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Protein aggregates in muscle cells are the morphological hallmark of myofibrillar myopathies, including myotilinopathies and desminopathies. The aim of the present study is to analyse the expression of mutant ubiquitin (UBB+1), an aberrant form of ubiquitin which accumulates in certain disorders characterized by intracellular aggregates of proteins, and p62, a multimeric signal protein which plays an active role in aggregate formation, in muscle biopsies from patients suffering from myotilinopathy and desminopathy in order to gain understanding of the mechanisms leading to protein aggregation in these disorders. Single immunohistochemistry, and single- and double-labelling immunofluorescence and confocal microscopy for UBB+1 and p62, has been performed in muscle biopsies from patients suffering from myotilinopathy and desminopathy. Strong UBB+1 immunoreactivity, colocalizing with myotilin aggregates, was found in muscle fibres in myotilinopathies. UBB+1 accumulation, colocalizing with desmin aggregates, also occurs in desminopathies. In addition, strong p62 immunoreactivity colocalizing with myotilin aggregates was observed in myotilinopathies. Similarly, p62 immunoreactivity colocalizing with desmin aggregates was found in desminopathies. The present findings suggest that accumulation of protein aggregates in myotilinopathies and in desminopathies may be related with UBB+1/abnormal protein complexes which are resistant to proteasome degradation. Furthermore, these observations suggest a relationship between the presence of p62 and the formation of inclusions in different subtypes of myofibrillar myopathies. [ABSTRACT FROM AUTHOR]

B. Puig, T. Gómez-Isla, E. Ribé, M. Cuadrado, B. Torrejón-Escribano, E. Dalfó and I. Ferrer (2004)Neuropathology and Applied Neurobiology, doi: 10.1111/j.1365-2990.2004.00569.xExpression of stress-activated kinases c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), andtauhyperphosphorylation in neurites surroundingβA plaques in APP Tg2576 miceHyperphosphorylatedtauin neurites surroundingβ-amyloid (βA) deposits, as revealed with phospho-specific anti-tauantibodies, are found in amyloid precursor protein (APP) Tg2576 mice. BecauseβA is a source of oxidative stress and may be toxic for cultured cells, the present study examines the expression of phosphorylated (active) stress-activated kinase c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), which have the capacity to phosphorylatetauat specific sites, and their specific substrates c-Jun and ATF-2, which are involved in cell death and survival in several paradigms, in Tg2576 mice. The study was planned to shed light about the involvement of these kinases intauphosphorylation in cell processes surrounding amyloid plaques, as well as in the possible phosphorylation (activation) of c-Jun and activating transcription factor-2 (ATF-2) in relation toβA deposition. Moderate increase in the expression of phosphorylated mitogen-activated protein kinase and extracelullar signal-regulated kinase (MAPK/ERK-P) occurs in a few amyloid plaques. However, strong expression of SAPK/JNK-P and p38-P is found in the majority of, if not all, amyloid plaques, as seen in serial consecutive sections stained forβA and stress kinases. Moreover, confocal microscopy reveals colocalization of phospho-tauand SAPK/JNK-P, and phospho-tauand p38-P in many dystrophic neurites surrounding amyloid plaques. Increased expression levels of nonboundtau, SAPK/JNK-P and p38-P are corroborated by Western blots of total cortical homogenate supernatants in Tg2576 mice when compared with age-matched controls. No increase in phosphorylated c-JunSer63 (c-Jun-P) and ATF-2Thr71 (ATF-2-P) is found in association withβA deposits. In addition, no expression of active (cleaved) caspase-3 (17 kDa) has been found in transgenic mice. Taken together, these observations provide a link betweenβA-induced oxidative stress, activation of stress kinases SAPK/JNK and p38, andtauhyperphosphorylation in neurites surrounding amyloid plaques, but activation of these kinases is not associated with accumulation of c-Jun-P and ATF-2-P, nor with activation of active caspase-3 in the vicinity ofβA deposits. [ABSTRACT FROM AUTHOR]

