FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C

Background: Glial Maturation Factor Beta (GMFB) is a highly conserved brain-enriched protein implicated in immunoregulation, neuroplasticity and apoptosis, processes central to neural injury and repair following cerebral ischaemia. Therefore, we examined if changes in neurocellular GMFB expression and release can be used to assess brain injury following ischaemia. Methods and Results: Immunofluorescence staining, Western blotting, immunohistochemistry and ELISA were used to measure GMFB in cultured neurons and astrocytes, rat brain tissues and plasma samples from stroke model rats and stroke patients, while cell viability assays, TTC staining and micro- PET were used to assess neural cell death and infarct severity. Immunofluorescence and immunohistochemistry revealed GMFB expression mainly in astrocyte and neuronal nuclei but also in neuronal axons and dendrites. Free GMFB concentration increased progressively in the culture medium during hypoxia-hypoglycaemia treatment. Plasma GMFB concentration increased in rats subjected to middle cerebral artery occlusion (MCAO, a model of stroke-reperfusion) and in stroke patients. Plasma GMFB in MCAO model rats was strongly correlated with infarct size (R2=0.9582). Plasma GMFB concentration was also markedly elevated in stroke patients within 24 h of onset and remained elevated for more than one week. Conversely, plasma GMFB elevations were not significant in myocardial infarct patients and stroke patients without infarction. Conclusion: GMFB has the prerequisite stability, expression specificity and response dynamics to serve as a reliable indicator of ischaemic injury in animal models and stroke patients. Plasma GMFB may be a convenient non-invasive adjunct to neuroimaging for stroke diagnosis and prognosis.

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Assessing the treatment of cannabidiolic acid methyl ester: a stable synthetic analogue of cannabidiolic acid on c-Fos and NeuN expression in the hypothalamus of rats

Journal of Cannabis Research ◽

10.1186/s42238-021-00081-1 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Eric Murillo-Rodríguez ◽

Diana Millán-Aldaco ◽

Gloria Arankowsky-Sandoval ◽

Tetsuya Yamamoto ◽

Roger G. Pertwee ◽

...

Keyword(s):

Methyl Ester ◽

Cannabis Sativa ◽

Brain Area ◽

Acid Methyl Ester ◽

Hypothalamic Nucleus ◽

Synthetic Analogue ◽

Neuronal Nuclei ◽

Cannabidiolic Acid ◽

Dorsomedial Hypothalamic Nucleus ◽

Psychotropic Compound

Abstract Background Cannabidiol (CBD), the non-psychotropic compound from Cannabis sativa, shows positive results on controlling several health disturbances; however, comparable data regarding additional chemical from C. sativa, such as cannabidiolic acid (CBDA), is scarce due to its instability. To address this limitation, a stable CBDA analogue, CBDA methyl ester (HU-580), was synthetized and showed CBDA-like effects. Recently, we described that HU-580 increased wakefulness and wake-related neurochemicals. Objective To extend the comprehension of HU-580´s properties on waking, the c-Fos and NeuN expression in a wake-linked brain area, the hypothalamus was evaluated. Methods c-Fos and NeuN expression in hypothalamic sections were analyzed after the injections of HU-580 (0.1 or 100 μg/kg, i.p.). Results Systemic administrations of HU-580 increased c-Fos and neuronal nuclei (NeuN) expression in hypothalamic nuclei, including the dorsomedial hypothalamic nucleus dorsal part, dorsomedial hypothalamic nucleus compact part, and dorsomedial hypothalamic nucleus ventral part. Conclusion HU-580 increased c-Fos and NeuN immunoreactivity in hypothalamus nuclei suggesting that this drug might modulate the sleep–wake cycle by engaging the hypothalamus.

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The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01228-0 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Alyssa N. Coyne ◽

Jeffrey D. Rothstein

Keyword(s):

Nuclear Pore Complex ◽

Mammalian Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Dependent Manner ◽

Structured Illumination Microscopy ◽

Neuronal Nuclei ◽

Complex Injury ◽

Sporadic Als ◽

Human Neurons

AbstractNuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

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DeadEasy neurons: Automatic counting of HB9 neuronal nuclei in Drosophila

Cytometry Part A ◽

10.1002/cyto.a.20877 ◽

2010 ◽

Vol 77A (4) ◽

pp. 371-378 ◽

Cited By ~ 5

Author(s):

Manuel G. Forero ◽

Jenny A. Pennack ◽

Alicia Hidalgo

Keyword(s):

Neuronal Nuclei ◽

Automatic Counting

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The Immunopathogenesis of Neuroinvasive Lesions of SARS-CoV-2 Infection in COVID-19 Patients

Frontiers in Neurology ◽

10.3389/fneur.2021.697079 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shamila D. Alipoor ◽

Esmaeil Mortaz ◽

Mohammad Varahram ◽

Johan Garssen ◽

Ian M. Adcock

Keyword(s):

Transport Mechanism ◽

Respiratory Rhythm ◽

Olfactory Nerve ◽

Severe Respiratory Distress ◽

Neuronal Nuclei ◽

The World ◽

Neurological Effects ◽

The Brain ◽

Trigger Cell Death

The new coronavirus disease COVID-19 was identified in December 2019. It subsequently spread across the world with over 125 M reported cases and 2.75 M deaths in 190 countries. COVID-19 causes severe respiratory distress; however, recent studies have reported neurological consequences of infection by the COVID-19 virus SARS-CoV-2 even in subjects with mild infection and no initial neurological effects. It is likely that the virus uses the olfactory nerve to reach the CNS and that this transport mechanism enables virus access to areas of the brain stem that regulates respiratory rhythm and may even trigger cell death by alteration of these neuronal nuclei. In addition, the long-term neuronal effects of COVID-19 suggest a role for SARS-CoV-2 in the development or progression of neurodegerative disease as a result of inflammation and/or hypercoagulation. In this review recent findings on the mechanism(s) by which SARS-CoV-2 accesses the CNS and induces neurological dysregulation are summarized.

