Cytophotometric Evidence of Non-S-Phase Extra-Dna In Human Neuronal Nuclei

After Feulgen staining with acriflavine-Schiff, the DNA content of glial and neuronal nuclei from various sites of the human CNS (pre- and post-central gyrus, cerebellar cortex and spinal cord) were determined by fluorescence cytophotometry. the specimens were obtained from twelve adult human autopsy cases. Glial cell nuclei always revealed a biomodal DNA distribution pattern with a large 2c and a smaller 4c peak. the 4c peak was most prominent in the cerebellum. A few 8c glial nuclei were found. Neuronal cell nuclei disclosed unimodal DNA histograms with hyperdiploid means in the range 2.2–2.5c (1.8–2.9c for the individual populations). Tetraploid 4c DNA values were not observed, neither in Purkinje cells, nor in pyramidal cells. In eleven out of a total of forty-four slides the higher DNA means of neuronal nuclei were found to be statistically significant (P < 0.05) when compared with a population of 2c hepatocytes on the same slide. The results indicate the existence of some ‘extra DNA’ in human neuronal cell nuclei, the biological significance of which has still to be elucidated. It is however, suggested that it may play an important role in the functional activity of the CNS.     

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells

Abstract: A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-associated protein-43 as well as neuronal polarity markers such as tau and microtubule-associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.     

Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

A quantitative comparison of the hominoid thalamus: II. Limbic nuclei anterior principalis and lateralis dorsalis

Structures in the limbic system are commonly thought to be similar in form and function in all mammalian brains. In the study reported here, two thalamic limbic nuclei, N. anterior principles and N. lateralis dorsalis, were compared among a group of extant of extant hominoids. The nuclear volumes, neuronal densities, number of neurons per nucleus, and volumes of neuronal perikarya were measured. Humans have much larger nuclei but the nuclei constitute a similar proportion of the whole thalamus as found in the other hominoids. Whereas the human limbic nuclei were observed to have a decrease in the densities of nerve cells compared with those of the other hominoids, this difference is less than that found in most other thalamic nuclei. Consequently the estimated number of neurons is much higher for humans. The total number of neurons best separates the human limbic nuclei from those of the other hominoids. This preliminary study suggests that during hominid evolution neurons were preferentially added to the limbic nuclei of the thalamus.     

Application of neuronal cell culture in human degenerative brain disorders

Neuronal cell culture system has been used for the study of pathochemical evaluations in human degenerative brain disorders, particularly for Krabbe's disease and neuronal ceroid lipofuscinosis. To understand the pathochemistry of Krabbe's disease, we added psychosine into neuronal cell cultures and psychosine treated cells showed the destruction of cytoskeleton and pathy intracellular changes. Electron microscopic finding showed the swelling of the mitochondria. Oligodendrocytes and Schwann cells were isolated from the brains and sciatic nerve of twitcher mouse as an authentic murine model of globoid cell leukodystrophy. Oligodendroglial cells cultured for 22 days were stained by anti-galactocerebroside antibodies. In twitcher oligodendrocyte processes were wirelike and progressively degenerated and there were few membranous expansion. Schwann cells from twitcher could not elongated their processes. These data suggest that psychosine might be important factor to result in these pathological conditions. Furthermore, we studied the effect of protease inhibitors, E-64 on dissociated primary cultures from fetal rat brain. After treated with E-64 in a concentration from 0.1-50 micrograms/ml, numerous cytoplasmic accumulations appeared in neuronal cells. These morphological pictures resemble with those of neuronal ceroid lipofuscinosis, Batten disease. We will discuss the relationship between the deficiency of catepsin H in Batten disease and inclusion bodies found in E-64.     

Nuclear tau, a key player in neuronal DNA protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.     

Neurotoxicity of 25-OH-Cholesterol on NGF-Differentiated PC12 Cells

PC12 cells induced to differentiate with nerve growth factor were used to study the neurotoxicity of 25-OH-cholesterol. This agent induced a dose- and time-dependent cell death in neuronal PC12 cells. Cells treated with this agent showed condensed nuclei, a morphology similar to that of cells dying of programmed cell death. However, agents known to prevent neuronal programmed cell death (cyclic AMP, KCl, aurintricarboxylic acid, and cycloheximide) failed to prevent the 25-OH-cholesterol-mediated cytotoxicity. On the other hand, cell death induced by 25-OH-cholesterol was prevented by treatment with vitamin E and methyl-beta-cyclodextrin. In contrast to observations made in other cell types, whole-cell patch clamp recording of neuronal PC12 cells revealed that treatment with 25-OH-cholesterol did not significantly alter calcium influx through voltage-dependent channels. These results provide the first characterization of the toxicity of cholesterol oxides toward neuronal PC12 cells, which should be useful in future studies on the interactions between cholesterol oxides and cells from the nervous system.     

Development of human cerebellar nuclei. Morphometric study

The morphometric development of the human cerebellar nuclei was examined in 9 fetuses (16-40 weeks of gestation; WG), an infant (2 months old) and 2 adults (16 and 63 years old). With the morphological observation of serial sections of the brain containing the cerebellar nuclei, the authors measured sections to get several morphometric parameters: the volume of nuclear column and number, packing density and cell body area of neurons. Each nucleus (dentate, emboliform, globose and fastigial nucleus) was recognized even at 16 WG. Nerve cells containing Nissl bodies were observed in all nuclei after 23 WG. Degenerative changes were detected in some neurons for every nucleus at 21 and 23 WG. Three stages were observed in the developmental course of nuclear volume and neuronal packing density: the primary or undifferentiated stage at 16 WG, the secondary stage with variability at 21-32 WG and the tertiary stage with monotonous increase (nuclear volume) or gradual decrease (neuronal packing density) after 35 WG. No significant correlation between neuronal number and gestational age was noticed for every nucleus. The analysis of cell body area (neuronal size) demonstrated that the dentate neurons developed after the intermediate or fastigial neurons. It is concluded that there is a critical period between slightly before 20 WG and slightly after 30 WG, matched with the secondary stage in the development of the cerebellar nuclei.     

Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts

We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.     

EGF and TGF-alpha stimulate retinal neuroepithelial cell proliferation in vitro.

Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-alpha, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces amyloid-β42 neurotoxicity in vitro

In this study, we found that expression and secretion of galectin-3 (GAL-3) were upregulated by amyloid-β42 (Aβ42) exposure in human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) without cell death. Aβ42-exposed rat primary cortical neuronal cells co-treated with recombinant GAL-3 were protected from neuronal death in a dose-dependent manner. hUCB-MSCs were cocultured with Aβ42-exposed rat primary neuronal cells or the neuroblastoma cell line, SH-SY5Y in a Transwell chamber. Coculture of hUCB-MSCs reduced cell death of Aβ42-exposed neurons and SH-SY5Y cells. This neuroprotective effect of hUCB-MSCs was reduced significantly by GAL-3 siRNA. These data suggested that hUCB-MSC-derived GAL-3 is a survival factor against Aβ42 neurotoxicity.     

Astroglial regulation of apolipoprotein E expression in neuronal cells. Implications for Alzheimer's disease

Although apolipoprotein (apo) E is synthesized in the brain primarily by astrocytes, neurons in the central nervous system express apoE, albeit at lower levels than astrocytes, in response to various physiological and pathological conditions, including excitotoxic stress. To investigate how apoE expression is regulated in neurons, we transfected Neuro-2a cells with a 17-kilobase human apoE genomic DNA construct encoding apoE3 or apoE4 along with upstream and downstream regulatory elements. The baseline expression of apoE was low. However, conditioned medium from an astrocytic cell line (C6) or from apoE-null mouse primary astrocytes increased the expression of both isoforms by 3-4-fold at the mRNA level and by 4-10-fold at the protein level. These findings suggest that astrocytes secrete a factor or factors that regulate apoE expression in neuronal cells. The increased expression of apoE was almost completely abolished by incubating neurons with U0126, an inhibitor of extracellular signal-regulated kinase (Erk), suggesting that the Erk pathway controls astroglial regulation of apoE expression in neuronal cells. Human neuronal precursor NT2/D1 cells expressed apoE constitutively; however, after treatment of these cells with retinoic acid to induce differentiation, apoE expression diminished. Cultured mouse primary cortical and hippocampal neurons also expressed low levels of apoE. Astrocyte-conditioned medium rapidly up-regulated apoE expression in fully differentiated NT2 neurons and in cultured mouse primary cortical and hippocampal neurons. Thus, neuronal expression of apoE is regulated by a diffusible factor or factors released from astrocytes, and this regulation depends on the activity of the Erk kinase pathway in neurons.     

Cytochrome c can be released into extracellular space and modulate functions of human astrocytes in a toll-like receptor 4-dependent manner

BackgroundChronic activation of glial cells contributes to neurodegenerative diseases. Cytochrome c (CytC) is a soluble mitochondrial protein that can act as a damage-associated molecular pattern (DAMP) when released into the extracellular space from damaged cells. CytC causes immune activation of microglia in a toll-like receptor (TLR) 4-dependent manner. The effects of extracellular CytC on astrocytes are unknown. Astrocytes, which are the most abundant glial cell type in the brain, express TLR 4 and secrete inflammatory mediators; therefore, we hypothesized that extracellular CytC can interact with the TLR 4 of astrocytes inducing their release of inflammatory molecules and cytotoxins.MethodExperiments were conducted using primary human astrocytes, U118 MG human astrocytic cells, BV-2 murine microglia, and SH-SY5Y human neuronal cells.ResultsExtracellularly applied CytC increased the secretion of interleukin (IL)-1β, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-12 p70 by cultured primary human astrocytes. Anti-TLR 4 antibodies blocked the CytC-induced secretion of IL-1β and GM-CSF by astrocytes. Supernatants from CytC-activated astrocytes were toxic to human SH-SY5Y neuronal cells. We also demonstrated CytC release from damaged glial cells by measuring CytC in the supernatants of BV-2 microglia after their exposure to cytotoxic concentrations of staurosporine, amyloid-β peptides (Aβ42) and tumor necrosis factor-α.ConclusionCytC can be released into the extracellular space from damaged glial cells causing immune activation of astrocytes in a TLR 4-dependent manner.General significanceAstrocyte activation by CytC may contribute to neuroinflammation and neuronal death in neurodegenerative diseases. Astrocyte TLR 4 could be a potential therapeutic target in these diseases.     

SYNTHESIS OF NUCLEAR RNA IN NERVE AND GLIAL CELLS

—Tritium-labelled RNA precursors were injected at 30 min intervals into the fourth ventricle of rats or rabbits. After 4 h the nuclei from neurones, astrocytes, and other glial cells were isolated and RNA extracted. Investigations were performed in order to establish optimum conditions for RNA extraction from this particular material. The sedimentation patterns obtained in sucrose gradients were similar to those of nuclear RNA from other mammalian tissues and showed the presence of RNA species with high specific activities in the region of the gradient between 10S and 16S and above 28S. All three types of nuclei contained a 45S and a 38S RNA. Moreover, a 32S component could be identified in astrocytic nuclei, a 35S fraction in neuronal nuclei, and both a 32S and 35S RNA in nuclei from glial cells. The nuclei from the various cell types also differ with respect to the rate of incorporation of the label into the nuclear RNA, being four times higher in astrocytic and neuronal nuclei than in those derived from the other glial cells.