Extracellular adenosine triphosphate (ATP) conducts a complex dynamic system of broadly represented cell signaling. Ectonucleotidases are the enzymes with nucleotide hydrolytic ability that regulate ATP levels in physiological and pathological conditions, thus playing a key role in the so-called purinergic signaling. Altered ectonucleotidase expression has been reported in cancer, and the ectonucleoside triphosphate diphosphohydrolase (NTPDase) family of enzymes, with its best-known form NTPDase1 (CD39), is targeted in cancer immunotherapy. The tandem of enzymes CD39-CD73 is responsible for the generation of immunosuppressive adenosine in the tumor microenvironment, and inhibition strategies are of great interest. Organoids have emerged as very convenient models for the study of tumors since they are three-dimensional cultures that retain many of the features of tissue. The present study aims to contribute to improving the methodology and the molecular tools needed for the study of ectonucleotidases in healthy and disease conditions. The study, performed in an endometrial cancer cell model, could be extended to other types of tumors and pathologies in which the purinergic system is involved. We generated organoids from endometrial cancer cells overexpressing NTPDase2 (CD39L1) and NTPDase3 (CD39L3) as fusion proteins with EGFP, and we performed functional assays by adapting in situ cytochemistry protocols. This allowed us to simultaneously detect enzyme activity and protein expression and to demonstrate that organoids can be used to test ectonucleotidase inhibitors-a result that can be used to develop new cancer treatment options., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood-brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. Myelin and lymphocyte protein (MAL) is widely considered to be the receptor for ETX as its presence is crucial for the effects of ETX on the plasma membrane of host cells that involve pore formation, resulting in cell death. To overcome the pores formed by PFTs, some host cells produce extracellular vesicles (EVs) to reduce the amount of pores inserted into the plasma membrane. The formation of EVs has not been studied for ETX in host cells. Here, we generated a highly sensitive clone from HeLa cells overexpressing the MAL-GFP protein in the plasma membrane. We observed that ETX induces the formation of EVs. Moreover, the MAL protein and ETX oligomers are found in these EVs, which are a very useful tool to decipher and study the mode of action of ETX and characterize the mechanisms involved in the binding of ETX to its receptor.

