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Current Alzheimer Research 2020We have recently identified Huntingtin (Htt), the pathogenic protein in Huntington's disease, as a mediator of Alzheimer's disease (AD) pathology in an amyloid precursor...
BACKGROUND
We have recently identified Huntingtin (Htt), the pathogenic protein in Huntington's disease, as a mediator of Alzheimer's disease (AD) pathology in an amyloid precursor protein (APP) knock-in mouse model of AD. That finding prompted us to examine if Htt is accumulated in the brains of AD patients and in which cell type Htt is present in the AD brain.
OBJECTIVE
To investigate whether location and levels of Htt are affected in hippocampus and frontal cortex in AD.
METHODS
Brains from AD patients (n=11) and controls (n=11) were stained for Htt using immunohistochemistry and signal intensity of Htt was quantified and localized in subregions and neurons. Confocal microscopy was used to characterize neuronal Htt localisation and its relationship with tau tangles and astrocytes.
RESULTS
Htt levels were increased in neuronal cells in the granular layer of the dentate gyrus, in CA1 and CA3 in hippocampus and in layer III of the frontal cortex. Htt was found in the soma, perinuclear space, thin neurites and nucleus of pyramidal neurons. Htt was present in neurons containing tau tangles but did not colocalize with astrocytes.
CONCLUSION
Htt accumulates in pyramidal neuron-rich areas including hippocampal subregions associated with memory and frontal cortex layer III. The accumulation of Htt in AD shows distinct cellular and morphological patterns and is not present in astrocytes. Clearly, further research is warranted to elucidate the role of Htt as a mediator of AD pathology and the potential use of Htt as a target in future therapeutic strategies.
Topics: Aged; Aged, 80 and over; Alzheimer Disease; Autopsy; Female; Frontal Lobe; Hippocampus; Humans; Huntingtin Protein; Male
PubMed: 33272184
DOI: 10.2174/1567205017666201203125622 -
Archives of Razi Institute Oct 2021The soft and delicate tissue of the brain, which is the center of our coordination, is protected by its surrounding layers. The disruption of these layers results in...
The soft and delicate tissue of the brain, which is the center of our coordination, is protected by its surrounding layers. The disruption of these layers results in complicated situations and serious health problems. The brain has three protective layers of bone or skull tissue, the blood tissue layer, and finally the meningeal layer. The layer of blood tissue contains the blood vessels that are located between the skull and the meningeal membranes. If germs or foreign matter enter the fluid through the blood vessels under any circumstances and cause infection, the bones that protect the meninges will break and cause tissue damage. The present study aimed to assess the histological and immunohistochemical characteristics of the brain of rats that underwent induced acute purulent pneumococcal meningitis after antibiotic therapy with Ceftriaxone. A number of 20 white adult male Wistar rats were assigned to three groups. The first group (n=5) regarded as the control were injected with a saline solution into the subarachnoid space in an equivalent amount. The second and third groups of rats (n=5 and 10, respectively) were infected with acute purulent meningitis by the injection of 10 μl of Streptococcus pneumoniae (S. pneumonia) suspension into the subarachnoid space of the brain using a 23-G needle. The various areas of the brains of rats after meningitis induced by S. pneumoniae were examined after the treatment with Ceftriaxone. The S. pneumoniae culture was injected into the subarachnoid space in the area of the rhomboid fossa. Treatment started 18 h after the injection. On day 10, a repeated puncture was performed with the analysis of cerebrospinal fluid in order to confirm the absence of meningitis; thereafter, the animals were taken out of the experiment. No signs of meningitis were found on histological examination. Mild perivascular and pericellular focal edema were revealed with signs of overload of the lymphatic system in the brain and focal ischemic changes in neurons. The investigation of expression with caspase-3 revealed a positive reaction of individual neurons. A positive reaction with antibodies to NeuN and Doublecortin was detected in most neurons; moreover, Glial fibrillary acidic protein (GFAP)-positive astrocytes and their processes were visualized in all layers of the brain substance. The reaction with neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), CD 31, and CD 34 was negative. Typical structure and pictures pointed to an intact brain and purulent meningitis in the first and second groups. The microscopic image and the changes revealed during immunohistochemistry by dual corticosteroid antibodies and neuronal nuclear protein were characterized by predominantly cytoplasmic and perinuclear reactions, respectively. Some neurons are positive for caspase-3 and are related to changes in the characteristic of premature aging.
