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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.
Laura Lossi; Adalberto Merighi; Vittorino Novello; Alessandra Ferrandino. Protective Effects of Some Grapevine Polyphenols against Naturally Occurring Neuronal Death. Molecules 2020, 25, 2925 .
AMA StyleLaura Lossi, Adalberto Merighi, Vittorino Novello, Alessandra Ferrandino. Protective Effects of Some Grapevine Polyphenols against Naturally Occurring Neuronal Death. Molecules. 2020; 25 (12):2925.
Chicago/Turabian StyleLaura Lossi; Adalberto Merighi; Vittorino Novello; Alessandra Ferrandino. 2020. "Protective Effects of Some Grapevine Polyphenols against Naturally Occurring Neuronal Death." Molecules 25, no. 12: 2925.
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.
Laura Lossi; Claudia Castagna; Alberto Granato; Adalberto Merighi. The Reeler Mouse: A Translational Model of Human Neurological Conditions, or Simply a Good Tool for Better Understanding Neurodevelopment? Journal of Clinical Medicine 2019, 8, 2088 .
AMA StyleLaura Lossi, Claudia Castagna, Alberto Granato, Adalberto Merighi. The Reeler Mouse: A Translational Model of Human Neurological Conditions, or Simply a Good Tool for Better Understanding Neurodevelopment? Journal of Clinical Medicine. 2019; 8 (12):2088.
Chicago/Turabian StyleLaura Lossi; Claudia Castagna; Alberto Granato; Adalberto Merighi. 2019. "The Reeler Mouse: A Translational Model of Human Neurological Conditions, or Simply a Good Tool for Better Understanding Neurodevelopment?" Journal of Clinical Medicine 8, no. 12: 2088.
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.
Claudia Castagna; Adalberto Merighi; Laura Lossi. Decreased Expression of Synaptophysin 1 (SYP1 Major Synaptic Vesicle Protein p38) and Contactin 6 (CNTN6/NB3) in the Cerebellar Vermis of reln Haplodeficient Mice. Cellular and Molecular Neurobiology 2019, 39, 833 -856.
AMA StyleClaudia Castagna, Adalberto Merighi, Laura Lossi. Decreased Expression of Synaptophysin 1 (SYP1 Major Synaptic Vesicle Protein p38) and Contactin 6 (CNTN6/NB3) in the Cerebellar Vermis of reln Haplodeficient Mice. Cellular and Molecular Neurobiology. 2019; 39 (6):833-856.
Chicago/Turabian StyleClaudia Castagna; Adalberto Merighi; Laura Lossi. 2019. "Decreased Expression of Synaptophysin 1 (SYP1 Major Synaptic Vesicle Protein p38) and Contactin 6 (CNTN6/NB3) in the Cerebellar Vermis of reln Haplodeficient Mice." Cellular and Molecular Neurobiology 39, no. 6: 833-856.
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.
Laura Lossi; Claudia Castagna; Adalberto Merighi. Caspase-3 Mediated Cell Death in the Normal Development of the Mammalian Cerebellum. International Journal of Molecular Sciences 2018, 19, 3999 .
AMA StyleLaura Lossi, Claudia Castagna, Adalberto Merighi. Caspase-3 Mediated Cell Death in the Normal Development of the Mammalian Cerebellum. International Journal of Molecular Sciences. 2018; 19 (12):3999.
Chicago/Turabian StyleLaura Lossi; Claudia Castagna; Adalberto Merighi. 2018. "Caspase-3 Mediated Cell Death in the Normal Development of the Mammalian Cerebellum." International Journal of Molecular Sciences 19, no. 12: 3999.
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.
Francesco Ferrini; Benjamin Dering; Andrea de Giorgio; Laura Lossi; Alberto Granato. Effects of Acute Alcohol Exposure on Layer 5 Pyramidal Neurons of Juvenile Mice. Cellular and Molecular Neurobiology 2017, 38, 955 -963.
AMA StyleFrancesco Ferrini, Benjamin Dering, Andrea de Giorgio, Laura Lossi, Alberto Granato. Effects of Acute Alcohol Exposure on Layer 5 Pyramidal Neurons of Juvenile Mice. Cellular and Molecular Neurobiology. 2017; 38 (4):955-963.