Lipid-based nanoparticles are a useful tool for nucleic acids delivery and have been regarded as a promising approach for diverse diseases. However, off-targets effects are a matter of concern and some strategies to improve selectivity of solid lipid nanoparticles (SLNs) were reported. The goal of this study was to test formulations of SLNs incorporating lipid cholesteryl-9-carboxynonanoate (9CCN) as "eat-me" signal to target antagomiR oligonucleotides to macrophages. We formulate four SLNs, and those with a mean diameter of 200 nm and a Z-potential values between 25 and 40 mV, which allowed the antagomiR binding, were selected for in vitro studies. Cell viability, transfection efficiency and cellular uptake assays were performed within in vitro macrophages using flow cytometry and confocal imaging and the SLNs incorporating 25 mg of 9CCN proved to be the best formulation. Subsequently, we used a labeled antagomiR to study tissue distribution in in-vivo ApoE-/- model of atherosclerosis. Using the ApoE-/- model we demonstrated that SLNs with phagocytic signal 9-CCN target macrophages and release the antagomiR cargo in a selective way., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The hippocampus of cases with neurofibrillary tangles (NFT) pathology classified as stages I-II, III-IV, and V-VI without comorbidities, and middle-aged (MA) individuals with no NFT pathology, were examined to learn about the composition of granulovacuolar degeneration (GVD). Our results confirm the presence of CK1-δ, p38-P Thr180/Tyr182, SAPK/JNK-P Thr183/Thr185, GSK-3α/β-P Tyr279/Tyr216, and GSK-3β Ser9 in the cytoplasmic granules in a subset of neurons of the CA1 and CA2 subfields of the hippocampus. Also, we identify the presence of PKA α/β-P Thr197, SRC-P Tyr416, PAK1-P Ser199/Ser204, CAMK2A-P Tyr197, and PKCG-P Thr655 in cytoplasmic granules in cases with NFT pathology, but not in MA cases. Our results also confirm the presence of β-catenin-P Ser45/Thr41, IREα-P Ser274, eIF2α-P Ser51, TDP-43-P Ser403-404 (but absent TDP-43), and ubiquitin in cytoplasmic granules. Other components of the cytoplasmic granules are MAP2-P Thr1620/1623, MAP1B-P Thr1265, ADD1-P Ser726, and ADD1/ADD1-P Ser726/Ser713, in addition to several tau species including 3Rtau, 4Rtau, and tau-P Ser262. The analysis of GVD at progressive stages of NFT pathology reveals the early appearance of phosphorylated kinases and proteins in cytoplasmic granules at stages I-II, before the appearance of pre-tangles and NFTs. Most of these granules are not surrounded by LAMP1-positive membranes. Markers of impaired ubiquitin-protesome system, abnormal reticulum stress response, and altered endocytic and autophagic pathways occur in a subpopulation of neurons containing cytoplasmic granules, and they appear later. These observations suggest early phosphorylation of kinases leading to their activation, and resulting in the abnormal phosphorylation of various substrates, including tau, as a main alteration at the first stages of GVD., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Introduction: Human tau seeding and spreading occur following intracerebral inoculation into different gray matter regions of brain homogenates obtained from tauopathies in transgenic mice expressing wild or mutant tau, and in wild-type (WT) mice. However, little is known about tau propagation following inoculation in the white matter. Objectives: The present study is geared to learning about the patterns of tau seeding and cells involved following unilateral inoculation in the corpus callosum of homogenates from sporadic Alzheimer's disease (AD), primary age-related tauopathy (PART: neuronal 4Rtau and 3Rtau), pure aging-related tau astrogliopathy (ARTAG: astroglial 4Rtau with thorn-shaped astrocytes TSAs), globular glial tauopathy (GGT: 4Rtau with neuronal tau and specific tau inclusions in astrocytes and oligodendrocytes, GAIs and GOIs, respectively), progressive supranuclear palsy (PSP: 4Rtau with neuronal inclusions, tufted astrocytes and coiled bodies), Pick's disease (PiD: 3Rtau with characteristic Pick bodies in neurons and tau containing fibrillar astrocytes), and frontotemporal lobar degeneration linked to P301L mutation (FTLD-P301L: 4Rtau familial tauopathy). Methods: Adult WT mice were inoculated unilaterally in the lateral corpus callosum with sarkosyl-insoluble fractions or with sarkosyl-soluble fractions from the mentioned tauopathies; mice were killed from 4 to 7 months after inoculation. Brains were fixed in paraformaldehyde, embedded in paraffin and processed for immunohistochemistry. Results: Tau seeding occurred in the ipsilateral corpus callosum and was also detected in the contralateral corpus callosum. Phospho-tau deposits were found in oligodendrocytes similar to coiled bodies and in threads. Moreover, tau deposits co-localized with active (phosphorylated) tau kinases p38 and ERK 1/2, suggesting active tau phosphorylation of murine tau. TSAs, GAIs, GOIs, tufted astrocytes, and tau-containing fibrillar astrocytes were not seen in any case. Tau deposits were often associated with slight myelin disruption and the presence of small PLP1-immunoreactive globules and dots in the ipsilateral corpus callosum 6 months after inoculation of sarkosyl-insoluble fractions from every tauopathy. Conclusions: Seeding and spreading of human tau in the corpus callosum of WT mice occurs in oligodendrocytes, thereby supporting the idea of a role of oligodendrogliopathy in tau seeding and spreading in the white matter in tauopathies. Slight differences in the predominance of threads or oligodendroglial deposits suggest disease differences in the capacity of tau seeding and spreading among tauopathies.

Glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2), a major modulator of glutamate homeostasis in astrocytes, is assessed in post-mortem human brain samples of frontal cortex area 8 in advanced stages of Alzheimer disease (AD) and terminal stages of dementia with Lewy bodies (DLB) in order to gain understanding of astrogliopathy in diseases manifested by dementia. Glial fibrillary acidic protein (GFAP) mRNA expression is significantly increased in AD but not in DLB, whereas GLT1 , vesicular glutamate transporter 1 ( vGLUT1 ) and aldehyde dehydrogenase 1 family member 1 ( ALDH1L1 ) are not modified in AD and DLB when compared with controls. GLT1 protein levels are not altered in AD and DLB but GFAP and ALDH1L1 are significantly increased in AD, and GFAP in DLB. As a result, a non-significant decrease in the ratio between GLT1 and GFAP, and between GLT1 and ALDH1L1, is found in both AD and DLB. Double-labeling immunofluorescence and confocal microscopy revealed no visible reduction of GLT1 immunoreactivity in relation to β-amyloid plaques in AD. These data suggest a subtle imbalance between GLT1, and GFAP and ALDH1L1 expression, with limited consequences in glutamate transport.

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those taking place in oviducts, including contraction, beating of cilia, and maintenance of fluid composition that, in turn, influences sperm capacitation and hyperactivation, as well as oocyte and embryo nourishing. Ecto-nucleotidases are the enzymes that regulate extracellular ATP and adenosine levels, thus playing a role in reproduction. We have optimized a convenient method for characterizing ecto-nucleotidases that simultaneously localizes the protein and its associated enzyme activity in the same tissue slice and characterizes ecto-nucleotidases in human oviducts. The technique combines immunofluorescence and in situ histochemistry, allowing precise identification of ecto-nucleotidases at a subcellular level. In oviducts, remarkably, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, with the ability to hydrolyze ATP to AMP, are expressed in ciliated epithelial cells but with different subcellular localization. Ecto-5'nucleotidase/CD73 is also expressed apically in ciliated cells. CD73, together with alkaline phosphatase, also expressed apically in oviductal epithelium, complete the hydrolysis sequence by dephosphorylating AMP to adenosine. The concerted action of these enzymes would contribute to the local increase of adenosine concentration necessary for sperm capacitation. The use of this method would be an asset for testing new potential therapeutic drugs with inhibitory potential, which is of great interest presently in the field of oncology and in other clinical disciplines.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare disease characterized by the deposition of multiple intracytoplasmic neuronal inclusions that contain mutated neuroserpin. Tg-Syracuse (Tg-Syr) mice express Ser49Pro mutated neuroserpin and develop clinical and neuropathological features of human FENIB. We used 8-, 34-, 45- and 80-week-old Tg-Syr mice to characterize neuroinflammation and the unfolded protein response (UPR) in a neurodegenerative disease in which abnormal protein aggregates accumulate within the endoplasmic reticulum (ER). There were scattered neuroserpin inclusions in Tg-Syr mice at 8 weeks of age; the numbers of neurons involved and the amount of neuroserpin per neuron increased with age throughout the CNS to 80 weeks of age; no similar inclusions were found in wild type (Tg-WT) mice at any age. Increases in numbers of astrocytes and microglia occurred at advanced disease stages. Among 22 markers in 80-week-old Tg-Syr mice, only II1b and II10rb mRNAs in the somatosensory cortex and CxCl10 and Il10rb mRNAs in the olfactory bulb were upregulated when compared with Tg-WT mice indicating a limited relationship between neuroserpin inclusions and inflammatory responses. The changes were accompanied by a transient increase in expression of Xbp1 spliced at 45 weeks and increased ERdJ4 mRNAs at 80 weeks. The sequestration of UPR activators GRP78 and GRP94 in neuroserpin inclusions might explain the limited UPR responses despite the accumulation of neuroserpin in the ER in this FENIB mouse model.

