be important to slow/treat the progression of neurodegeneration. Finally, a model time line is presented for neurodegeneration starting at the initial injurious events up to protein aggregation and cell death, with potential time points for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer’s disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases are featured by progressive dysfunction and death of cells in selected areas in the nervous system, determining clinical presentation. Neuronal loss is associated with conformational changes in proteins that result in extra- and intra-cellular accumulation of misfolded proteins, representing the hallmarks of many neurodegenerative disorders, summarized as proteinopathies. Intermediate forms such as oligomers and protofibrils are thought to have cytotoxic effects to neurons. Major basic processes, caused by genetic, environmental, and endogenous factors, in addition to abnormal protein dynamics with defective degradation due to deficiency of the ubiquitin–proteosomal–autophagy system, include oxidative stress and free radical formation, impaired bioenergetics and mitochondrial dysfunction, disruption of neuronal Golgi apparatus and transport, molecular chaperones, neurotrophins and “neuroinflammatory” processes. These mechanisms are interrelated in vicious circles finally leading to programmed cell death. A common feature of these conditions is a long run until sufficient protein accumulates, followed by a cascade of symptoms over many years with increasing disability leading to death. This provides a wide therapeutic window, especially in groups at risk identified early and preclinical diagnosis becomes feasible. Neurodegenerative disorders are classified according to known genetic mechanisms or to the major components of protein deposits. Although this has been a productive paradigm for the development of diagnostic consensus criteria, recent molecular biologic and genetic approaches have revealed that there are both overlap and intraindividual diversities between different phenotypes, related to synergistic mechanisms between major pathologic proteins (β-amyloid, tau, α-synuclein, TDP-43), suggesting common pathogenic mechanisms. The nature, time course, and molecular causes of cell degeneration and demise, and the role of various pathogenic factors are a matter of considerable debate, but recent studies have provided insight into the basic processes in neurodegeneration and the roles of cell death programs common to this complex group of disorders, offering new ways for future prevention and treatment strategies. [ABSTRACT FROM AUTHOR]

Commercially available antisera against five subtypes of muscarinic receptors and nine subtypes of adrenoceptors showed highly distinct immunohistochemical staining patterns in rat ureter and stomach. However, using the M1–4 muscarinic receptor subtypes and α2B-, β2-, and β3-adrenoceptors as examples, Western blots with membranes prepared from cell lines stably expressing various subtypes of muscarinic receptors or adrenoceptors revealed that each of the antisera recognized a set of proteins that differed between the cell lines used but lacked specificity for the claimed target receptor. We propose that receptor antibodies need better validation before they can reliably be used. [ABSTRACT FROM AUTHOR]

Most neurodegenerative disorders are characterised by deposits of aggregated proteins that are readily visualised by light microscopy. Although the presence of such a bulky structure inside the cell or in the extracellular space is likely not to be healthy, over recent years the idea has emerged that these end-stage aggregates are a relatively safe way to deposit harmful aberrant proteins. Protein quality control is a multi-level security system to safeguard cells from aberrant proteins and is therefore a protective response. However, protein quality control may turn destructive in case of impairment of protein quality control for example by aging or because of overflow of the quality control systems due to prolonged exposure. In many cases the medicine is worse than the cause and the “protective” response of the cell to aggregates kills the cell, rather than the aggregate itself. Here we review the role of protein quality control in neurodegeneration and aim to distinguish protective and destructive responses to aggregates in order to find targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Neurodegenerative disorders share a process of aggregation of insoluble protein. Frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) is characterized by the presence of ubiquitin and TDP-43 positive aggregates which are likely related to specific gene expression profiles. We carried out gene expression microarray analysis on post-mortem brain tissue from FTLD-U, FTLD-MND, and controls. Using total RNA from carefully dissected frontal cortical layer II, we obtained gene expression profiles showing that FTLD-U and controls differ in over 100 networks, including those involved in synapse formation, the ubiquitin-proteasome system, endosomal sorting, and apoptosis. We performed qRT-PCR validation for three genes, representative of three different networks. Dynein axonemal light intermediate chain 1 (DNALI1) (microtubule/cytoskeleton network associated) expression was 3-fold higher and myeloid differentiation primary response gene 88 (MYD88) (signal transduction network) was 3.3 times higher in FTLD-U than FTLD-MND and controls; annexin A2 (ANXA2) (endosomal sorting) expression was 11.3-fold higher in FTLD-U than FTLD-MND and 2.3-fold higher than controls. The identification of progranulin ( PGRN) gene mutations and TDP-43 as the major protein component of the ubiquitinated inclusions, are two recent landmark discoveries in the field of FTLD-U. We found 1.5-fold increase in TDP-43 in both FTLD-MND and FTLD-U while progranulin showed no gene expression differences between controls and FTLD-MND. However, one of the FTLD-U cases tested by Affymetrix microarray showed “absence call” of this transcript, suggesting absent or decreased gene expression. Our findings point to specific gene-linked-pathways which may be influenced by neurodegenerative disease process and may be targeted for further exploration. [ABSTRACT FROM AUTHOR]

