A model of alcoholic liver disease based on different hepatotoxics leading to liver cancer.

The worst-case scenario related to alcoholic liver disease (ALD) arises after a long period of exposure to the harmful effect of alcohol consumption along with other hepatotoxics. ALD encompasses a broad spectrum of liver-associated disorders, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Based on the chronic administration of different hepatotoxics, including ethanol, sucrose, lipopolysaccharide, and low doses of diethylnitrosamine over a short period, here we aimed to develop a multiple hepatotoxic (MHT)-ALD model in the mouse that recapitulates the human ALD-associated disorders. We demonstrated that the MHT-ALD model induces ADH1A and NXN, an ethanol metabolizer and a redox-sensor enzyme, respectively; promotes steatosis associated with the induction of the lipid droplet forming FSP27, inflammation identified by the infiltration of hepatic neutrophils-positive to LY-6G marker, and the increase of MYD88 level, a protein involved in inflammatory response; and stimulates the early appearance of cellular senescence identified by the senescence markers SA-ß-gal activity and p-H2A.XSer139. It also induces fibrosis associated with increased desmin, a marker of hepatic stellate cells whose activation leads to the deposition of collagen fibers, accompanied by cell death and compensatory proliferation revealed by increased CASP3-mediated apoptosis, and KI67- and PCNA-proliferation markers, respectively. It also induces histopathological traits of malignancy and the level of the HCC marker, GSTP1. In conclusion, we provide a useful model for exploring the chronological ALD-associated alterations and stages, and addressing therapeutic approaches.

Association of α-1-Antichymotrypsin Expression with the Development of Conformational Changes of Tau Protein in Alzheimer's Disease Brain.

In Alzheimer's disease (AD), two mutually exclusive amino-terminal-dependent conformations have been reported to occur during the aggregation of Tau protein into neurofibrillary tangles (NFTs). An early conformation of full-length Tau, involving the bending of the amino terminus over the third repeated domain, is recognized by the Alz-50 antibody, followed by a second conformation recognized by Tau-66 antibody that depends on the folding of the proline-rich region over the third repeated domain in a molecule partially truncated at the amino- and carboxyl-termini. α-1-antichymotrypsin (ACT) is an acute phase serum glycoprotein that accumulates abnormally in the brain of AD patients, and since it is considered to promote the in vitro and in vivo aggregation of amyloid-ß, we here seek further evidence that ACT may also contribute to the abnormal aggregation of Tau in AD. By analyzing brain samples from a population of AD cases under immunofluorescence and high-resolution confocal microscopy, we demonstrate here the abundant expression of ACT in hippocampal neurons, visualized as a granular diffuse accumulation, frequently reaching the nuclear compartment. In a significant number of these neurons, intracellular NFTs composed of abnormally phosphorylated and truncated Tau at Asp421 were also observed to coexist in separated regions of the cytoplasm. However, we found strong colocalization between ACT and diffuse aggregates of Tau-66-positive granules, which was not observed with Alz-50 antibody. These results suggest that ACT may play a role during the development of Tau conformational changes facilitating its aggregation during the formation of the neurofibrillary pathology in AD.

An Extremely Low-Frequency Vortex Magnetic Field Modifies Protein Expression, Rearranges the Cytoskeleton, and Induces Apoptosis of a Human Neuroblastoma Cell Line.

Homogeneous extremely low-frequency electromagnetic fields (ELF-EMFs) alter biological phenomena, including the cell phenotype and proliferation rate. Heterogenous vortex magnetic fields (VMFs), a new approach of exposure to magnetic fields, induce systematic movements on charged biomolecules from target cells; however, the effect of VMFs on living systems remains uncertain. Here, we designed, constructed, and characterized an ELF-VMF-modified Rodin's coil to expose SH-SY5Y cells. Samples were analyzed by performing 2D-differential-gel electrophoresis, identified by MALDI-TOF/TOF, validated by western blotting, and characterized by confocal microscopy. A total of 106 protein spots were differentially expressed; 40 spots were downregulated and 66 were upregulated in the exposed cell proteome, compared to the control cell proteome. The identified spots are associated with cytoskeleton and cell viability proteins, and according to the protein-protein interaction network, a significant interaction among them was found. Our data revealed a decrease in cell survival associated with apoptotic cells without effects on the cell cycle, as well as evident changes in the cytoskeleton. We demonstrated that ELF-VMFs, at a specific frequency and exposure time, alter the cell proteome and structurally affect the target cells. This is the first report showing that VMF application might be a versatile system for testing different hypotheses in living systems, using appropriate exposure parameters.© 2022 Bioelectromagnetics Society.