Tauopathies are degenerative diseases characterized by the accumulation of phosphorylated tau in neurons and glial cells. With some exceptions, tau deposits in neurons are mainly manifested as pretangles and tangles unrelated to the tauopathy. It is thought that abnormal tau deposition in neurons occurs following specific steps, but little is known about the progression of tau pathology in glial cells in tauopathies. We compared tau pathology in different astrocyte phenotypes and oligodendroglial inclusions with that in neurons in a large series of tauopathies, including progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick disease, frontotemporal lobar degenerations (FTLD) associated with mutations in the tau gene, globular glial tauopathy (GGT), and tauopathy in the elderly. Our findings indicate that disease-specific astroglial phenotypes depend on i) the primary amino acid sequence of tau (mutated tau, 3Rtau, and 4Rtau); ii) phospho-specific sites of tau phosphorylation, tau conformation, tau truncation, and ubiquitination in that order (which parallel tau modifications related to pretangle and tangle stages in neurons); and iii) modifications of the astroglial cytoskeleton. In contrast to astrocytes, coiled bodies in oligodendrocytes have similar characteristics whatever the tauopathy, except glial globular inclusions in GGT, and coiled bodies and globular oligodendroglial inclusions in FTLD-tau/K317M. These observations indicate that tau pathology in glial cells largely parallels, but is not identical to, that in neurons in many tauopathies.

Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5'-nucleotidase (5'NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility.

Parkinson disease (PD) is no longer considered a complex motor disorder but rather a systemic disease with variable nonmotor deficits that may include impaired olfaction, depression, mood and sleep disorders, and altered cortical function. Increasing evidence indicates that multiple metabolic defects occur in regions outside the substantia nigra, including the cerebral cortex, even at premotor stages of the disease. We investigated changes in gene expression in the frontal cortex in PD patient brains using a transcriptomics approach. Functional genomics analysis indicated that cortical olfactory receptors (ORs) and taste receptors (TASRs) are altered in PD patients. Olfactory receptors OR2L13, OR1E1, OR2J3, OR52L1, and OR11H1 and taste receptors TAS2R5 and TAS2R50 were downregulated, but TAS2R10 and TAS2R13 were upregulated at premotor and parkinsonian stages in the frontal cortex area 8 in PD patient brains. Furthermore, we present novel evidence that, in addition to the ORs, obligate downstream components of OR function adenylyl cyclase 3 and olfactory G protein (Gαolf), OR transporters, receptor transporter proteins 1 and 2 and receptor expression enhancing protein 1, and OR xenobiotic removing UDP-glucuronosyltransferase 1 family polypeptide A6 are widely expressed in neurons of the cerebral cortex and other regions of the adult human brain. Together, these findings support the concept that ORs and TASRs in the cerebral cortex may have novel physiologic functions that are affected in PD patients.

Prolonged exposure to high glucose concentration alters the expression of a set of proteins in pancreatic β-cells and impairs their capacity to secrete insulin. The cellular and molecular mechanisms that lie behind this effect are poorly understood. In this study, three either in vitro or in vivo models (cultured rat pancreatic islets incubated in high glucose media, partially pancreatectomized rats, and islets transplanted to streptozotozin-induced diabetic mice) were used to evaluate the dependence of the biological model and the treatment, together with the cell location (insulin granule or plasma membrane) of the affected proteins and the possible effect of sustained insulin secretion, on the glucose-induced changes in protein expression. In all three models, islets exposed to high glucose concentrations showed a reduced expression of secretory granule-associated vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptobrevin/vesicle-associated membrane protein 2 and cellubrevin but minor or no significant changes in the expression of the membrane-associated target-SNARE proteins syntaxin1 and synaptosomal-associated protein-25 and a marked increase in the expression of synaptosomal-associated protein-23 protein. The inhibition of insulin secretion by the L-type voltage-dependent calcium channel nifedipine or the potassium channel activator diazoxide prevented the glucose-induced reduction in islet insulin content but not in vesicle-SNARE proteins, indicating that the granule depletion due to sustained exocytosis was not involved in the changes of protein expression induced by high glucose concentration. Altogether, the results suggest that high glucose has a direct toxic effect on the secretory pathway by decreasing the expression of insulin granule SNARE-associated proteins.

