Satellite cells of te dorsal root ganglia in neuronal hypertrophy]

Amputation of the lizard tail is followed by its complete regeneration over a period of six-eight months. The new tail is innervated only by the last three pairs of spinal nerves upstream from the plane of amputation, since no nerve cells are present in the regenerated. The corresponding dorsal root ganglia increase in volume (hypertrophic ganglia) and most of their sensory neurons become hypertrophic. Satellite cells belonging to this hypertrophic ganglia increase in number. This paper describes an autoradiographic study, after administration of tritiated thymidine, of the hypertrophic dorsal root ganglia of the lizard during tail regeneration. We evaluated the number of satellite cells which neo-synthetize DNA ("labeling index = LI%) and are therefore suitable to undergo cell division. The LI% was significatively increased in hypertrophic ganglia when compared to internal control ganglia (not directly involved in the reinnervation process) and normal ganglia (lizards with intact tails). The comparison between internal control ganglia and normal ganglia showed higher LI% values in the formers, although this difference was not statistically significative. These results are in line with those obtained by other authors and suggest that satellite cells of dorsal root ganglia can undergo cellular proliferation also in the adult, especially in particular experimental conditions.     

Reduced tail regeneration in the Common Lizard, Lacerta vivipara, parasitized by blood parasites

1. Many lizards will lose their tail through autotomy as an antipredator device even though there must be significant costs during tail regeneration.
2. Parasites are energetically costly to the host, and may reduce the rate of cell regeneration. The relation between the presence of haemogregarines (phylum Sporozoa) and the rate of tail regeneration in the Common Lizard Lacerta vivipara (Jacquin) was examined.
3. Experimentally induced autotomy in parasitized lizards resulted in a significantly reduced rate of tail regeneration compared with non-parasitized lizards. On the other hand, tail loss was not associated with an abnormal increase of parasite load, suggesting that the physiological stress (induced by tail loss) did not cause a decrease in parasite defence.     

Differentiation of liquor contacting neurons in the regenerating spinal cord of the newt Triturus

This paper reports a detailed electron microscopic study of the ventricular cells in the growing spinal cord during the first two months of tail regeneration in Triturus vulgaris and T. cristatus. In the proliferating ependyma some cells undergo a neural differentiation in order to produce "Cerebro-Spinal-Fluid-Contacting-Neurons (CSFCNs). These round or pear-like cells store dense core vesicles, 80-100 nm in diameter, and show less electron-density than ependymal elements. The stereociliar apparatus is the last cytological differentiation of these elements and after two months of regeneration, synapse-like endings were occasionally found.     

Microscopical observations on the regenerating tail in the tuatara Sphenodon punctatus indicate a tendency to scarring,but also influence from somatic growth

The process of tail regeneration in the tuatara (Sphenodon punctatus) is not entirely known. Similarity to and differences from lizard tail regenerations are indicated in the present histological and ultrastructural study. Regeneration is influenced by the animal's age and ambient temperature, but in comparison to that of lizards it is very slow and tends to produce outgrowths that do not reach the length of the original tail. Although microscopically similar to lizard blastemas, the mesenchyme rapidly gives rise to a dense connective tissue that contains few muscle bundles, nerves, and fat cells. The unsegmented cartilaginous tube forming the axial skeleton is not calcified after 5 months of regeneration, but calcification in the inner region of the cartilage, present after 10 months, increases thereafter. Amyelinic and myelinic peripheral nerves are seen within the regenerating tails of 2–3 mm in length and the spinal cord forms an ependymal tube inside a cartilaginous casing. Tissues of the original tail, like muscles, vertebrae and the adipose mass, are largely replaced by dense connective tissue that occupies most of the volume of the new tail at 5 and 10 months of regeneration. It is unknown whether the differentiation of the dense connective tissue is caused by the relatively low temperature that this species lives under or stems from a genetic predisposition toward scarring as with most other amniotes. Increases of muscle and adipose tissues seen in older regenerated tails derive from somatic growth of the new tail in the years following tail loss and not from a rapid regeneration process like that in lizards.     