Inhibitors of DNA topoisomerases I and II applied to Candida dubliniensis reduce growth, viability, the generation of petite mutants and toxicity, while acting synergistically with fluconazole.

The increasing resistance of Candida species to azoles emphasizes the urgent need for new antifungal agents with novel mechanisms of action. The aim of this study was to examine the effect of three DNA topoisomerase inhibitors of plant origin (camptothecin, etoposide and curcumin) on the growth of Candida dubliniensis. The phylogenetic analysis showed a close relationship between the topoisomerase enzymes of C. dubliniensis and Candida albicans. The alignment of the amino acid sequences of topoisomerase I and II of yeasts and humans evidenced conserved domains. The docking study revealed affinity of the test compounds for the active site of topoisomerase I and II in C. dubliniensis. Curcumin and camptothecin demonstrated a stronger in vitro antifungal effect than the reference drugs (fluconazole and itraconazole). Significant synergistic activity between the topoisomerase inhibitors and fluconazole at the highest concentration (750 µM) was observed. Fluconazole induced the petite phenotype to a greater degree than the topoisomerase inhibitors, indicating a tendency to generate resistance. Lower toxicity was found for such inhibitors versus reference drugs on Galleria mellonella larva. The topoisomerase inhibitors exhibited promising antifungal activity, and the DNA topoisomerase enzymes of C. dubliniensis proved to be an excellent model for evaluating new antifungal compounds.

Current Evidence on the Protective Effects of Recombinant Human Erythropoietin and Its Molecular Variants against Pathological Hallmarks of Alzheimer's Disease.

Substantial evidence in the literature demonstrates the pleiotropic effects of the administration of recombinant human erythropoietin (rhEPO) and its molecular variants in different tissues and organs, including the brain. Some of these reports suggest that the chemical properties of this molecule by itself or in combination with other agents (e.g., growth factors) could provide the necessary pharmacological characteristics to be considered a potential protective agent in neurological disorders such as Alzheimer's disease (AD). AD is a degenerative disorder of the brain, characterized by an aberrant accumulation of amyloid ß (Aß) and hyperphosphorylated tau (tau-p) proteins in the extracellular and intracellular space, respectively, leading to inflammation, oxidative stress, excitotoxicity, and other neuronal alterations that compromise cell viability, causing neurodegeneration in the hippocampus and the cerebral cortex. Unfortunately, to date, it lacks an effective therapeutic strategy for its treatment. Therefore, in this review, we analyze the evidence regarding the effects of exogenous EPOs (rhEPO and its molecular variants) in several in vivo and in vitro Aß and tau-p models of AD-type neurodegeneration, to be considered as an alternative protective treatment to this condition. Particularly, we focus on analyzing the differential effect of molecular variants of rhEPO when changes in doses, route of administration, duration of treatment or application times, are evaluated for the improved cellular alterations generated in this disease. This narrative review shows the evidence of the effectiveness of the exogenous EPOs as potential therapeutic molecules, focused on the mechanisms that establish cellular damage and clinical manifestation in the AD.

Early but not late conformational changes of tau in association with ubiquitination of neurofibrillary pathology in Alzheimer's disease brains.

In Alzheimer's disease, tau protein undergoes post-translational modifications including hyperphosphorylation and truncation, which promotes two major conformational changes associated with progressive N-terminal folding. Along with the development of the disease, tau ubiquitination was previously shown to emerge in the early and intermediate stages of the disease, which is closely associated with early tau truncation at aspartic acid 421, but not with a subsequently truncated tau molecule at glutamic acid 391. In the same group of cases, using multiple immunolabeling and confocal microscopy, a possible relationship between the ubiquitin-targeting of tau and the progression of conformational changes adopted by the N-terminus of this molecule was further studied. A comparable number of neurofibrillary tangles was found displaying ubiquitin, an early conformation recognized by the Alz-50 antibody, and a phosphorylation. However, a more reduced number of neurofibrillary tangles were immunoreactive to Tau-66 antibody, a late tau conformational change marker. When double-labeling profiles of neurofibrillary tangles were assessed, ubiquitination was clearly demonstrated in tau molecules undergoing early N-terminal folding, but was barely observed in late conformational changes of the N-terminus adopted by tau. The same pattern of colocalization was visualized in neuritic pathology. Overall, these results indicate that a more intact conformation of the N-terminus of tau may facilitate tau ubiquitination, but this modification may not occur in a late truncated and more compressed folding of the N-terminus of the tau molecule.

Do BDNF and NT-4/5 exert synergistic or occlusive effects on corticostriatal transmission in a male mouse model of Huntington's disease?