Protein aggregate myopathies, including myofibrillar myopathies and sporadic inclusion body myositis (sIBM), are characterized by abnormal protein aggregates composed of various muscular and ectopic proteins. Previous studies have shown the crucial role ofdysregulated transcription factors such as neuron-restrictive silencerfactor in the expression of aberrant proteins in myotilinopathies. Here, we assessed possible aberrant expression of TAR DNA-bindingprotein 43 (TDP-43), another factor involved in transcription regulation. TDP-43-immunoreactive intracytoplasmic inclusions were seen in all cases examined of myotilinopathy, desminopathy, and sIBM, and in 1 case of inclusion body myositis with Paget disease of bone and frontotemporal degeneration (IBMPFD). TAR DNA-binding protein 43 colocalized with myotilin and valosin in myotilinopathies and IBMPFD, respectively, but only occasionally colocalized with ubiquitin in myotilinopathies, desminopathies, sIBM, and IBMPFD; this indicates that accumulated TDP-43 is largely not ubiquitinated. Moreover, phosphorylated TDP-43 at Ser403/404 and Ser409/410 accumulated in the cytoplasm of vulnerable fibers but did not always colocalize with nonphosphorylated TDP-43. Cytoplasmic deposition was accompanied by decreased TDP-43 localization in the nuclei of affected fibers. These findings indicate that TDP-43 not only is another protein accumulated in myofibrillar myopathies, sIBM, and IBMPFD but also likely has a role through altered microRNA processing in the abnormal protein production, modification, and accumulation in protein aggregate myopathies.

Double-labeling immunofluorescence and confocal microscopy have been used to learn about the local relationship between amyloid, mitochondria, and cytochrome c oxidase (COX) in dystrophic neurites of senile plaques in the frontal cortex in Alzheimer's disease (AD). Dystrophic neurites surrounding amyloid plaques are filled with mitochondrial porin-immunoreactive structures. In contrast with tangle-bearing and non-tangle-bearing neurons, which express mitochondrial porin and COX subunit 4, porin-immunoreactive neurites of senile plaques lack COX subunit 4. Parallel western blot studies in mitochondria-enriched fractions of the frontal cortex in the same cases disclosed reduced expression levels of COX, but not of prohibitin, in AD stages VB/C of Braak. Co-localization of porin and lysosomal associated protein 1, as revealed by double-labeling immunofluorescence and confocal microscopy, suggests that mitochondria may be engulfed by lysosomes in dystrophic neurites. These findings support a local link between amyloid deposition, abnormal mitochondria and impaired respiratory chain function (resulting from decrease of COX expression) in dystrophic neurites of senile plaques in AD.

Recent studies have shown the co-existence of alpha-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 alpha-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 alpha-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 alpha-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 alpha-synuclein and UBB+1 was found in only a few neurons. These findings indicate that alpha-synuclein and phosphorylated tau co-occur in certain brain regions in cases of combined MSA and AD and that the proteasomal pathways differ between alpha-synuclein- and pTau-bearing neurons.

Abnormalities in the cerebral microvasculature are common in Alzheimer disease (AD). Expression levels of the water channels aquaporin 1 and aquaporin 4 (AQP1, AQP4) were examined in AD cases by gel electrophoresis and Western blotting, and densitometric values normalized with beta-actin were compared with corresponding values in age-matched controls processed in parallel. In addition, samples of cases with Pick disease (PiD) were examined for comparative purposes. A significant increase in the expression levels of AQP1 was observed in AD stage II (following Braak and Braak classification). Individual variations were seen in advanced stages which resulted in non-significant differences between AD stages V-VI and age-matched controls. No differences in AQP1 levels were observed between familial AD cases (FAD, all of them at advanced stages) and corresponding age-matched controls. Immunohistochemistry showed increased AQP1 in astrocytes at early stages of AD. Double-labelling immunofluorescence and confocal microscopy disclosed AQP1 immunoreactivity at the cell surface of astrocytes which were recognized with anti-glial fibrillary acidic protein antibodies. No differences in the levels of AQP4 were observed in AD, FAD and PiD when compared with corresponding controls. These results indicate abnormal expression of AQP1 in astrocytes in AD, and they add support to the idea that abnormal regulation of mechanisms involved in the control of water fluxes occurs at early stages in AD.

During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.

