An Overview of the Epigenetic Modifications in the Brain under Normal and Pathological Conditions.

Epigenetic changes are changes in gene expression that do not involve alterations to the DNA sequence. These changes lead to establishing a so-called epigenetic code that dictates which and when genes are activated, thus orchestrating gene regulation and playing a central role in development, health, and disease. The brain, being mostly formed by cells that do not undergo a renewal process throughout life, is highly prone to the risk of alterations leading to neuronal death and neurodegenerative disorders, mainly at a late age. Here, we review the main epigenetic modifications that have been described in the brain, with particular attention on those related to the onset of developmental anomalies or neurodegenerative conditions and/or occurring in old age. DNA methylation and several types of histone modifications (acetylation, methylation, phosphorylation, ubiquitination, sumoylation, lactylation, and crotonylation) are major players in these processes. They are directly or indirectly involved in the onset of neurodegeneration in Alzheimer's or Parkinson's disease. Therefore, this review briefly describes the roles of these epigenetic changes in the mechanisms of brain development, maturation, and aging and some of the most important factors dynamically regulating or contributing to these changes, such as oxidative stress, inflammation, and mitochondrial dysfunction.

The concept of intrinsic versus extrinsic apoptosis.

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer.

Endoplasmic Reticulum Stress Signaling and Neuronal Cell Death.

Besides protein processing, the endoplasmic reticulum (ER) has several other functions such as lipid synthesis, the transfer of molecules to other cellular compartments, and the regulation of Ca2+ homeostasis. Before leaving the organelle, proteins must be folded and post-translationally modified. Protein folding and revision require molecular chaperones and a favorable ER environment. When in stressful situations, ER luminal conditions or chaperone capacity are altered, and the cell activates signaling cascades to restore a favorable folding environment triggering the so-called unfolded protein response (UPR) that can lead to autophagy to preserve cell integrity. However, when the UPR is disrupted or insufficient, cell death occurs. This review examines the links between UPR signaling, cell-protective responses, and death following ER stress with a particular focus on those mechanisms that operate in neurons.

The Phosphorylated Form of the Histone H2AX (γH2AX) in the Brain from Embryonic Life to Old Age.

The γ phosphorylated form of the histone H2AX (γH2AX) was described more than 40 years ago and it was demonstrated that phosphorylation of H2AX was one of the first cellular responses to DNA damage. Since then, γH2AX has been implicated in diverse cellular functions in normal and pathological cells. In the first part of this review, we will briefly describe the intervention of H2AX in the DNA damage response (DDR) and its role in some pivotal cellular events, such as regulation of cell cycle checkpoints, genomic instability, cell growth, mitosis, embryogenesis, and apoptosis. Then, in the main part of this contribution, we will discuss the involvement of γH2AX in the normal and pathological central nervous system, with particular attention to the differences in the DDR between immature and mature neurons, and to the significance of H2AX phosphorylation in neurogenesis and neuronal cell death. The emerging picture is that H2AX is a pleiotropic molecule with an array of yet not fully understood functions in the brain, from embryonic life to old age.

Protective Effects of Some Grapevine Polyphenols against Naturally Occurring Neuronal Death.

The interest in the biological properties of grapevine polyphenols (PPs) in neuroprotection is continuously growing in the hope of finding translational applications. However, there are several concerns about the specificity of action of these molecules that appear to act non-specifically on the permeability of cellular membranes. Naturally occurring neuronal death (NOND) during cerebellar maturation is a well characterized postnatal event that is very useful to investigate the death and rescue of neurons. We here aimed to establish a baseline comparative study of the potential to counteract NOND of certain grapevine PPs of interest for the oenology. To do so, we tested ex vivo the neuroprotective activity of peonidin- and malvidin-3-O-glucosides, resveratrol, polydatin, quercetin-3-O-glucoside, (+)-taxifolin, and (+)-catechin. The addition of these molecules (50 µM) to organotypic cultures of mouse cerebellum explanted at postnatal day 7, when NOND reaches a physiological peak, resulted in statistically significant (two-tailed Mann-Whitney test-p < 0.001) reductions of the density of dead cells (propidium iodide+ cells/mm2) except for malvidin-3-O-glucoside. The stilbenes were less effective in reducing cell death (to 51-60%) in comparison to flavanols, (+)-taxifolin and quercetin 3-O-glucoside (to 69-72%). Thus, molecules with a -OH group in ortho position (taxifolin, quercetin 3-O-glucoside, (+)-catechin, and peonidin 3-O-glucoside) have a higher capability to limit death of cerebellar neurons. As NOND is apoptotic, we speculate that PPs act by inhibiting executioner caspase 3.