Topics: Animals; Male; Rats; Anti-Bacterial Agents; Brain; Ceftriaxone; Meningitis, Pneumococcal; Rats, Wistar
PubMed: 35096336
DOI: 10.22092/ari.2021.355885.1733 -
Journal of Virology Dec 2019The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the...
The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the cytoplasm by facilitating the budding of capsids into the perinuclear space (PNS). The NEC of alphaherpesvirus can induce the formation of virion-like vesicles from the nuclear membrane in the absence of other viral proteins. However, whether the NEC of gammaherpesvirus harbors the ability to do so in mammalian cells remains to be determined. In this study, we first constructed open reading frame 67 (ORF67)-null and ORF69-null mutants of murine gammaherpesvirus 68 (MHV-68) and demonstrated that both ORF67 and ORF69 play critical roles in nuclear egress and hence viral lytic replication. Biochemical and bioimaging analyses showed that ORF67 and ORF69 interacted with each other and were sufficient to induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. Thus, we designated ORF67 and ORF69 components of MHV-68 NEC. Furthermore, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 through homology modeling and verified their function in nuclear egress, providing insights into the molecular basis of NEC formation in gammaherpesviruses. Increasing amounts of knowledge indicate that the nuclear egress complex (NEC) is critical for the nuclear egress of herpesvirus capsids, which can be viewed as a vesicle-mediated transport pathway through the nuclear membrane. In this study, we identified open reading frame 67 (ORF67) and ORF69 as components of the NEC in murine gammaherpesvirus 68 (MHV-68) and demonstrated that they efficiently induce virion-like vesicles from the nuclear membrane in mammalian cells. This is the first time that the NEC of a gammaherpesvirus has been found to demonstrate such an essential characteristic. In addition, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 as well as nuclear egress. Notably, these amino acids are conserved in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), providing a structural basis to design antigammaherpesvirus drugs.
Topics: Active Transport, Cell Nucleus; Animals; Capsid; Cell Nucleus; Cytoplasm; DNA, Viral; Gammaherpesvirinae; HEK293 Cells; HeLa Cells; Herpesviridae; Herpesviridae Infections; Herpesvirus 4, Human; Herpesvirus 8, Human; Humans; Loss of Function Mutation; Mice; Nuclear Envelope; Open Reading Frames; Viral Envelope Proteins; Viral Proteins; Virion; Virus Replication
PubMed: 31554685
DOI: 10.1128/JVI.01422-19 -
Biochimica Et Biophysica Acta.... Jan 2022Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to...
Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.
Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Chick Embryo; Intrinsically Disordered Proteins; Lysosomes; Microscopy, Electron, Transmission; Mitochondria; Nuclear Envelope; Proteome; Zebrafish
PubMed: 34655689
DOI: 10.1016/j.bbamcr.2021.119161 -
Genetics Jul 2021The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each...
The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.
Topics: Animals; Cell Nucleus; Chromatin; Chromatin Immunoprecipitation Sequencing; Drosophila melanogaster; Green Fluorescent Proteins; Histone Code; Organ Specificity
PubMed: 34022041
DOI: 10.1093/genetics/iyab079 -
Histochemistry and Cell Biology Nov 2019Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's...
Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time-space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.