Hyper-phosphorylated tau deposition in Pick bodies and neuron loss are major hallmarks of Pick's disease (PiD). However, there is no regional correlation between neuron loss and Pick bodies, as illustrated in dentate gyrus, where Pick bodies are present in almost every neuron, whereas cell death, if present, is not a major event. In order to better understand the possible role of selected transcription factors and members of the caspase family in cell death and cell survival, immunohistochemistry to c-Fos, c-Jun, CREB-1, ATF-2; c-Fos(P), c-Jun(P) and CREB-1(P); and procaspase-8, procaspase-3 and active (cleaved) caspase-3 immunohistochemistry was carried out in the frontal cortex and hippocampus. Increased expression of c-Fos, c-Jun, CREB-1 and ATF-2 was observed in PiD cases. Increased c-Fos(P), c-Jun(P) and CREB-1(P) was also found in the nuclei of neurons in diseased brains. Interestingly, c-Fos but not c-Fos(P) co-localized in many Pick bodies, as observed by double labelling-immunofluorescence and confocal microscopy. Pro-caspase-8 and pro-caspase-3 were increased in PiD. Moreover, granular active caspase-3 was observed in the nuclei as was aggregated active caspase-3 in the cytoplasm of neurons in PiD. Finally, double-labelling immunofluorescence and confocal microscopy disclosed co-localization of cytoplasmic active caspase-3 only in neurons with Pick bodies. Together, these findings show an increased expression of selected transcription factors and active (phosphorylated) forms in PiD, c-Fos sequestration in Pick bodies, and increased active caspase-3 expression in relation with Pick bodies. Since all these findings were observed equally in neurons of both vulnerable regions (frontal cortex) and resistant regions (dentate gyrus), it may be suggested that transcription factors are only barely related with cell death. Active caspase-3 is associated with tau deposition in Pick bodies, but it is not a marker of cell death in the dentate gyrus in PiD. The present findings are in line with the previous studies showing tau products cleaved by caspase-3, as recognized by specific tau-cleaved antibodies, in Alzheimer's disease and other tauopathies.

The synaptobrevin/vesicle-associated membrane protein (VAMP) family of proteins, which are essential for neurotransmitter release, are the vesicle donor soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins first described in synaptic vesicles at nerve terminals. Two synaptobrevin/VAMP isoforms are involved in calcium-dependent synaptic vesicle exocytosis, synaptobrevin/VAMP 1 and synaptobrevin/VAMP 2. However, the functional significance of these two highly homologous isoforms remains to be elucidated. Here, we used immunohistochemical, immunofluorescence and confocal microscope techniques to localize the two synaptobrevin/VAMP isoforms in rat brain areas, particularly in nerve terminals. Our results show that the two isoforms are present in the rat central nervous system and that their expression overlaps in some areas. However, a distinct distribution pattern was detected. Synaptobrevin/VAMP 2 is the most abundant isoform in the rat brain and is widely distributed. Although synaptobrevin/VAMP 1 is less abundant, it is the main isoform in particular brain areas (e.g. zona incerta at the subthalamus or nerve terminals surrounding thalamic neurons). The colocalization of synaptophysin with synaptobrevin/VAMP 1 demonstrates the presence of this isoform in subsets of nerve terminals. These results indicate that each synaptic vesicle donor SNARE protein isoform could have a specialized role in the neurosecretory process.

Differentiation of 3T3-L1 cells into adipocytes presupposes the expression of the glucose transporter isoform GLUT4 and the acquisition of insulin-dependent GLUT4 translocation from intracellular storage vesicles to plasma membrane. This ability to translocate GLUT4 depends on the presence of a set of proteins of the SNARE category that are essential in the fusion step. The expression and levels of some of these SNARE proteins are altered during 3T3-L1 differentiation. Levels of the v-SNARE protein cellubrevin and of the t-SNARE protein syntaxin 4 were increased in this process in parallel to GLUT4. However, the levels of SNAP-23, another t-SNARE, were maintained during differentiation. Immunofluorescence images of SNAP-23 showed the initial distribution of this protein in a perinuclear region before differentiation and its redistribution towards plasma membrane in the adipocyte form. These results suggest a capital role in the expression levels and cellular distribution, during 3T3-L1 differentiation, of SNARE proteins involved in the late steps of GLUT4 translocation.