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) are trophic factors belonging to the neurotrophin family; in addition to their trophic role, both neurotrophins play an important role in modulating corticostriatal synaptic transmission. Failures in BDNF supply and mitochondrial dysfunction are among the factors involved in the striatal degeneration that occurs in Huntington's disease (HD). While the effects of BDNF have been widely studied in striatal degeneration, the role of NT-4/5 has been less addressed. NT-4/5 does not appear to exert effects similar to those of BDNF in HD. The physiological roles of these molecules in corticostriatal transmission have been evaluated separately, and we have demonstrated that sequential exposure to both neurotrophins results in different modulatory effects on corticostriatal transmission depending on the exposure order. In the present study, we evaluated the effects of BDNF followed by NT-4/5 or NT-4/5 followed by BDNF on corticostriatal synaptic transmission with field recordings in a male mouse model of HD produced by in vivo treatment with the mitochondrial toxin 3-nitropropionic acid. Here, we show that these neurotrophins elicit an antagonistic or synergistic effect that depends on the activation of the truncated isoform or the stimulation of the full-length isoform of the tropomyosin receptor kinase B.

NT-4/5 antagonizes the BDNF modulation of corticostriatal transmission: Role of the TrkB.T1 receptor.

Neurotrophins are related to survival, growth, differentiation and neurotrophic maintenance as well as modulation of synaptic transmission in different regions of the nervous system. BDNF effects have been studied in the striatum due to the trophic role of BDNF in medium spiny neurons; however, less is known about the effects of NT-4/5, which is also present in the striatum and activates the TrkB receptor along with BDNF. If both neurotrophins are present in the striatum, the following question arises: What role do they play in striatal physiology? Thus, the aim of this study was to determine the physiological effect of the sequential application and coexistence of BDNF and NT-4/5 on the modulation of corticostriatal synapses. Our data demonstrated that neurotrophins exhibit differential effects depending on exposure order. BDNF did not modify NT-4/5 effect; however, NT-4/5 inhibited the effects of BDNF. Experiments carried out in COS-7 cells to understand the mechanisms of this antagonism, indicated that NT-4/5 exerts its inhibitory effect on BDNF by upregulating the TrkB.T1 and downregulating the TrkB-FL isoforms of the TrkB receptor.

Fragmentation of the Golgi Apparatus in Neuroblastoma Cells Is Associated with Tau-Induced Ring-Shaped Microtubule Bundles.

Abnormal fibrillary aggregation of tau protein is a pathological condition observed in Alzheimer's disease and other tauopathies; however, the presence and pathological significance of early non-fibrillary aggregates of tau remain under investigation. In cell and animal models expressing normal or modified tau, toxic effects altering the structure and function of several membranous organelles have also been reported in the absence of fibrillary structures; however, how these abnormalities are produced is an issue yet to be addressed. In order to obtain more insights into the mechanisms by which tau may disturb intracellular membranous elements, we transiently overexpressed human full-length tau and several truncated tau variants in cultured neuroblastoma cells. After 48 h of transfection, either full-length or truncated tau forms produced significant fragmentation of the Golgi apparatus (GA) with no changes in cell viability. Noteworthy is that in the majority of cells exhibiting dispersion of the GA, a ring-shaped array of cortical or perinuclear microtubule (Mt) bundles was also generated under the expression of either variant of tau. In contrast, Taxol treatment of non-transfected cells increased the amount of Mt bundles but not sufficiently to produce fragmentation of the GA. Tau-induced ring-shaped Mt bundles appeared to be well-organized and stable structures because they were resistant to Nocodazole post-treatment and displayed a high level of tubulin acetylation. These results further indicate that a mechanical force generated by tau-induced Mt-bundling may be responsible for Golgi fragmentation and that the repeated domain region of tau may be the main promoter of this effect.

Effect of in vivo exposure to ambient fine particles (PM2.5) on the density of dopamine D2-like receptors and dopamine-induced [35S]-GTPγS binding in rat prefrontal cortex and striatum membranes.

Male Sprague-Dawley rats (8-9 weeks-old) were exposed for three days (acute exposure) or eight weeks (subchronic exposure) to purified air or concentrated ambient fine particles, PM2.5 (≤2.5 µm; 15 to 18-fold of ambient air; 370-445 µg/m3). In membranes from rat prefrontal cortex (PFC) or striatum, the density and function of dopamine D2-like receptors (D2Rs) were assessed by [3H]-spiperone binding and dopamine-stimulated [35S]-GTPγS binding, respectively. Glial activation was evaluated by immunoperoxidase labeling of the glial fibrillary acidic protein (GFAP). In the PFC, no significant changes in D2R density or signaling were observed after the acute and subchronic exposure to PM2.5. In the striatum, acute exposure to PM2.5 decreased D2R density, with no effect on signaling efficacy, whereas subchronic exposure did not affect D2R density but reduced signaling efficacy. Both acute and subchronic exposure to PM2.5 induced reactive gliosis in the striatum but not in the PFC. These results indicate that exposure to PM2.5 induces astrocyte activation and alters striatal dopaminergic transmission.