Recent studies have shown the co-existence of α-synuclein and phosphorylated tau (pTau) in several neurodegenerative diseases. Here, we report two autopsy cases of combined multiple system atrophy (MSA) and Alzheimer’s disease (AD). In both cases, abundant α-synuclein-positive glial and neuronal cytoplasmic inclusions were found in the brainstem, amygdala and hippocampal formation. pTau-positive neurofibrillary tangles (NFTs) were widely distributed in case 1 (Braak stage VI) and moderate in case 2 (Braak stage III). Although α-synuclein and pTau pathology co-occurred in the hippocampus and entorhinal cortex, only a few neurons showed co-existence of these two proteins. Immunoreactivity for p62, a ubiquitin proteasome system related protein, was found in the majority of NFTs, but in only a small proportion of neuronal α-synuclein inclusions. In addition, UBB+1, a mutant form of ubiquitin and a marker for proteasomal dysfunction, was present in the majority of NFTs, whereas co-existence of α-synuclein and UBB+1 was found in only a few neurons. These findings indicate that α-synuclein and phosphorylated tau co-occur in certain brain regions in cases of combined MSA and AD and that the proteasomal pathways differ between α-synuclein- and pTau-bearing neurons. [ABSTRACT FROM AUTHOR]

Some probabilistic results on simple sequence repeats (SSRs) in DNA sequences are derived and used to quantify the nonrandomness of SSRs as an index of nonrandomness. The applicability of the index of nonrandomness is illustrated using several examples from the literature on selected human diseased genes. [ABSTRACT FROM AUTHOR]

Proteins as a product from genetic information execute and determine how development, growth, aging and disease factors are orchestrated within the lifetime of an organism. Differential protein expression and/or modification are always context dependent i.e. they happen within a specific context of a tissue, organ, environmental situation and individual fate. Consequently, the function/dysfunction (in a certain disease) of a specific gene cannot be predicted comprehensively by its sequence only. Genetic information can only be understood when genes and proteins are analyzed in the context of the biological system and specific networks they are involved in. In regard to neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s disease (PD) many proteins are known for long years to be the cause or the consequence of the pathomechanism of the respective disease. The treatment of these neurodegenerative diseases represents a major challenge for the pharmaceutical industry, whereas the understanding of their pathogenesis is still in its infancy. With the development of several powerful techniques for proteome analysis it is now possible to investigate the expression of thousands of proteins in single cells, tissues or whole organisms at the same time. These developments opened new doors in medical sciences, and identification of cellular alterations associated with e.g. neurodegeneration will result in the identification of novel diagnostic as well as therapeutic targets. In this review, general considerations and strategies of proteomics technologies, the advantages and challenges as well as the special needs for analyzing brain tissue in the context of AD and AD are described and summarized. [ABSTRACT FROM AUTHOR]

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 × 10−2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy. [ABSTRACT FROM AUTHOR]

The discovery of glial cytoplasmic inclusions (GCIs) in 1989 helped to define multiple system atrophy (MSA) as a clinicopathological entity, and drew attention to the prominent role played by these inclusions in the pathogenesis of the disorder. Subsequently, GCIs were shown to be highly positive for a-synuclein, a neuronal protein that is normally absent in oligodendroglia except during embryonic development. The source of oligodendroglial a-synuclein aggregation in MSA is unknown. Since genetic overexpression has been excluded, active uptake from dying neurons remains a possibility. The similar topography of oligodendroglial and neuronal pathology in MSA suggests a fundamental disturbance of the functional unit between oligodendroglia, axon, and neuron. Transgenic MSA mouse models are now available to determine these aspects of cellular disturbance experimentally. [ABSTRACT FROM AUTHOR]

Ubiquitin-positive tau-negative inclusions were initially described in the rare form of frontotemporal dementia (FTD) associated with motor neuron disease. However, recent studies have indicated that these inclusions are also present in typical FTD, which is usually characterized by nonspecific histological changes. To examine the contribution of these inclusions to neuronal loss and to explore their relationship with disease duration, we performed a quantitative immunocytochemical analysis of 38 typical FTD cases. Relationships between neuron and ubiquitin inclusion densities as well as between duration of illness and neuropathological parameters was studied using linear regression in both univariate and multivariate models. Ubiquitin-positive tau-negative intracytoplasmic inclusions were present in 65.8% of cases in the dentate gyrus, 57.9% in temporal cortex and 31.6% in frontal cortex. The highest densities of ubiquitin-positive inclusions were consistently observed in the dentate gyrus, followed by the temporal and frontal cortex. There was no statistically significant relationship between neuron and ubiquitin-positive inclusion densities in any of the areas studied. In contrast, ubiquitin-positive inclusion densities in the dentate gyrus were negatively related to the duration of illness. Our data suggest that the development of ubiquitin-related pathology is the rule and not the exception in typical FTD, yet is not causally related to neuronal loss. They also reveal that the development of ubiquitin-positive inclusion densities in the dentate gyrus may be associated with a more aggressive form of the disease. [ABSTRACT FROM AUTHOR]

The introduction of cloned and manipulated genetic material into the germline of an experimental organism is one of the most powerful tools of modern biology. In the case of the fruit fly, Drosophila melanogaster, there is also an unparalleled range of sophisticated genetic tools to facilitate subsequent analysis. In consequence, Drosophila remains a most favourable model organism for the dissection of gene structure and function in vivo. In this review we look at some of the achievements to date in Drosophila genome manipulation, and at what may be possible in the near future. [ABSTRACT FROM AUTHOR]

In most reports in which the activity of numerous Drosophila transposon families has been studied, only a subset of the families tested appears mobile. A comparison of these data shows that there are no transposons inherently more unstable than others and suggests that host factors regulate the activity of transposable elements. Consistent with this conclusion are the properties of Bari 1 elements, which are the only ones of the 14 families tested to be unstable in Charolles stock. Instability is persistent over 53 generations and appears to affect recurrent insertion sites. [ABSTRACT FROM AUTHOR]