Non-Newly Generated, "Immature" Neurons in the Sheep Brain Are Not Restricted to Cerebral Cortex.

A newly proposed form of brain structural plasticity consists of non-newly generated, "immature" neurons of the adult cerebral cortex. Similar to newly generated neurons, these cells express the cytoskeletal protein Doublecortin (DCX), yet they are generated prenatally and then remain in a state of immaturity for long periods. In rodents, the immature neurons are restricted to the paleocortex, whereas in other mammals, they are also found in neocortex. Here, we analyzed the DCX-expressing cells in the whole sheep brain of both sexes to search for an indicator of structural plasticity at a cellular level in a relatively large-brained, long-living mammal. Brains from adult and newborn sheep (injected with BrdU and analyzed at different survival times) were processed for DCX, cell proliferation markers (Ki-67, BrdU), pallial/subpallial developmental origin (Tbr1, Sp8), and neuronal/glial antigens for phenotype characterization. We found immature-like neurons in the whole sheep cortex and in large populations of DCX-expressing cells within the external capsule and the surrounding gray matter (claustrum and amygdala). BrdU and Ki-67 detection at neonatal and adult ages showed that all of these DCX+ cells were generated during embryogenesis, not after birth. These results show that the adult sheep, unlike rodents, is largely endowed with non-newly generated neurons retaining immature features, suggesting that such plasticity might be particularly important in large-brained, long-living mammals.SIGNIFICANCE STATEMENT Brain plasticity is important in adaptation and brain repair. Structural changes span from synaptic plasticity to adult neurogenesis, the latter being highly reduced in large-brained, long-living mammals (e.g., humans). The cerebral cortex contains "immature" neurons, which are generated prenatally and then remain in an undifferentiated state for long periods, being detectable with markers of immaturity. We studied the distribution and developmental origin of these cells in the whole brain of sheep, relatively large-brained, long-living mammals. In addition to the expected cortical location, we also found populations of non-newly generated neurons in several subcortical regions (external capsule, claustrum, and amygdala). These results suggests that non-neurogenic, parenchymal structural plasticity might be more important in large mammals with respect to adult neurogenesis.

Methylation of cytokines gene promoters in IL-1ß-treated human intestinal epithelial cells.

OBJECTIVE AND DESIGN: Epigenetic regulation is important in the activation of inflammatory cells. In the present study, we evaluated if DNA-methylation variations are involved in Interleukin-1ß (IL-1ß)-induced intestinal epithelial cells activation. MATERIALS AND METHODS: Differentiated Caco-2 cells were exposed to IL-1ß or to 5-azadeoxycytidine (5-azadC) for 24 or 48 h. Genome-wide methylation status was evaluated, while DNA methylation status at the promoter region of the gene encoding interleukin-6, 8 and 10 (IL-6, 8 and 10) was estimated. The levels of the corresponding gene products as well as DNA methyltransferases (DNMTs) quantity were assessed. RESULTS: IL-1ß decreased genomic methylation of human intestinal epithelial cells and induced demethylation at cg-specific sites at the promoter of pro-inflammatory genes IL6 and IL8; conversely it did not change the methylation of the IL10 promoter. IL-1ß also increased the release of IL-6 and IL-8 but did not change the IL-10 expression. Finally, cell exposure to IL-1ß decreased the DNMT3b expression, increased DNMT3a and was not able to change DNMT1 expression. CONCLUSIONS: Our results suggest a potential role of IL-1ß as modulator of DNA methylation in activated differentiated Caco-2 cell line.

Adult neurogenesis and "immature" neurons in mammals: an evolutionary trade-off in plasticity?