Topics: 3T3 Cells; Animals; Cells, Cultured; Dental Enamel Proteins; Hypoxia-Inducible Factor 1; Immunohistochemistry; Mice; Osteogenesis; Transcription Factors
PubMed: 31520138
DOI: 10.1007/s00418-019-01813-4 -
Toxicology and Industrial Health Feb 2020In this study, the effects of a potent antioxidant, selenium, on apoptosis induced by acrolein, a cytotoxic and genotoxic environmental pollutant, were investigated by... (Comparative Study)
Comparative Study
In this study, the effects of a potent antioxidant, selenium, on apoptosis induced by acrolein, a cytotoxic and genotoxic environmental pollutant, were investigated by immunohistochemical and electron microscopic methods. One hundred adult male Wistar albino rats were used in the study. The rats were divided into four main groups: control, acrolein, selenium, and acrolein + selenium. The animals in the experimental groups were given 1 mg/kg/day selenium and 4 mg/kg/day acrolein daily for 7 days by gavage. After drug administration, each group was divided into subgroups according to the time they were to be euthanized: 12th hour, 1st, 2nd, 3rd, and 5th day. The rats in each group at the determined time were euthanized and their livers were removed. Routine histological procedures were performed for light and electron microscopy examinations. After applying the Terminal Deoxynucleotidyl Transferase dUTP nick end labeling assay on the liver sections, apoptotic index values were calculated. Comparing the liver sections of the rats in the acrolein group and the control group, acrolein was found to cause a significant increase in the apoptotic index. The apoptotic index values of the acrolein + selenium group decreased compared to the acrolein group. In the electron microscopic examinations, apoptotic findings were observed in the liver tissues of the rats given acrolein, such as chromatin condensation in the nucleus of hepatocytes, dilatations in the perinuclear space, and cytoplasmic vacuolization. These apoptotic findings were not observed in the acrolein + selenium group after the 12th hour. These findings show that selenium may potentially be useful as a protective agent for people exposed to acrolein.
Topics: Acrolein; Animals; Antioxidants; Apoptosis; Euthanasia, Animal; Liver; Male; Rats; Rats, Wistar; Selenium
PubMed: 32279646
DOI: 10.1177/0748233720909043 -
Cellular and Molecular Neurobiology Nov 2023Isolated exposure to intermittent hypoxia and permissive hypercapnia activates signaling mechanisms that induce ultrastructural changes in mitochondria and endoplasmic...
Isolated exposure to intermittent hypoxia and permissive hypercapnia activates signaling mechanisms that induce ultrastructural changes in mitochondria and endoplasmic reticulum, accompanied by the development of maximal ischemic tolerance in neurons under the combined influence of these factors. However, there are a lack of data on the combined impact of these factors on the ultrastructure of neuronal organelles. The present study aims to comparatively assess the ultrastructural changes in neurons following isolated and combined exposure to hypoxia and hypercapnia, as well as to correlate these changes with the neuroprotective potential previously observed for these factors. Following a 15-session course of 30-min exposures to permissive hypercapnia (P ≈ 50 mmHg) and/or normobaric hypoxia (P ≈ 150 mmHg), morphometric assessment was conducted to evaluate the extent of ultrastructural changes in hippocampal neurons (mitochondria, perinuclear space, and granular endoplasmic reticulum). It was found that in hippocampal neurons from the CA1 region, permissive hypercapnia resulted in increased mitochondrial size, expansion of membranous compartments of the granular endoplasmic reticulum, and perinuclear space. Normobaric hypoxia affected only mitochondrial size, while hypercapnic hypoxia specifically widened the perinuclear space. These ultrastructural changes objectively reflect varying degrees of the influence of hypoxia and hypercapnia on organelles responsible for energy metabolism, anti-apoptotic, and synthetic functions of neurons. This confirms the effect of potentiation of their neuroprotective effects under combined exposure and highlights the dominant role of the hypercapnic component in this mechanism.
Topics: Humans; Hypercapnia; Hypoxia; Neurons; Cerebral Cortex; Hippocampus
PubMed: 37716927
DOI: 10.1007/s10571-023-01407-8 -
Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus.The Journal of Cell Biology Feb 2022In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and...
In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.
Topics: Autophagy; Autophagy-Related Proteins; Chromosomes, Fungal; Nuclear Envelope; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35061008
DOI: 10.1083/jcb.202103178 -
Pharmaceuticals (Basel, Switzerland) Jun 2022Linearolactone (LL) is a -clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the...
Linearolactone (LL) is a -clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand-protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from . LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.
PubMed: 35890108
DOI: 10.3390/ph15070809