Chicago/Turabian StyleFrancesco Ferrini; Benjamin Dering; Andrea de Giorgio; Laura Lossi; Alberto Granato. 2017. "Effects of Acute Alcohol Exposure on Layer 5 Pyramidal Neurons of Juvenile Mice." Cellular and Molecular Neurobiology 38, no. 4: 955-963.
Vascular cognitive impairment (VCI) is a heterogeneous entity with multiple aetiologies, all linked to underlying vascular disease. Among these, VCI related to subcortical small vessel disease (SSVD) is emerging as a major homogeneous subtype. Its progressive course raises the need for biomarker identification and/or development for adequate therapeutic interventions to be tested. In order to shed light in the current status on biochemical markers for VCI-SSVD, experts in field reviewed the recent evidence and literature data. The group conducted a comprehensive search on Medline, PubMed and Embase databases for studies published until 15.01.2017. The proposal on current status of biochemical markers in VCI-SSVD was reviewed by all co-authors and the draft was repeatedly circulated and discussed before it was finalized. This review identifies a large number of biochemical markers derived from CSF and blood. There is a considerable overlap of VCI-SSVD clinical symptoms with those of Alzheimer’s disease (AD). Although most of the published studies are small and their findings remain to be replicated in larger cohorts, several biomarkers have shown promise in separating VCI-SSVD from AD. These promising biomarkers are closely linked to underlying SSVD pathophysiology, namely disruption of blood-CSF and blood–brain barriers (BCB-BBB) and breakdown of white matter myelinated fibres and extracellular matrix, as well as blood and brain inflammation. The leading biomarker candidates are: elevated CSF/blood albumin ratio, which reflects BCB/BBB disruption; altered CSF matrix metalloproteinases, reflecting extracellular matrix breakdown; CSF neurofilment as a marker of axonal damage, and possibly blood inflammatory cytokines and adhesion molecules. The suggested SSVD biomarker deviations contrasts the characteristic CSF profile in AD, i.e. depletion of amyloid beta peptide and increased phosphorylated and total tau. Combining SSVD and AD biomarkers may provide a powerful tool to identify with greater precision appropriate patients for clinical trials of more homogeneous dementia populations. Thereby, biomarkers might promote therapeutic progress not only in VCI-SSVD, but also in AD.
A. Wallin; E. Kapaki; M. Boban; S. Engelborghs; D. M. Hermann; B. Huisa; M. Jonsson; M. G. Kramberger; L. Lossi; B. Malojcic; S. Mehrabian; A. Merighi; E. B. Mukaetova-Ladinska; G. P. Paraskevas; B. O. Popescu; R. Ravid; L. Traykov; G. Tsivgoulis; G. Weinstein; A. Korczyn; M. Bjerke; G. Rosenberg. Biochemical markers in vascular cognitive impairment associated with subcortical small vessel disease - A consensus report. BMC Neurology 2017, 17, 1 -12.
AMA StyleA. Wallin, E. Kapaki, M. Boban, S. Engelborghs, D. M. Hermann, B. Huisa, M. Jonsson, M. G. Kramberger, L. Lossi, B. Malojcic, S. Mehrabian, A. Merighi, E. B. Mukaetova-Ladinska, G. P. Paraskevas, B. O. Popescu, R. Ravid, L. Traykov, G. Tsivgoulis, G. Weinstein, A. Korczyn, M. Bjerke, G. Rosenberg. Biochemical markers in vascular cognitive impairment associated with subcortical small vessel disease - A consensus report. BMC Neurology. 2017; 17 (1):1-12.
Chicago/Turabian StyleA. Wallin; E. Kapaki; M. Boban; S. Engelborghs; D. M. Hermann; B. Huisa; M. Jonsson; M. G. Kramberger; L. Lossi; B. Malojcic; S. Mehrabian; A. Merighi; E. B. Mukaetova-Ladinska; G. P. Paraskevas; B. O. Popescu; R. Ravid; L. Traykov; G. Tsivgoulis; G. Weinstein; A. Korczyn; M. Bjerke; G. Rosenberg. 2017. "Biochemical markers in vascular cognitive impairment associated with subcortical small vessel disease - A consensus report." BMC Neurology 17, no. 1: 1-12.