Fine observation on nerves colonizing the regenerating tail of the lizard Podarcis sicula

During the regeneration of lizard tail, nerves sprouting from ganglia and the spinal cord invade the blastema as far as the apical epidermis. Electron microscopical observations reveal axons storing dense granules (dg) and dense core vesicles (dcv) which are concentrated in nerve terminals or in axoplasmatic regions. In the regenerating spinal cord (SC) these terminals resemble aminergic-peptidergic endings and grow as far as the distal portion of the SC, which is made up of irregularly arranged ependymal cells. Some axons storing dcv contact blastematic cells and other nerve terminals show a plasma membrane incomplete or broken. Whether this latter aspect is due to fixation artifacts or physiological rupture is unknown. Nerves containing dcv and a few dg also originate from spinal ganglia innervating the regenerating tail. The accumulation of material into these endings is probably slow and a possible trophic influence on the regeneration of lizard tail is discussed.     

The role of tanycytes in hypothalamic glucosensing

Tanycytes are elongated hypothalamic glial cells that cover the basal walls of the third ventricle; their apical regions contact the cerebrospinal fluid (CSF), and their processes reach hypothalamic neuronal nuclei that control the energy status of an organism. These nuclei maintain the balance between energy expenditure and intake, integrating several peripheral signals and triggering cellular responses that modify the feeding behaviour and peripheral glucose homeostasis. One of the most important and well‐studied signals that control this process is glucose; however, the mechanism by which this molecule is sensed remains unknown. We along with others have proposed that tanycytes play a key role in this process, transducing changes in CSF glucose concentration to the neurons that control energy status. Recent studies have demonstrated the expression and function of monocarboxylate transporters and canonical pancreatic β cell glucose sensing molecules, including glucose transporter 2 and glucokinase, in tanycytes. These and other data, which will be discussed in this review, suggest that hypothalamic glucosensing is mediated through a metabolic interaction between tanycytes and neurons through lactate. This article will summarize the recent evidence that supports the importance of tanycytes in hypothalamic glucosensing, and discuss the possible mechanisms involved in this process. Finally, it is important to highlight that a detailed analysis of this mechanism could represent an opportunity to understand the evolution of associated pathologies, including diabetes and obesity, and identify new candidates for therapeutic intervention.     

The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema

Review. The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema. Acta Zoologica (Stockolm). Molecular studies on lizard tail regeneration indicate that the blastema stage is a tumour-like outgrowth capable of self-regulate to produce a new tail. Various oncogenes and tumour suppressors are expressed, and their proteins are localized in specific regions of the growing blastema. SnoRNAs are exclusively overexpressed in the tail blastema suggesting changes in ribosome translation efficiency in blastema cells, like in cancer. Blastema cells secrete high levels of hyaluronate and adopt an anaerobic metabolism (Warburg effect). These studies indicate that the lizard blastema represents a unique case among terrestrial vertebrates of physiological tumour remission. Mesenchymal cells and fibroblasts forming the blastema are turned within 1–2 months into a functional organ, the tail. In vitro studies on isolated mesenchymal cells from the regenerative blastema shows that these cells do not undergo contact inhibition but continue proliferation after confluence, and contain nestin, vimentin and K17. After 2–3 weeks they stratify into 5–7 layers forming a pellicle of loose connective tissue. Future molecular studies on genes and proteins that allow the control of growth in the lizard blastema may help to determine how lizards turn a tumour into a new organ with numerous differentiated and functional tissues, providing clues on cancer growth regulation.     

Immunolocalization of Wnts in the lizard blastema supports a key role of these signaling proteins for tail regeneration