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DNA methylation analysis of neuronal nuclei derived from bipolar disorder

Neuroscience Research ◽

10.1016/j.neures.2011.07.020 ◽

2011 ◽

Vol 71 ◽

pp. e7

Author(s):

Kazuya Iwamoto ◽

Miki Bundo ◽

Kiyoto Kasai ◽

Tadafumi Kato

Keyword(s):

Dna Methylation ◽

Bipolar Disorder ◽

Methylation Analysis ◽

Neuronal Nuclei ◽

Dna Methylation Analysis

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Cholinephosphotransferase activities in microsomes and neuronal nuclei isolated from immature rabbit cerebral cortex: the use of endogenously generated diacylglycerols as substrate

Canadian Journal of Biochemistry ◽

10.1139/o82-089 ◽

1982 ◽

Vol 60 (7) ◽

pp. 724-733 ◽

Cited By ~ 19

Author(s):

R. Roy Baker ◽

Huu-Yi Chang

Keyword(s):

Phospholipase C ◽

Microsomal Fraction ◽

Neuronal Development ◽

Nuclear Fraction ◽

Neuronal Nuclei ◽

Nuclear Membranes ◽

Choline Phosphotransferase ◽

Rabbit Cerebral Cortex ◽

Low Levels ◽

Triton X

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities were much more profoundly inhibited at low levels of Triton X-100 (0.01–0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.

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Early deficits in olfaction are associated with structural and molecular alterations in the olfactory system of a Huntington disease mouse model

Human Molecular Genetics ◽

10.1093/hmg/ddaa099 ◽

2020 ◽

Vol 29 (13) ◽

pp. 2134-2147

Author(s):

M Laroche ◽

M Lessard-Beaudoin ◽

M Garcia-Miralles ◽

C Kreidy ◽

E Peachey ◽

...

Keyword(s):

Huntington Disease ◽

Olfactory System ◽

Piriform Cortex ◽

Olfactory Dysfunction ◽

Altered Expression ◽

Neuronal Nuclei ◽

Molecular Alterations ◽

Cell Death Pathways ◽

Structural Abnormalities

Abstract Olfactory dysfunction and altered neurogenesis are observed in several neurodegenerative disorders including Huntington disease (HD). These deficits occur early and correlate with a decline in global cognitive performance, depression and structural abnormalities of the olfactory system including the olfactory epithelium, bulb and cortices. However, the role of olfactory system dysfunction in the pathogenesis of HD remains poorly understood and the mechanisms underlying this dysfunction are unknown. We show that deficits in odour identification, discrimination and memory occur in HD individuals. Assessment of the olfactory system in an HD murine model demonstrates structural abnormalities in the olfactory bulb (OB) and piriform cortex, the primary cortical recipient of OB projections. Furthermore, a decrease in piriform neuronal counts and altered expression levels of neuronal nuclei and tyrosine hydroxylase in the OB are observed in the YAC128 HD model. Similar to the human HD condition, olfactory dysfunction is an early phenotype in the YAC128 mice and concurrent with caspase activation in the murine HD OB. These data provide a link between the structural olfactory brain region atrophy and olfactory dysfunction in HD and suggest that cell proliferation and cell death pathways are compromised and may contribute to the olfactory deficits in HD.

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Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400613 ◽

2020 ◽

Vol 10 (5) ◽

pp. 1647-1655

Author(s):

Benjamin C. Reiner ◽

Glenn A. Doyle ◽

Andrew E. Weller ◽

Rachel N. Levinson ◽

Esin Namoglu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Restriction Enzyme ◽

Postmortem Brain ◽

Whole Genome ◽

Mobile Dna ◽

Neuronal Nuclei ◽

Dna Elements ◽

Long Interspersed Element ◽

Generation Sequencing ◽

Human Specific

Long interspersed element-1 retrotransposons (LINE-1 or L1) are ∼6 kb mobile DNA elements implicated in the origins of many Mendelian and complex diseases. The actively retrotransposing L1s are mostly limited to the L1 human specific (L1Hs) transcriptional active (Ta) subfamily. In this manuscript, we present REBELseq as a method for the construction of Ta subfamily L1Hs-enriched next-generation sequencing libraries and bioinformatic identification. REBELseq was performed on DNA isolated from NeuN+ neuronal nuclei from postmortem brain samples of 177 individuals and empirically-driven bioinformatic and experimental cutoffs were established. Putative L1Hs insertions passing bioinformatics cutoffs were experimentally validated. REBELseq reliably identified both known and novel Ta subfamily L1Hs insertions distributed throughout the genome. Differences in the proportion of individuals possessing a given reference or non-reference retrotransposon insertion were identified. We conclude that REBELseq is an unbiased, whole genome approach to the amplification and detection of Ta subfamily L1Hs retrotransposons.

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Cerebral Ischemia Causes Dysregulation of Synaptic Adhesion in Mouse Synaptosomes

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600510 ◽

2007 ◽

Vol 28 (1) ◽

pp. 99-110 ◽

Cited By ~ 15

Author(s):

Willard J Costain ◽

Ingrid Rasquinha ◽

Jagdeep K Sandhu ◽

Peter Rippstein ◽

Bogdan Zurakowski ◽

...

Keyword(s):

Cell Death ◽

Cerebral Ischemia ◽

Adhesion Molecules ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Artery Occlusion ◽

Synaptic Proteins ◽

Mrna Levels ◽

Neuronal Nuclei ◽

Protein Levels

Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.

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