The Reeler heterozygous mice (reln+/−) are haplodeficient in the gene (reln) encoding for the reelin glycoprotein (RELN) and display reductions in brain/peripheral RELN similar to autistic or schizophrenic patients. Cytoarchitectonic alterations of the reln+/− brain may be subtle, and are difficult to demonstrate by current histological approaches. We analyzed the number and topological organization of the Purkinje neurons (PNs) in five vermal lobules - central (II-III), culmen (IV-V), tuber (VIIb), uvula (IX), and nodulus (X) - that process different types of afferent functional inputs in reln+/+ and reln+/− adult mice (P60) of both sexes (n = 24). Animals were crossed with L7GFP mice so that the GFP-tagged PNs could be directly identified in cryosections. Digital images from these sections were processed with different open source software for quantitative topological and statistical analyses. Diversity indices calculated were: maximum caliper, density, area of soma, dispersion along the XZ axis, and dispersion along the YZ axis. We demonstrate: i. reduction in density of PNs in reln+/− males (14.37%) and reln+/− females (17.73%) compared to reln+/+ males; ii. that reln+/− males have larger PNs than other genotypes, and females (irrespective of the reln genetic background) have smaller PNs than reln+/+ males; iii. PNs are more chaotically arranged along the YZ axis in reln+/− males than in reln+/+ males and, except in central lobulus, reln+/− females. Therefore, image processing and statistics reveal previously unforeseen gender and genotype-related structural differences in cerebellum that may be clues for the definition of novel biomarkers in human psychiatric disorders.
Chiara Magliaro; Carolina Cocito; Stefano Bagatella; Adalberto Merighi; Arti Ahluwalia; Laura Lossi. The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse. Annals of Anatomy - Anatomischer Anzeiger 2016, 207, 68 -75.
AMA StyleChiara Magliaro, Carolina Cocito, Stefano Bagatella, Adalberto Merighi, Arti Ahluwalia, Laura Lossi. The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse. Annals of Anatomy - Anatomischer Anzeiger. 2016; 207 ():68-75.
Chicago/Turabian StyleChiara Magliaro; Carolina Cocito; Stefano Bagatella; Adalberto Merighi; Arti Ahluwalia; Laura Lossi. 2016. "The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse." Annals of Anatomy - Anatomischer Anzeiger 207, no. : 68-75.
A mutation of the reln gene gives rise to the Reeler mouse (reln-/-) displaying an ataxic phenotype and cerebellar hypoplasia. We have characterized the neurochemistry of postnatal (P0-P60) reln-/- mouse cerebella with specific attention to the intervention of cell proliferation and apoptosis in the P0-P25 interval. Homozygous reln-/- mice and age-matched controls were analyzed by immunofluorescence using primary antibodies against NeuN, calbindin, GFAP, vimentin, SMI32, and GAD67. Proliferation and apoptosis were detected after a single intraperitoneal BrdU injection and by the TUNEL assay with anti-digoxigenin rhodamine-conjugated antibodies. Quantitative analysis with descriptive and predictive statistics was used to calculate cell densities (number/mm2) after fluorescent nuclear stain (TCD, total cell density), labeling with BrdU (PrCD, proliferating cell density), or TUNEL (ApoCD, apoptotic cell density). By this approach we first have shown that the temporal pattern of expression of neuronal/glial markers in postnatal cerebellum is not affected by the Reeler mutation. Then, we have demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to reduction of cortical size and cellularity (TCD), and that TCD is, in turn, linked to quantitative differences in the extent of cell proliferation and apoptosis, as well as derangements in their temporal trends during postnatal maturation. Finally, we have calculated that PrCD is the most important predictive factor to determine TCD in the cerebellar cortex of the mutants. These results support the notion that, beside the well-known consequences onto the migration of the cerebellar neurons, the lack of Reelin results in a measurable deficit in neural proliferation.
Carolina Cocito; Adalberto Merighi; Mario Giacobini; Laura Lossi. Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum. Frontiers in Cellular Neuroscience 2016, 10, 141 .
AMA StyleCarolina Cocito, Adalberto Merighi, Mario Giacobini, Laura Lossi. Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum. Frontiers in Cellular Neuroscience. 2016; 10 ():141.
Chicago/Turabian StyleCarolina Cocito; Adalberto Merighi; Mario Giacobini; Laura Lossi. 2016. "Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum." Frontiers in Cellular Neuroscience 10, no. : 141.
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.
Laura Lossi; Livia D’Angelo; Paolo De Girolamo; Adalberto Merighi. Anatomical features for an adequate choice of experimental animal model in biomedicine: II. Small laboratory rodents, rabbit, and pig. Annals of Anatomy - Anatomischer Anzeiger 2016, 204, 11 -28.