Neuronal plasticity can vary remarkably in its form and degree across animal species. Adult neurogenesis, namely the capacity to produce new neurons from neural stem cells through adulthood, appears widespread in non-mammalian vertebrates, whereas it is reduced in mammals. A growing body of comparative studies also report variation in the occurrence and activity of neural stem cell niches between mammals, with a general trend of reduction from small-brained to large-brained species. Conversely, recent studies have shown that large-brained mammals host large amounts of neurons expressing typical markers of neurogenesis in the absence of cell division. In layer II of the cerebral cortex, populations of prenatally generated, non-dividing neurons continue to express molecules indicative of immaturity throughout life (cortical immature neurons; cINs). After remaining in a dormant state for a very long time, these cINs retain the potential of differentiating into mature neurons that integrate within the preexisting neural circuits. They are restricted to the paleocortex in small-brained rodents, while extending into the widely expanded neocortex of highly gyrencephalic, large-brained species. The current hypothesis is that these populations of non-newly generated "immature" neurons might represent a reservoir of developmentally plastic cells for mammalian species that are characterized by reduced stem cell-driven adult neurogenesis. This indicates that there may be a trade-off between various forms of plasticity that coexist during brain evolution. This balance may be necessary to maintain a "reservoir of plasticity" in brain regions that have distinct roles in species-specific socioecological adaptations, such as the neocortex and olfactory structures.

Age-related changes in layer II immature neurons of the murine piriform cortex.

The recent identification of a population of non-newly born, prenatally generated "immature" neurons in the layer II of the piriform cortex (cortical immature neurons, cINs), raises questions concerning their maintenance or depletion through the lifespan. Most forms of brain structural plasticity progressively decline with age, a feature that is particularly prominent in adult neurogenesis, due to stem cell depletion. By contrast, the entire population of the cINs is produced during embryogenesis. Then these cells simply retain immaturity in postnatal and adult stages, until they "awake" to complete their maturation and ultimately integrate into neural circuits. Hence, the question remains open whether the cINs, which are not dependent on stem cell division, might follow a similar pattern of age-related reduction, or in alternative, might leave a reservoir of young, undifferentiated cells in the adult and aging brain. Here, the number and features of cINs were analyzed in the mouse piriform cortex from postnatal to advanced ages, by using immunocytochemistry for the cytoskeletal marker doublecortin. The abundance and stage of maturation of cINs, along with the expression of other markers of maturity/immaturity were investigated. Despite a marked decrease in this neuronal population during juvenile stages, reminiscent of that observed in hippocampal neurogenesis, a small amount of highly immature cINs persisted up to advanced ages. Overall, albeit reducing in number with increasing age, we report that the cINs are present through the entire animal lifespan.

Indicaxanthin Induces Autophagy in Intestinal Epithelial Cancer Cells by Epigenetic Mechanisms Involving DNA Methylation.

Autophagy is an evolutionarily conserved process critical in maintaining cellular homeostasis. Recently, the anticancer potential of autophagy inducers, including phytochemicals, was suggested. Indicaxanthin is a betalain pigment found in prickly pear fruit with antiproliferative and pro-apoptotic activities in colorectal cancer cells associated with epigenetic changes in selected methylation-silenced oncosuppressor genes. Here, we demonstrate that indicaxanthin induces the up-regulation of the autophagic markers LC3-II and Beclin1, and increases autophagolysosome production in Caco-2 cells. Methylomic studies showed that the indicaxanthin-induced pro-autophagic activity was associated with epigenetic changes. In addition to acting as a hypermethylating agent at the genomic level, indicaxanthin also induced significant differential methylation in 39 out of 47 autophagy-related genes, particularly those involved in the late stages of autophagy. Furthermore, in silico molecular modelling studies suggested a direct interaction of indicaxanthin with Bcl-2, which, in turn, influenced the function of Beclin1, a key autophagy regulator. External effectors, including food components, may modulate the epigenetic signature of cancer cells. This study demonstrates, for the first time, the pro-autophagic potential of indicaxanthin in human colorectal cancer cells associated with epigenetic changes and contributes to outlining its potential healthy effect in the pathophysiology of the gastrointestinal tract.

Brain Structural Plasticity: From Adult Neurogenesis to Immature Neurons.