Decreased Expression of Synaptophysin 1 (SYP1 Major Synaptic Vesicle Protein p38) and Contactin 6 (CNTN6/NB3) in the Cerebellar Vermis of reln Haplodeficient Mice.

Reeler heterozygous mice (reln+/-) are seemingly normal but haplodeficient in reln, a gene implicated in autism. Structural/neurochemical alterations in the reln+/- brain are subtle and difficult to demonstrate. Therefore, the usefulness of these mice in translational research is still debated. As evidence implicated several synapse-related genes in autism and the cerebellar vermis is structurally altered in the condition, we have investigated the expression of synaptophysin 1 (SYP1) and contactin 6 (CNTN6) within the vermis of reln+/- mice. Semi-thin plastic sections of the vermis from adult mice of both sexes and different genotypes (reln+/- and reln+/+) were processed with an indirect immunofluorescence protocol. Immunofluorescence was quantified on binary images and statistically analyzed. Reln+/- males displayed a statistically significant reduction of 11.89% in the expression of SYP1 compared to sex-matched wild-type animals, whereas no differences were observed between reln+/+ and reln+/- females. In reln+/- male mice, reductions were particularly evident in the molecular layer: 10.23% less SYP1 than reln+/+ males and 5.84% < reln+/+ females. In reln+/- females, decrease was 9.84% versus reln+/+ males and 5.43% versus reln+/+ females. Both reln+/- males and females showed a stronger decrease in CNTN6 expression throughout all the three cortical layers of the vermis: 17-23% in the granular layer, 24-26% in the Purkinje cell layer, and 9-14% in the molecular layer. Altogether, decrease of vermian SYP1 and CNTN6 in reln+/- mice displayed patterns compatible with the structural modifications of the autistic cerebellum. Therefore, these mice may be a good model in translational studies.

Effects of Acute Alcohol Exposure on Layer 5 Pyramidal Neurons of Juvenile Mice.

Early-onset drinking during childhood or preadolescence is a serious social problem. Yet, most of the basic neurobiological research on the acute effects of ethanol has been carried out on adult or early postnatal animals. We studied the effect of alcohol exposure on the basic electrophysiological properties and cell viability of layer 5 pyramidal neurons from the somatosensory cortex of juvenile (P21-P23) C57BL/6N mice. After bath application of 50 mM ethanol to acute slices of the somatosensory cortex, no adverse effects were detected on cells survival, whereas the input resistance and firing rate of layer 5 neurons were significantly reduced. While the effect on the input resistance was reversible, the depressing effect on cell firing remained stable after 6 min of alcohol exposure. Ethanol application did not result in any significant change of mIPSC frequency, amplitude, and rise time. A slight increase of mIPSC decay time was observed after 6 min of ethanol exposure. The molecular mechanisms leading to these alterations and their significance for the physiology of the cerebral cortex are briefly discussed.

Caspase-3 Mediated Cell Death in the Normal Development of the Mammalian Cerebellum.

Caspase-3, onto which there is a convergence of the intrinsic and extrinsic apoptotic pathways, is the main executioner of apoptosis. We here review the current literature on the intervention of the protease in the execution of naturally occurring neuronal death (NOND) during cerebellar development. We will consider data on the most common altricial species (rat, mouse and rabbit), as well as humans. Among the different types of neurons and glia in cerebellum, there is ample evidence for an intervention of caspase-3 in the regulation of NOND of the post-mitotic cerebellar granule cells (CGCs) and Purkinje neurons, as a consequence of failure to establish proper synaptic contacts with target (secondary cell death). It seems possible that the GABAergic interneurons also undergo a similar type of secondary cell death, but the intervention of caspase-3 in this case still remains to be clarified in full. Remarkably, CGCs also undergo primary cell death at the precursor/pre-migratory stage of differentiation, in this instance without the intervention of caspase-3. Glial cells, as well, undergo a process of regulated cell death, but it seems possible that expression of caspase-3, at least in the Bergmann glia, is related to differentiation rather than death.

Phosphorylation of histone H2AX in the mouse brain from development to senescence.

Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.

Anatomical features for an adequate choice of the experimental animal model in biomedicine: III. Ferret, goat, sheep, and horse.

The anatomical characteristics of each of the many species today employed in biomedical research are very important when selecting the correct animal model(s), especially for conducting translational research. In previous papers, these features have been considered for fish (D'Angelo et al., 2016), the most common laboratory rodents, rabbits, and pigs (Lossi et al. 2016). I here follow this line of discussion by dealing with the importance of proper knowledge of ferrets, goats, sheep, and horses' main anatomical features in translational research.

Co-cultures of cerebellar slices from mice with different reelin genetic backgrounds as a model to study cortical lamination.

Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler phenotype in mice, the first described cerebellar mutation. In homozygous mutants missing the Reelin gene ( reln -/-), neurons are incapable of correctly positioning themselves in layered brain areas such as the cerebral and cerebellar cortices. We here demonstrate that by employing ex vivo cultured cerebellar slices one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs). Methods: We generated mouse hybrids (L7-GFP relnF1/) with green fluorescent protein (GFP)-tagged PNs, directly visible under fluorescence microscopy. We then cultured the slices obtained from mice with different reln genotypes and demonstrated that when the slices from reln -/- mutants were co-cultured with those from reln +/- mice, the Reelin produced by the latter induced migration of the PNs to partially rescue the normal layered cortical histology. We have confirmed this observation with Voronoi tessellation to analyze PN dispersion. Results: In images of the co-cultured slices from reln -/- mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than those generated from slices of reln +/- animals. The mean roundness factor, area disorder, and roundness factor homogeneity were different when slices from reln -/- mice were cultivated singularly or co-cultivated, supporting mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied ex vivo. Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it for the possibility to study and manipulate ex vivo the activity of a brain-secreted or genetically engineered protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the total avoidance of severe surgery (3Rs perspective).

Association of Caspase 3 Activation and H2AX γ Phosphorylation in the Aging Brain: Studies on Untreated and Irradiated Mice.

Phosphorylation of H2AX is a response to DNA damage, but γH2AX also associates with mitosis and/or apoptosis. We examined the effects of X-rays on DNA integrity to shed more light on the significance of H2AX phosphorylation and its relationship with activation of caspase 3 (CASP3), the main apoptotic effector. After administration of the S phase marker BrdU, brains were collected from untreated and irradiated (10 Gray) 24-month-old mice surviving 15 or 30 min after irradiation. After paraffin embedding, brain sections were single- or double-stained with antibodies against γH2AX, p53-binding protein 1 (53BP1) (which is recruited during the DNA damage response (DDR)), active CASP3 (cCASP3), 5-Bromo-2-deoxyuridine (BrdU), and phosphorylated histone H3 (pHH3) (which labels proliferating cells). After statistical analysis, we demonstrated that irradiation not only induced a robust DDR with the appearance of γH2AX and upregulation of 53BP1 but also that cells with damaged DNA attempted to synthesize new genetic material from the rise in BrdU immunostaining, with increased expression of cCASP3. Association of γH2AX, 53BP1, and cCASP3 was also evident in normal nonirradiated mice, where DNA synthesis appeared to be linked to disturbances in DNA repair mechanisms rather than true mitotic activity.

Mesenchymal stem cell conditioned medium increases glial reactivity and decreases neuronal survival in spinal cord slice cultures.

Ex vivo spinal cord slice cultures (SCSC) allow study of spinal cord circuitry, maintaining stimuli responses comparable to live animals. Previously, we have shown that mesenchymal stem/stromal cell (MSC) transplantation in vivo reduced inflammation and increased nerve regeneration but MSC survival was short-lived, highlighting that beneficial action may derive from the secretome. Previous in vitro studies of MSC conditioned medium (CM) have also shown increased neuronal growth. In this study, murine SCSC were cultured in canine MSC CM (harvested from the adipose tissue of excised inguinal fat) and cell phenotypes analysed via immunohistochemistry and confocal microscopy. SCSC in MSC CM displayed enhanced viability after propidium iodide staining. GFAP immunoreactivity was significantly increased in SCSC in MSC CM compared to controls, but with no change in proteoglycan (NG2) immunoreactivity. In contrast, culture in MSC CM significantly decreased the prevalence of ßIII-tubulin immunoreactive neurites, whilst Ca2+ transients per cell were significantly increased. These ex vivo results contradict previous in vitro and in vivo reports of how MSC and their secretome may affect the microenvironment of the spinal cord after injury and highlight the importance of a careful comparison of the different experimental conditions used to assess the potential of cell therapies for the treatment of spinal cord injury.