A highly upregulated gene during tail regeneration in lizards is Wnt2b, a gene broadly expressed during development. The present study examines the distribution of Wnt proteins, most likely wnt2b, by western blotting and immunofluorescence in the blastema-cone of lizards using a specific antibody produced against a lizard Wnt2b protein. Immunopositive bands at 48–50 and 18 kDa are present in the regenerative blastema, the latter likely as a degradation product. Immunofluorescence is mainly observed in the wound epidermis, including in the Apical Epidermal Peg where the protein appears localized in intermediate and differentiating keratinocytes. Labeling is more intense along the perimeter of keratinocytes, possibly as a secretory product, and indicates that the high epidermal proliferation of the regenerating epidermis is sustained by Wnt proteins. The regenerating spinal cord forms an ependymal tube within the blastema and shows immunolabeling especially in the cytoplasm of ependymal cells contacting the central canal where some secretion might occur. Also, regenerating nerves and proximal spinal ganglia innervating the regenerating blastema contain this signaling protein. In contrast, the blastema mesenchyme, muscles and cartilage show weak immunolabeling that tends to disappear in tissues located in more proximal regions, close to the original tail. However, a distal to proximal gradient of Wnt proteins was not detected. The present study supports the hypothesis that Wnt proteins, in particular Wnt2b, are secreted by the apical epidermis covering the blastema and released into the mesenchyme where they stimulate cell multiplication.     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

Hyperphosphorylation and cleavage at D421 enhance tau secretion

It is well established that tau pathology propagates in a predictable manner in Alzheimer's disease (AD). Moreover, tau accumulates in the cerebrospinal fluid (CSF) of AD's patients. The mechanisms underlying the propagation of tau pathology and its accumulation in the CSF remain to be elucidated. Recent studies have reported that human tau was secreted by neurons and non-neuronal cells when it was overexpressed indicating that tau secretion could contribute to the spreading of tau pathology in the brain and could lead to its accumulation in the CSF. In the present study, we showed that the overexpression of human tau resulted in its secretion by Hela cells. The main form of tau secreted by these cells was cleaved at the C-terminal. Surprisingly, secreted tau was dephosphorylated at several sites in comparison to intracellular tau which presented a strong immunoreactivity to all phospho-dependent antibodies tested. Our data also revealed that phosphorylation and cleavage of tau favored its secretion by Hela cells. Indeed, the mimicking of phosphorylation at 12 sites known to be phosphorylated in AD enhanced tau secretion. A mutant form of tau truncated at D421, the preferential cleavage site of caspase-3, was also significantly more secreted than wild-type tau. Taken together, our results indicate that hyperphosphorylation and cleavage of tau by favoring its secretion could contribute to the propagation of tau pathology in the brain and its accumulation in the CSF.     

Spinal cord is required for proper regeneration of the tail in Xenopus tadpoles

Tail regeneration in urodeles is dependent on the spinal cord (SC), but it is believed that anuran larvae regenerate normal tails without the SC. To evaluate the precise role of the SC in anuran tail regeneration, we developed a simple operation method to ablate the SC completely and minimize the damage to the tadpole using Xenopus laevis . The SC-ablated tadpole regenerated a twisted and smaller tail. These morphological abnormalities were attributed to defects in the notochord (NC), as the regenerated NC in the SC-ablated tail was short, slim and twisted. The SC ablation never affected the early steps of the regeneration, including closure of the amputated surface with epidermis and accumulation of the NC precursor cells. The proliferation rate of the NC precursor cells, however, was reduced, and NC cell maturation was retarded in the SC-ablated tail. These results show that the SC has an essential role in the normal tail regeneration of Xenopus larvae, especially in the proliferation and differentiation of the NC cells. Gene expression analysis and implantation of a bead soaked with growth factor showed that fibroblast growth factor-2 and -10 were involved in the signaling molecules, which were expressed in the SC and stimulated growth of the NC cells.     

Caudal autotomy and regeneration in lizards

Caudal autotomy, or the voluntary self-amputation of the tail, is an anti-predation strategy in lizards that depends on a complex array of environmental, individual, and species-specific characteristics. These factors affect both when and how often caudal autotomy is employed, as well as its overall rate of success. The potential costs of autotomy must be weighed against the benefits of this strategy. Many species have evolved specialized behavioral and physiological adaptations to minimize or compensate for any negative consequences. One of the most important steps following a successful autotomous escape involves regeneration of the lost limb. In some species, regeneration occurs rapidly; such swift regeneration illustrates the importance of an intact, functional tail in everyday experience. In lizards and other vertebrates, regeneration is a highly ordered process utilizing initial developmental programs as well as regeneration-specific mechanisms to produce the correct types and pattern of cells required to sufficiently restore the structure and function of the sacrificed tail. In this review, we discuss the behavioral and physiological features of self-amputation, with particular reference to the costs and benefits of autotomy and the basic mechanisms of regeneration. In the process, we identify how these behaviors could be used to explore the neural regulation of complex behavioral responses within a functional context.     

