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.
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 Ca
...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.
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.
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.
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.
Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the ...glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro. We will discuss here the main advantages/disadvantages linked to the use of CNS slices for histological analysis of neuronal proliferation and death, as well as the main findings obtained in the most popular types of preparations, i.e. the cortical, hippocampal, cerebellar and retinal slices.
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/mm
) 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.
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).
The principles of the 3Rs-
-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
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
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.
The anatomical features distinctive to each of the very large array of species used in today's biomedical research must be born in mind when considering the correct choice of animal model(s), ...particularly when translational research is concerned. In this paper we take into consideration and discuss the most important anatomical and histological features of the commonest species of laboratory rodents (rat, mouse, guinea pig, hamster, and gerbil), rabbit, and pig related to their importance for applied research.
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