AMA StyleLaura Lossi, Livia D’Angelo, Paolo De Girolamo, Adalberto Merighi. Anatomical features for an adequate choice of experimental animal model in biomedicine: II. Small laboratory rodents, rabbit, and pig. Annals of Anatomy - Anatomischer Anzeiger. 2016; 204 ():11-28.
Chicago/Turabian StyleLaura Lossi; Livia D’Angelo; Paolo De Girolamo; Adalberto Merighi. 2016. "Anatomical features for an adequate choice of experimental animal model in biomedicine: II. Small laboratory rodents, rabbit, and pig." Annals of Anatomy - Anatomischer Anzeiger 204, no. : 11-28.
After more than 70 years from its initial development, immunocytochemistry (ICC ) has become a fundamental technique in the study of the nervous system . After a brief excursus along the history of the different techniques that led to substantial amelioration of the original indirect immunofluorescence protocol, we discuss here the main advantages and disadvantages of the individual techniques for the study of central and peripheral neurons, in parallel with standardization , quantification , and reaction bias . Particular attention is given to immunofluorescence and its novel developments that allow high-resolution imaging at the light microscope level. The possibility of combining ICC with other fundamental techniques for analysis of neuronal circuitry such as neurotracing , electrophysiology , and molecular biology is also discussed, as well as a series of approaches for correlative light and electron microscopic studies.
Adalberto Merighi; Laura Lossi. The Evolution of Immunocytochemistry in the Dissection of Neural Complexity. Animal Models of Drug Addiction 2015, 101, 1 -35.
AMA StyleAdalberto Merighi, Laura Lossi. The Evolution of Immunocytochemistry in the Dissection of Neural Complexity. Animal Models of Drug Addiction. 2015; 101 ():1-35.
Chicago/Turabian StyleAdalberto Merighi; Laura Lossi. 2015. "The Evolution of Immunocytochemistry in the Dissection of Neural Complexity." Animal Models of Drug Addiction 101, no. : 1-35.
Transfection techniques in vitro and ex vivo (organotypic cultures ) offer an array of possibilities to investigate the consequence(s) of the introduction of foreign nucleic acids (DNA but also RNA ) and or other biologically active molecules into neurons , and to combine observations with immunocytochemistry . In particular, a wide number of fluorescent reporter proteins (FRPs ) can be employed for multicolor fluorescence imaging . Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy . These protocols allow analysis of live transfected cells , and, after fixation, correlative neurochemical studies.
Silvia Alasia; Adalberto Merighi; Laura Lossi. Transfection Techniques and Combined Immunocytochemistry in Cell Cultures and Organotypic Slices. Animal Models of Drug Addiction 2015, 101, 329 -355.
AMA StyleSilvia Alasia, Adalberto Merighi, Laura Lossi. Transfection Techniques and Combined Immunocytochemistry in Cell Cultures and Organotypic Slices. Animal Models of Drug Addiction. 2015; 101 ():329-355.
Chicago/Turabian StyleSilvia Alasia; Adalberto Merighi; Laura Lossi. 2015. "Transfection Techniques and Combined Immunocytochemistry in Cell Cultures and Organotypic Slices." Animal Models of Drug Addiction 101, no. : 329-355.
The discovery of neuronal cell death dates back to the nineteenth century. Nowadays, after a very long period of conceptual difficulties, the notion that cell death is a phenomenon occurring during the entire life course of the nervous system , from neurogenesis to adulthood and senescence , is fully established. The dichotomy between apoptosis , as the prototype of programmed cell death (PCD ), and necrosis , as the prototype of death caused by an external insult, must be carefully reconsidered, as different types of PCD: apoptosis, autophagy , pyroptosis , and oncosis have all been demonstrated in neurons (and glia ). These modes of PCD may be triggered by different stimuli, but share some intracellular pathways such that different types of cell death may affect the same population of neurons according to several intrinsic and extrinsic factors. Therefore, a mixed morphology is often observed also depending on degrees of differentiation , activity, and injury . The main histological and ultrastructural features of the different types of cell death in neurons are described and related to the cellular pathways that are specifically activated in any of these types of PCD.
Laura Lossi; Claudia Castagna; Adalberto Merighi. Neuronal Cell Death: An Overview of Its Different Forms in Central and Peripheral Neurons. Advanced Structural Safety Studies 2014, 1254, 1 -18.