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Background

Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius.

Results

Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway.

Conclusions

We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.     

Relationship between urotensin II- and somatostatin-immunoreactive spinal cord neurons of Catostomus commersoni and Oncorhynchus kisutch (Teleostei)

Summary This immunocytochemical study describes the presence of separate immunoreactive (IR)-urotensin II (UII) and IR-somatostatin (SOM) systems in the spinal cord of two species of teleost fish. Both systems are arranged in a close spatial interrelationship in which IR-SOM fibres apparently innervate cerebrospinal fluid (CSF)-contacting IR-UII neurons. Specimens of Oncorhynchus kisutch also display CSF-contacting IR-SOM neurons located in the lateral ependymal walls of the central canal, in addition to CSF-contacting IR-UII neurons located ventrally. It is suggested that, in this species, CSF-contacting IR-SOM and IR-UII neurons perceive different stimuli from the CSF and are integrated in such a way that one peptidergic system may modulate the function of the other.     

The caudal spinal cord of coho salmon (Oncorhynchus kisutch): Immunocytochemical evidence of a “caudal serotoninergic system”

Summary The caudal spinal cord of the coho salmon was investigated by means of immunocytochemistry using antisera against serotonin, urotensin I, urotensin II, somatostatin and a urea-extract of bovine Reissner's fiber (AFRU). Populations of serotonin-immunoreactive (IR) neurons were found rostral and dorsal to the urophysis in close spatial association with caudal secretory neurons. Thick, smooth serotonin-IR processes extended toward the external surface of the spinal cord where they displayed conspicuous terminal dilatations. Thin, beaded serotonin-IR fibers appeared to innervate populations of caudal secretory and somatostatin-IR cerebrospinal fluid-contacting neurons. Most caudal neurosecretory cells displayed both urotensin I and urotensin II immunoreactivities; only a minority reacted exclusively with either urotensin I or urotensin II antisera. Urotensin II-IR and somatostatin-IR cerebrospinal fluid (CSF)-contacting neurons were found as an integral component of the central canal wall in the caudal spinal cord and filum terminale; their dendritic processes appeared to contact Reissner's fiber, which displayed a weak AFRU-immunoreactivity while inside the central canal, but became strongly reactive in the interior of the terminal ventricle as it formed the massa caudalis. The distribution of serotoninergic processes points to a regulatory role in the function of caudal secretory and CSF-contacting neurons and to a putative serotonin release into the subarachnoid space and/or meningeal vasculature. It is also suggested that the CSF-contacting neurons of the central canal may participate in a feedback mechanism controlling the secretory activity of the subcommissural organ.Supported by Grant A/1095-1 from the International Foundation for Science, Sweden, to C.Y.; Grant I/63-476 from Volkswagen-Stiftung to E.R.; and Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile     

Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.     

The system of cerebrospinal fluid-contacting neurons. Its supposed role in the nonsynaptic signal transmission of the brain