Brain structural plasticity is an extraordinary tool that allows the mature brain to adapt to environmental changes, to learn, to repair itself after lesions or disease, and to slow aging. A long history of neuroscience research led to fascinating discoveries of different types of plasticity, involving changes in the genetically determined structure of nervous tissue, up to the ultimate dream of neuronal replacement: a stem cell-driven "adult neurogenesis" (AN). Yet, this road does not seem a straight one, since mutable dogmas, conflicting results and conflicting interpretations continue to warm the field. As a result, after more than 10,000 papers published on AN, we still do not know its time course, rate or features with respect to other kinds of structural plasticity in our brain. The solution does not appear to be behind the next curve, as differences among mammals reveal a very complex landscape that cannot be easily understood from rodents models alone. By considering evolutionary aspects, some pitfalls in the interpretation of cell markers, and a novel population of undifferentiated cells that are not newly generated [immature neurons (INs)], we address some conflicting results and controversies in order to find the right road forward. We suggest that considering plasticity in a comparative framework might help assemble the evolutionary, anatomical and functional pieces of a very complex biological process with extraordinary translational potential.

Phylogenetic variation in cortical layer II immature neuron reservoir of mammals.

The adult mammalian brain is mainly composed of mature neurons. A limited amount of stem cell-driven neurogenesis persists in postnatal life and is reduced in large-brained species. Another source of immature neurons in adult brains is cortical layer II. These cortical immature neurons (cINs) retain developmentally undifferentiated states in adulthood, though they are generated before birth. Here, the occurrence, distribution and cellular features of cINs were systematically studied in 12 diverse mammalian species spanning from small-lissencephalic to large-gyrencephalic brains. In spite of well-preserved morphological and molecular features, the distribution of cINs was highly heterogeneous, particularly in neocortex. While virtually absent in rodents, they are present in the entire neocortex of many other species and their linear density in cortical layer II generally increased with brain size. These findings suggest an evolutionary developmental mechanism for plasticity that varies among mammalian species, granting a reservoir of young cells for the cerebral cortex.

To acquire new skills or recover after injuries, the mammalian brain relies on plasticity, the ability for the brain to change its architecture and its connections during the lifetime of an animal. Creating new nerve cells is one way to achieve plasticity, but this process is rarer in humans than it is in mammals with smaller brains. In particular, it is absent in the human cortex: this region is enlarged in species with large brains, where it carries out complex tasks such as learning and memory. Producing new cells in the cortex would threaten the stability of the structures that retain long-term memories. Another route to plasticity is to reshape the connections between existing, mature nerve cells. This process takes place in the human brain during childhood and adolescence, as some connections are strengthened and others pruned away. An alternative mechanism relies on keeping some nerve cells in an immature, 'adolescent' state. When needed, these nerve cells emerge from their state of arrested development and 'grow up', connecting with the appropriate brain circuits. This mechanism does not involve producing new nerve cells, and so it would be suitable to maintain plasticity in the cortex. Consistent with this idea, in mice some dormant nerve cells are present in a small, primitive part of the cortex. La Rosa et al. therefore wanted to determine if the location and number of immature cells in the cortex differed between mammals, and if so, whether these differences depended on brain size. The study spanned 12 mammal species, from small-brained species like mice to larger-brained animals including sheep and non-human primates. Microscopy imaging was used to identify immature nerve cells in brain samples, which revealed that the cortex in larger-brained species contained more adolescent cells than its mouse counterpart. The difference was greatest in a region called the neocortex, which has evolved most recently. This area is most pronounced in primates ­ especially humans ­ where it carries out high-level cognitive tasks. These results identify immature nerve cells as a potential mechanism for plasticity in the cortex. La Rosa et al. hope that the work will inspire searches for similar reservoirs of young cells in humans, which could perhaps lead to new treatments for brain disorders like dementia.

Newly Generated and Non-Newly Generated "Immature" Neurons in the Mammalian Brain: A Possible Reservoir of Young Cells to Prevent Brain Aging and Disease?