BDNF as a pain modulator.

At least some neurotrophins may be powerful modulators of synapses, thereby influencing short- and long-term synaptic efficiency. BDNF acts at central synapses in pain pathways both at spinal and supraspinal levels. Neuronal synthesis, subcellular storage/co-storage and release of BDNF at these synapses have been characterized on anatomical and physiological grounds, in parallel with trkB (the high affinity BDNF receptor) distribution. Histological and functional evidence has been provided, mainly from studies on acute slices and intact animals, that BDNF modulates fast excitatory (glutamatergic) and inhibitory (GABAergic/glycinergic) signals, as well as slow peptidergic neurotrasmission in spinal cord. Recent studies have unraveled some of the neuronal circuitries and mechanisms involved, highlighting the key role of synaptic glomeruli in lamina II as the main sites for such a modulation.

Autophagy regulates the post-translational cleavage of BCL-2 and promotes neuronal survival.

B-cell lymphoma 2 protein (BCL-2) is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs) that are known to undergo massive programmed cell death (apoptosis) during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice.

The Reeler Mouse: A Translational Model of Human Neurological Conditions, or Simply a Good Tool for Better Understanding Neurodevelopment?

The first description of the Reeler mutation in mouse dates to more than fifty years ago, and later, its causative gene (reln) was discovered in mouse, and its human orthologue (RELN) was demonstrated to be causative of lissencephaly 2 (LIS2) and about 20% of the cases of autosomal-dominant lateral temporal epilepsy (ADLTE). In both human and mice, the gene encodes for a glycoprotein referred to as reelin (Reln) that plays a primary function in neuronal migration during development and synaptic stabilization in adulthood. Besides LIS2 and ADLTE, RELN and/or other genes coding for the proteins of the Reln intracellular cascade have been associated substantially to other conditions such as spinocerebellar ataxia type 7 and 37, VLDLR-associated cerebellar hypoplasia, PAFAH1B1-associated lissencephaly, autism, and schizophrenia. According to their modalities of inheritances and with significant differences among each other, these neuropsychiatric disorders can be modeled in the homozygous (reln-/-) or heterozygous (reln+/-) Reeler mouse. The worth of these mice as translational models is discussed, with focus on their construct and face validity. Description of face validity, i.e., the resemblance of phenotypes between the two species, centers onto the histological, neurochemical, and functional observations in the cerebral cortex, hippocampus, and cerebellum of Reeler mice and their human counterparts.

The gastrointestinal hormone ghrelin modulates inhibitory neurotransmission in deep laminae of mouse spinal cord dorsal horn.

Ghrelin is mainly described for its effects on feeding behavior and metabolism. However, central nervous system distribution of its receptor [type 1a GH secretagogue receptor (GHSR)] and modulation of neurotransmission in the hypothalamus suggest broader effects than originally predicted. Systemically administrated ghrelin inhibits inflammatory pain after behavioral observations. Therefore, we investigated the expression and function of type 1a GHSR in mouse spinal cord by molecular biology, biochemistry, histology, and electrophysiology. The mRNA and protein were detected in tissue extracts by RT-PCR and Western blotting. In situ, receptor mRNA and immunoreactivity were localized to cell bodies within the medial aspect of the deep dorsal horn. Patch clamp recordings on laminae IV-VI demonstrated that bath-applied ghrelin (100 nm) induced a strong increase of spontaneous gamma-aminobutyric acid/glycine-mediated current frequency (463 +/- 93% of the control) and amplitude (150 +/- 16% of the control) in about 60% of recorded neurons. Specificity of type 1a GHSR activation was confirmed by the lack of effect of the deacylated form of ghrelin (des-acyl-ghrelin) and after preincubation with the specific receptor antagonist [d-Lys(3)]GHRP-6. In the presence of tetrodotoxin, the effect of the peptide was strongly reduced, mainly indicating an action potential-dependent mechanism. The functional link between ghrelin and pain was confirmed by inhibition in vitro of the c-fos response to capsaicin activation of nociceptive fibers, after quantification of Fos-immunoreactive nuclei in laminae IV-VI. Our results are the first demonstration of the presence of functional type 1a GHSRs in the spinal cord and indicate that ghrelin may exert antinociceptive effects by directly increasing inhibitory neurotransmission in a subset of deep dorsal horn neurons.