AMA StyleLaura Lossi, Claudia Castagna, Adalberto Merighi. Neuronal Cell Death: An Overview of Its Different Forms in Central and Peripheral Neurons. Advanced Structural Safety Studies. 2014; 1254 ():1-18.
Chicago/Turabian StyleLaura Lossi; Claudia Castagna; Adalberto Merighi. 2014. "Neuronal Cell Death: An Overview of Its Different Forms in Central and Peripheral Neurons." Advanced Structural Safety Studies 1254, no. : 1-18.
As apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple cell points, different methods exist to evaluate the activity of the proteins involved in the intracellular apoptotic pathways and the phenotype of apoptotic neurons . Detention of the activity of the enzyme caspase-3 , the key executioner caspase in programmed cell death , by laser scanning confocal fluorescence microscopy and the fluorescence resonance energy transfer technology is an alternative approach to classical standard techniques, such as Western blotting , activity assays, or histological techniques, and allows working with both fixed and living cells . This technique combined with the organotypic culture approach ex vivo represents a valid tool for the study of the mechanisms of neuronal survival /death and neuroprotection.
Silvia Alasia; Carolina Cocito; Adalberto Merighi; Laura Lossi. Real-Time Visualization of Caspase-3 Activation by Fluorescence Resonance Energy Transfer (FRET). Methods in Molecular Biology 2014, 1254, 99 -113.
AMA StyleSilvia Alasia, Carolina Cocito, Adalberto Merighi, Laura Lossi. Real-Time Visualization of Caspase-3 Activation by Fluorescence Resonance Energy Transfer (FRET). Methods in Molecular Biology. 2014; 1254 ():99-113.
Chicago/Turabian StyleSilvia Alasia; Carolina Cocito; Adalberto Merighi; Laura Lossi. 2014. "Real-Time Visualization of Caspase-3 Activation by Fluorescence Resonance Energy Transfer (FRET)." Methods in Molecular Biology 1254, no. : 99-113.
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.
Serena Barral; Riccardo Beltramo; Chiara Salio; Patrizia Aimar; Laura Lossi; Adalberto Merighi. Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence. International Journal of Molecular Sciences 2014, 15, 1554 -1573.
AMA StyleSerena Barral, Riccardo Beltramo, Chiara Salio, Patrizia Aimar, Laura Lossi, Adalberto Merighi. Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence. International Journal of Molecular Sciences. 2014; 15 (1):1554-1573.
Chicago/Turabian StyleSerena Barral; Riccardo Beltramo; Chiara Salio; Patrizia Aimar; Laura Lossi; Adalberto Merighi. 2014. "Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence." International Journal of Molecular Sciences 15, no. 1: 1554-1573.
Alzheimer's disease (AD) is the major cause of dementia in old people. AD pathology is characterized by amyloid-β (Aβ) deposits in several regions of the brain, and links have been hypothesized between Aβ toxicity and apoptosis. Cerebellar granule cells (CGCs) have been widely used as in vitro tools for molecular studies correlating apoptosis with AD, although the cerebellum is a relatively spared area of the brain in vivo. We have used mixed neuronal-glial cerebellar cultures (NGCCs) and organotypic cerebellar cultures (OCCs) obtained from postnatal mice to assess the toxic effect of the Aβ oligomer 1-40 (Aβ1-40) after propidium iodide uptake in vitro. Our results demonstrate that NGCCs, which are primarily composed of CGCs, are resistant to Aβ1-40 challenge (5-10 μM) when cultured in physiological (5 mM) extracellular KCl ([K+]e) concentrations, i.e., in a condition in which CGCs undergo full maturation. Conversely, when 10 μM Aβ1-40 is given to NGCCs cultured in elevated (25 mM) [K+]e (and thus maintained in an immature state), there is a statistically significant increase in cell death. Cell death is by apoptosis, as demonstrated by ultrastructural examination. OCCs are resistant to Aβ challenge in any of the conditions tested (variation of [K+]e, presence or absence of serum, or addition of the neprilysin blocker phosphoramidon). Altogether these observations lead us to conclude that cerebellar cells in an organotypic context may be less susceptible to damage by Aβ, raising the question whether isolated CGCs are a reliable assay in drug discovery studies of AD.
Silvia Alasia; Patrizia Aimar; Adalberto Merighi; Laura Lossi. Context-Dependent Toxicity of Amyloid-β Peptides on Mouse Cerebellar Cells. Journal of Alzheimer's Disease 2012, 30, 41 -51.