Plasmonic/Magnetic Multifunctional nanoplatform for Cancer Theranostics.

A multifunctional magneto-plasmonic CoFe2O4@Au core-shell nanoparticle was developed by iterative-seeding based method. This nanocargo consists of a cobalt ferrite kernel as a core (Nk) and multiple layers of gold as a functionalizable active stratum, (named as Nk@A after fifth iteration). Nk@A helps in augmenting the physiological stability and enhancing surface plasmon resonance (SPR) property. The targeted delivery of Doxorubicin using Nk@A as a nanopayload is demonstrated in this report. The drug release profile followed first order rate kinetics optimally at pH 5.4, which is considered as an endosomal pH of cells. The cellular MR imaging showed that Nk@A is an efficient T2 contrast agent for both L6 (r2-118.08 mM-1s-1) and Hep2 (r2-217.24 mM-1s-1) cells. Microwave based magnetic hyperthermia studies exhibited an augmentation in the temperature due to the transformation of radiation energy into heat at 2.45 GHz. There was an enhancement in cancer cell cytotoxicity when hyperthermia combined with chemotherapy. Hence, this single nanoplatform can deliver 3-pronged theranostic applications viz., targeted drug-delivery, T2 MR imaging and hyperthermia.

Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling.

Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

Proteolytic cleavage of polymeric tau protein by caspase-3: implications for Alzheimer disease.

Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.

Alterations in the nuclear architecture produced by the overexpression of tau protein in neuroblastoma cells.

Abnormal intracellular aggregation of tau protein is a pathological condition leading to neuronal death in Alzheimer's disease. Fibrillar and nonfibrillar aggregates of tau protein alter the normal functioning of neurons by disturbing important cellular processes and distinct membranous organelles. However, tau-caused alterations in the nuclear compartment are not totally established so far. In our study we evaluated whether tau protein and its Asp421-truncated variant produce alterations in the normal architecture of the nucleus when expressed in cultured neuroblastoma cells. After 48 hours of transfection, significant deformity of the nuclear compartment with extensive lobulations along the nuclear envelope was observed in SH-SY5Y cells expressing either full-length tau or Asp421-truncated tau. This aberrant formation did not involve either nuclear fragmentation or cell death. The lobulated nuclei were devoid of tau protein, which mostly remained in the cytoplasm in a nonfibrillar state. Degradation of nuclear Lamins was not observed in tau-expressing SH-SY5Y cells, and a cell-cycle analysis did not show aberrant chromosome accumulation. Thus multiple division defects leading to multinucleation were discarded. The lobulated nuclei in tau-expressing SH-SY5Y cells seem to more resemble the multilobular phenotype of the nuclear envelope seen in Lamin-mutated cells from those pathological conditions leading to premature aging. Nevertheless, in our tau-expressing cells, the abnormal formation of cortical and perinuclear rings of tubulin generated by tau binding may be a more feasible mechanism of a nuclear-cytoskeleton generating force that causes the nuclear deformation.

Structure and pathology of tau protein in Alzheimer disease.

Alzheimer's disease (AD) is the most common type of dementia. In connection with the global trend of prolonging human life and the increasing number of elderly in the population, the AD becomes one of the most serious health and socioeconomic problems of the present. Tau protein promotes assembly and stabilizes microtubules, which contributes to the proper function of neuron. Alterations in the amount or the structure of tau protein can affect its role as a stabilizer of microtubules as well as some of the processes in which it is implicated. The molecular mechanisms governing tau aggregation are mainly represented by several posttranslational modifications that alter its structure and conformational state. Hence, abnormal phosphorylation and truncation of tau protein have gained attention as key mechanisms that become tau protein in a pathological entity. Evidences about the clinicopathological significance of phosphorylated and truncated tau have been documented during the progression of AD as well as their capacity to exert cytotoxicity when expressed in cell and animal models. This paper describes the normal structure and function of tau protein and its major alterations during its pathological aggregation in AD.

Ubiquitin is associated with early truncation of tau protein at aspartic acid(421) during the maturation of neurofibrillary tangles in Alzheimer's disease.