The Use of ex Vivo Rodent Platforms in Neuroscience Translational Research With Attention to the 3Rs Philosophy.

The principles of the 3Rs-Replacement, Reduction, and Refinement-are at the basis of most advanced national and supranational (EU) regulations on animal experimentation and welfare. In the perspective to reduce and refine the use of these animals in translational research, we here discuss the use of rodent acute and organotypically cultured central nervous system slices. We describe novel applications of these ex vivo platforms in medium-throughput screening of neuroactive molecules of potential pharmacological interest, with particular attention to more recent developments that permit to fully exploit the potential of direct genetic engineering of organotypic cultures using transfection techniques. We then describe the perspectives for expanding the use ex vivo platforms in neuroscience studies under the 3Rs philosophy using the following approaches: (1) Use of co-cultures of two brain regions physiologically connected to each other (source-target) to analyze axon regeneration and reconstruction of circuitries; (2) Microinjection or co-cultures of primary cells and/or cell lines releasing one or more neuroactive molecules to screen their physiological and/or pharmacological effects onto neuronal survival and slice circuitry. Microinjected or co-cultured cells are ideally made fluorescent after transfection with a plasmid construct encoding green or red fluorescent protein under the control of a general promoter such as hCMV; (3) Use of "sniffer" cells sensing the release of biologically active molecules from organotypic cultures by means of fluorescent probes. These cells can be prepared with activatable green fluorescent protein, a unique chromophore that remains in a "dark" state because its maturation is inhibited, and can be made fluorescent (de-quenched) if specific cellular enzymes, such as proteases or kinases, are activated.

Spongiform neurodegenerative disease in a Persian kitten.

A congenital encephalopathy with spongiform degeneration and prominent neuronal apoptosis was observed in a 4-month-old Persian male cat with a history of depressed mental status and ataxia. On clinical examination, signs included right head tilt, ventroflexion of the head and neck, and tetraparesis. Histological examination of the central nervous system revealed multifocal, bilateral and symmetrical vacuolar degeneration of the neuropil, mainly involving the cerebellar and vestibular nuclei area, the caudal colliculi, the mesencephalic nuclei, the tegmental area and the deeper layer of the cerebral cortex. Accumulation of phosphorylated neurofilaments was detected in neuronal perikarya of the deep cortical layers, hippocampus and thalamus. Numerous pyknotic and apoptotic neurons were also observed in the cerebral cortex. These neuropathological changes differ from those observed in previous reports of spongiform degeneration of the grey matter in cats and were suggestive of a congenital neurodegenerative disease.

Cell death and neurodegeneration in the postnatal development of cerebellar vermis in normal and Reeler mice.

Programmed cell death (PCD) was demonstrated in neurons and glia in normal brain development, plasticity, and aging, but also in neurodegeneration. (Macro)autophagy, characterized by cytoplasmic vacuolization and activation of lysosomal hydrolases, and apoptosis, typically entailing cell shrinkage, chromatin and nuclear condensation, are the two more common forms of PCD. Their underlying intracellular pathways are partly shared and neurons can die following both modalities, according to the type of death-triggering stimulus. Reelin is an extracellular protein necessary for proper neuronal migration and brain lamination. In the mutant Reeler mouse, its absence causes neuronal mispositioning, with a notable degree of cerebellar hypoplasia that was tentatively related to an increase in PCD. We have carried out an ultrastructural analysis on the occurrence and type of postnatal PCD affecting the cerebellar neurons in normal and Reeler mice. In the forming cerebellar cortex, PCD took the form of apoptosis or autophagy and mainly affected the cerebellar granule cells (CGCs). Densities of apoptotic CGCs were comparable in both mouse strains at P0-P10, while, in mutants, they increased to become significantly higher at P15. In WT mice the density of autophagic neurons did not display statistically significant differences in the time interval examined in this study, whereas it was reduced in Reeler in the P0-P10 interval, but increased at P15. Besides CGCs, the Purkinje neurons also displayed autophagic features in both WT and Reeler mice. Therefore, cerebellar neurons undergo different types of PCD and a Reelin deficiency affects the type and degree of neuronal death during postnatal development of the cerebellum.