Brain plasticity is important for translational purposes since most neurological disorders and brain aging problems remain substantially incurable. In the mammalian nervous system, neurons are mostly not renewed throughout life and cannot be replaced. In humans, the increasing life expectancy explains the increase in brain health problems, also producing heavy social and economic burden. An exception to the "static" brain is represented by stem cell niches leading to the production of new neurons. Such adult neurogenesis is dramatically reduced from fish to mammals, and in large-brained mammals with respect to rodents. Some examples of neurogenesis occurring outside the neurogenic niches have been reported, yet these new neurons actually do not integrate in the mature nervous tissue. Non-newly generated, "immature" neurons (nng-INs) are also present: Prenatally generated cells continuing to express molecules of immaturity (mostly shared with the newly born neurons). Of interest, nng-INs seem to show an inverse phylogenetic trend across mammals, being abundant in higher-order brain regions not served by neurogenesis and providing structural plasticity in rather stable areas. Both newly generated and nng-INs represent a potential reservoir of young cells (a "brain reserve") that might be exploited for preventing the damage of aging and/or delay the onset/reduce the impact of neurological disorders.

Brain Plasticity in Mammals: An Example for the Role of Comparative Medicine in the Neurosciences.

Comparative medicine deals with similarities and differences between veterinary and human medicine. All mammals share most basic cellular and molecular mechanisms, thus justifying murine animal models in a translational perspective; yet "mice are not men," thus some biases can emerge when complex biological processes are concerned. Brain plasticity is a cutting-edge, expanding topic in the field of Neurosciences with important translational implications, yet, with remarkable differences among mammals, as emerging from comparative studies. In particular, adult neurogenesis (the genesis of new neurons from brain stem cell niches) is a life-long process in laboratory rodents but a vestigial, mostly postnatal remnant in humans and dolphins. Another form of "whole cell" plasticity consisting of a population of "immature" neurons which are generated prenatally but continue to express markers of immaturity during adulthood has gained interest more recently, as a reservoir of young neurons in the adult brain. The distribution of the immature neurons also seems quite heterogeneous among different animal species, being confined within the paleocortex in rodents while extending into neocortex in other mammals. A recent study carried out in sheep, definitely showed that gyrencephalic, large-sized brains do host higher amounts of immature neurons, also involving subcortical, white, and gray matter regions. Hence, "whole cell" plasticity such as adult neurogenesis and immature neurons are biological processes which, as a whole, cannot be studied exclusively in laboratory rodents, but require investigation in comparative medicine, involving large-sized, long-living mammals, in order to gain insights for translational purposes.

Clusters of DCX+ cells "trapped" in the subcortical white matter of early postnatal Cetartiodactyla (Tursiops truncatus, Stenella coeruloalba and Ovis aries).

The cytoskeletal protein doublecortin (DCX) is a marker for neuronal cells retaining high potential for structural plasticity, originating from both embryonic and adult neurogenic processes. Some of these cells have been described in the subcortical white matter of neonatal and postnatal mammals. In mice and humans it has been shown they are young neurons migrating through the white matter after birth, reaching the cortex in a sort of protracted neurogenesis. Here we show that DCX+ cells in the white matter of neonatal and young Cetartiodactyla (dolphin and sheep) form large clusters which are not newly generated (in sheep, and likely neither in dolphins) and do not reach the cortical layers, rather appearing "trapped" in the white matter tissue. No direct contact or continuity can be observed between the subventricular zone region and the DCX+ clusters, thus indicating their independence from any neurogenic source (in dolphins further confirmed by the recent demonstration that periventricular neurogenesis is inactive since birth). Cetartiodactyla include two orders of large-brained, relatively long-living mammals (cetaceans and artiodactyls) which were recognized as two separate monophyletic clades until recently, yet, despite the evident morphological distinctions, they are monophyletic in origin. The brain of Cetartiodactyla is characterized by an advanced stage of development at birth, a feature that might explain the occurrence of "static" cell clusters confined within their white matter. These results further confirm the existence of high heterogeneity in the occurrence, distribution and types of structural plasticity among mammals, supporting the emerging view that multiple populations of DCX+, non-newly generated cells can be abundant in large-brained, long-living species.