Recent investigations confirm the importance of nonsynaptic signal transmission in several functions of the nervous tissue. Present in various periventricular brain regions of vertebrates, the system of cerebrospinal fluid (CSF)-contacting neurons seems to have a special role in taking up, transforming and emitting nonsynaptic signals mediated by the internal and external CSF and intercellular fluid of the brain. Most of the CSF-contacting nerve cells send dendritic processes into the internal CSF of the brain ventricles or central canal where they form terminals bearing stereocilia and a 9+0-, or 9+2-type cilium. Some of these neurons resemble known sensory cells of chemoreceptor-type, others may be sensitive to the pressure or flow of the CSF, or to the illumination of the brain tissue. The axons of the CSF-contacting neurons transmit information taken up by dendrites and perikarya to synaptic zones of various brain areas. By forming neurohormonal terminals, axons also contact the external CSF space and release various bioactive substances there. Some perikarya send their axons into the internal CSF, and form free endings there, or synapses on intraventricular dendrites, perikarya and/or on the ventricular surface of ependymal cells. Contacting the intercellular space, sensory-type cilia were also demonstrated on nerve cells situated in the brain tissue subependymally or farther away from the ventricles. Among neuronal elements entering the internal CSF-space, the hypothalamic CSF-contacting neurons are present in the magnocellular and parvicellular nuclei and in some circumventricular organs like the paraventricular organ and the vascular sac. The CSF-contacting dendrites of all these areas bear a solitary 9 x 2+0-type cilium and resemble chemoreceptors cytologically. In electrophysiological experiments, the neurons of the paraventricular organ are highly sensitive to the composition of the ventricular CSF. The axons of the CSF-contacting neurons terminate not only in the hypothalamic synaptic zones but also in tel-, mes- and rhombencephalic nuclei and reach the spinal cord as well. The supposed chemical information taken up by the CSF-contacting neurons from the ventricular CSF may influence the function of these areas of the central nervous system. Some nerve cells of the photoreceptor areas form sensory terminals similar to those of the hypothalamic CSF-contacting neurons. Special secondary neurons of the retina and pineal organ contact the retinal photoreceptor space and pineal recess respectively, both cavities being embryologically derived from the 3rd ventricle. The composition of these photoreceptor spaces is important in the photochemical transduction and may modify the activity of the secondary neurons. Septal and preoptic CSF-contacting neurons contain various opsins and other compounds of the phototransduction cascade and represent deep encephalic photoreceptors detecting the illumination of the brain tissue and play a role in the regulation of circadian and reproductive responses to light. The medullo-spinal CSF-contacting neurons present in the oblongate medulla, spinal cord and terminal filum, send their dendrites into the fourth ventricle and central canal. Resembling mechanoreceptors of the lateral line organ, the spinal CSF-contacting neurons may be sensitive to the pressure or flow of the CSF. The axons of these neurons terminate at the external CSF-space of the oblongate medulla and spinal cord and form neurohormonal nerve endings. Based on information taken up from the CSF, a regulatory effect on the production or composition of CSF was supposed for bioactive materials released by these terminals. Most of the axons of the medullospinal CSF-contacting neurons and the magno- and parvicellular neurosecretory nuclei running to neurohemal areas (neurohypophysis, median eminence, terminal lamina, vascular sac and urophysis) do not terminate directly on vessels, instead they form neurohormonal nerve terminals attached by half-desmosomes on the basal lamina of the external and vascular surface of the brain tissue. Therefore, the bioactive materials released from these terminals primarily enter the external CSF and secondarily, by diffusion into vessels and the composition of the external CSF, may have a modulatory effect on the bioactive substances released by the neurohormonal terminals. Contacting the intercellular space, sensory-type cilia were also demonstrated on nerve cells situated subependymally or farther away from the ventricles, among others in the neurosecretory nuclei. Since tight-junctions are lacking between ependymal cells of the ventricular wall, not only CSF-contacting but also subependymal ciliated neurons may be influenced by the actual composition of the CSF besides that of the intercellular fluid of the brain tissue. According to the comparative histological data summarised in this review, the ventricular CSF-contacting neurons represent the phylogenetically oldest component detecting the internal fluid milieu of the brain. The neurohormonal terminals on the external surface of the brain equally represent an ancient form of nonsynaptic signal transmission.     

Frameshifted beta-amyloid precursor protein (APP+1) is a secretory protein,and the level of APP+1 in cerebrospinal fluid is linked to Alzheimer pathology

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP+1 proficiently secreted APP+1. Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP+1 is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP+1 level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) (p < 0.001), and the level of APP+1 in CSF was inversely correlated with the severity of the neuropathology. Moreover the earliest neuropathological changes are already reflected in a significant decrease of the APP+1 level in CSF. These data show that APP+1 is normally secreted by neurons, preventing intra-neuronal accumulation of APP+1 in brains of non-demented controls without neurofibrillary pathology.