AMA StyleSilvia Alasia, Patrizia Aimar, Adalberto Merighi, Laura Lossi. Context-Dependent Toxicity of Amyloid-β Peptides on Mouse Cerebellar Cells. Journal of Alzheimer's Disease. 2012; 30 (1):41-51.
Chicago/Turabian StyleSilvia Alasia; Patrizia Aimar; Adalberto Merighi; Laura Lossi. 2012. "Context-Dependent Toxicity of Amyloid-β Peptides on Mouse Cerebellar Cells." Journal of Alzheimer's Disease 30, no. 1: 41-51.
Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.
Laura Lossi; Graziana Gambino; Francesco Ferrini; Silvia Alasia; Adalberto Merighi. Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival. Developmental Neurobiology 2009, 69, 855 -870.
AMA StyleLaura Lossi, Graziana Gambino, Francesco Ferrini, Silvia Alasia, Adalberto Merighi. Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival. Developmental Neurobiology. 2009; 69 (13):855-870.
Chicago/Turabian StyleLaura Lossi; Graziana Gambino; Francesco Ferrini; Silvia Alasia; Adalberto Merighi. 2009. "Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival." Developmental Neurobiology 69, no. 13: 855-870.
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.
Laura Lossi; Silvia Alasia; Chiara Salio; Adalberto Merighi. Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Progress in Neurobiology 2009, 88, 221 -245.
AMA StyleLaura Lossi, Silvia Alasia, Chiara Salio, Adalberto Merighi. Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Progress in Neurobiology. 2009; 88 (4):221-245.
Chicago/Turabian StyleLaura Lossi; Silvia Alasia; Chiara Salio; Adalberto Merighi. 2009. "Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS." Progress in Neurobiology 88, no. 4: 221-245.
Laura Lossi; Graziana Gambino. Apoptosis of the cerebellar neurons. Histol. Histopathol. 2008, 23, 1 .
AMA StyleLaura Lossi, Graziana Gambino. Apoptosis of the cerebellar neurons. Histol. Histopathol.. 2008; 23 (3):1.
Chicago/Turabian StyleLaura Lossi; Graziana Gambino. 2008. "Apoptosis of the cerebellar neurons." Histol. Histopathol. 23, no. 3: 1.
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.
Claudia Salvadori; Laura Lossi; Mario Arispici; Carlo Cantile. Spongiform neurodegenerative disease in a Persian kitten. Journal of Feline Medicine and Surgery 2007, 9, 242 -245.
AMA StyleClaudia Salvadori, Laura Lossi, Mario Arispici, Carlo Cantile. Spongiform neurodegenerative disease in a Persian kitten. Journal of Feline Medicine and Surgery. 2007; 9 (3):242-245.
Chicago/Turabian StyleClaudia Salvadori; Laura Lossi; Mario Arispici; Carlo Cantile. 2007. "Spongiform neurodegenerative disease in a Persian kitten." Journal of Feline Medicine and Surgery 9, no. 3: 242-245.
It is generally assumed that about half of the neurons produced during neurogenesis die before completion of maturation of the central nervous system (CNS). Neural cell death is also relevant in aging and several neurodegenerative diseases. Among the modalities by which neurons die, apoptosis has very much attracted the interest of investigators because in this type of cell death neurons are actively responsible for their own demise by switching on a number of genes and activating a series of specific intracellular pathways. This review focuses on the cellular and molecular mechanisms of apoptosis in normal and transgenic animal models related to naturally occurring neuronal death within the CNS. We will also consider some examples of apoptotic cell death in canine neuropathologies. A thorough analysis of naturally occurring neuronal death in vivo will offer a basis for parallel and future studies involving secondary neuronal loss such as those in neurodegenerative disorders, trauma or ischaemia.
L. Lossi; C. Cantile; I. Tamagno; Adalberto Merighi. Apoptosis in the mammalian CNS: Lessons from animal models. The Veterinary Journal 2005, 170, 52 -66.
AMA StyleL. Lossi, C. Cantile, I. Tamagno, Adalberto Merighi. Apoptosis in the mammalian CNS: Lessons from animal models. The Veterinary Journal. 2005; 170 (1):52-66.
Chicago/Turabian StyleL. Lossi; C. Cantile; I. Tamagno; Adalberto Merighi. 2005. "Apoptosis in the mammalian CNS: Lessons from animal models." The Veterinary Journal 170, no. 1: 52-66.