Pathological processing of tau protein during the formation and maturation of neurofibrillary tangles (NFTs) includes abnormal phosphorylation, conformational changes and truncation of the C-terminus at aspartic-acid(421) (apoptotic product) and glutamic-acid(391) residues. Abnormal phosphorylation and misfolding may serve as recognition signals for ubiquitin-targeting and proteosomal processing. For this reason, we sought to determine whether ubiquitin-targeting of tau is associated with particular tau modifications that herald specific stages of NFTs maturation in the hippocampus of Alzheimer's disease cases. Using multiple tau antibodies, we found that 30% of the total load of NFTs is ubiquitin-associated. As reported previously ubiquitin immunoreactivity was associated with markers of phosphorylated tau in certain NFTs; however, a strong association was also found between ubiquitin and the earliest known truncation event at aspartic-acid(421) . These findings indicate that tau protein in the NFTs may be dually subjected to both apoptotic and proteosomal processing. By contrast ubiquitin immunoreactivity was poorly associated with truncation of tau at glutamic-acid(391) , suggesting that this proteolytic event may be independent of proteosomal activity. It would appear, therefore, that ubiquitin targeting of tau protein occurs at NFTs in the early and intermediate stages of the maturation.

Myotonic dystrophy type 1-associated CTG repeats disturb the expression and subcellular distribution of microtubule-associated proteins MAP1A, MAP2, and MAP6/STOP in PC12 cells.

To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3'-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.

Modeling familial Danish dementia in mice supports the concept of the amyloid hypothesis of Alzheimer's disease.

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between beta-amyloid (Abeta)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Abeta peptides, the lack of codeposition of the two peptides in crosses between ADan- and Abeta-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain.

Lewy body variant of Alzheimer's disease: selective neocortical loss of t-SNARE proteins and loss of MAP2 and alpha-synuclein in medial temporal lobe.

Lewy bodies (LBs) appear in the brains of nondemented individuals and also occur in a range of neurodegenerative disorders, such as dementia with Lewy bodies (DLB) and Parkinson's disease. A number of people with a definite diagnosis of Alzheimer's disease (AD) also exhibit these intraneuronal inclusions in allo- and/or neocortical areas. The latter, referred to as Lewy body variant of AD (LBV), bears a clinical resemblance to AD in terms of age at onset, duration of illness, cognitive impairment, and illness severity. Since the presence of LBs is accompanied by neuronal cytoskeleton changes, it is possible that the latter may influence neuronal connectivity via alterations to the synaptic network. To address this, we examined the expression of synaptic proteins (synaptophysin, syntaxin, SNAP-25, and alpha-synuclein) and two cytoskeletal proteins (tau and MAP2) in the brain tissue of subjects enrolled in a population-based autopsy study (n = 47). They were divided into groups with no memory problems (control group, n = 15), LBV (n = 5), AD devoid of LBs (n = 17), cerebrovascular dementia (n = 3), and mixed dementia (n = 7). The LBV and AD groups had a similar degree of cognitive impairment and neuropathological staging in terms of Braak staging and CERAD score. In comparison with the control group and the dementia groups without LBs, the LBV group had significantly lower levels of syntaxin and SNAP-25 (23%) in the neocortex, and depletion of MAP2 (64%), SNAP-25 (34%), and alpha-synuclein (44%) proteins in the medial temporal lobes. These findings suggest that the t-SNARE complex deficit present in LBV may be associated with the presence of LB-related pathology and may explain the more profound cholinergic loss seen in these patients.

Dystrophin Dp71f associates with components of the beta1-integrin adhesion complex in PC12 cell neurites.

Dystrophin Dp71 has been implicated with cognitive impairment shown by Duchenne muscular dystrophy patients. To study Dp71 neural role, we used PC12 cell line, since these cells differentiate into sympathetic like neurons when stimulated with nerve growth factor Previously in undiferentiated PC12 cells, it was demonstrated that dystrophin Dp71f is a key component of the beta1-integrin adhesion complex that confers proper complex assembly. Since integrin based mediated adhesion is important during neuronal differentiation, it was important to know if dystrophin Dp71f was a structural component of the beta1-integrin adhesion complex in neurites of nerve growth factor stimulated PC12 cells. In the present work, by performing immunofluorescence assays, we determined the association of dystrophin Dp71f with some components of the beta1-integrin adhesion complex such as beta1-integrin subunit, talin, alpha-actinin and vinculin in neurites of nerve growth factor stimulated PC12 cells seeded onto the extracellular matrix protein laminin. The association was stronger in neural growth cones suggesting that dystrophin Dp71f is important for the function that the beta1-integrin complex has during neurite outgrowth.