Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.     

Seasonal changes in neuronal turnover in a forebrain nucleus in adult songbirds

Neuronal death and replacement, or neuronal turnover, in the adult brain are one of many fundamental processes of neural plasticity. The adult avian song control circuit provides an excellent model for exploring mature neuronal death and replacement by new neurons. In the song control nucleus, HVC of adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli) nearly 68,000 neurons are added each breeding season and die during the subsequent nonbreeding season. To accommodate large seasonal differences in HVC neuron number, the balance between neuronal addition and death in HVC must differ between seasons. To determine whether maintenance of new HVC neurons changes within and between breeding and nonbreeding conditions, we pulse-labeled two different cohorts of new HVC neurons under both conditions and quantified their maintenance. We show that the maintenance of new HVC neurons, as well as new nonneuronal cells, was higher at the onset of breeding conditions than at the onset of nonbreeding conditions. Once a steady-state HVC volume and neuronal number were attained in either breeding or nonbreeding conditions, neuronal and nonneuronal maintenance were similarly low. We found that new neuronal number correlated with a new nonneuronal number within each cohort of new neurons. Together, these data suggest that sex steroids promote the survival of an initial population of new neurons and nonneuronal cells entering HVC. However, once HVC is fully grown or regressed, neuronal and nonneuronal cell turnover is regulated by a common mechanism likely independent of direct sex steroid signaling.     

2′-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons

Recently, we have demonstrated that adenosine and 2′-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2′-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2′-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2′-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2′-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2′-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2′-deoxyadenosine kinase with iodotubercidin and 5′-amino-5′-deoxyadenosine had no protective effect against 2′-deoxyadenosine toxicity. [³H]-thymidine incorporation was inhibited over 90% by 2′-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2′-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2′-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.     

Neuronal phenotypes in mouse dorsal root ganglion cell cultures: Enrichment of substance P and calbindin D-28k expressing neurons in a defined medium

The primary sensory neurons in mouse dorsal root ganglia consist of diversified subpopulations which express distinct phenotypic characteristics such as substance P or calbindin D-28k. To determine whether neuronal phenotypes are altered or not in in vitro cultures carried out in a defined synthetic medium, dissociated dorsal root ganglion cells from newborn mice were grown in the alpha-modified minimum essential medium either supplemented with 10% fetal calf serum or serum-free. About 80% of the neurons survived after 5 days of culture in both media, but only 35% or 65% were rescued after 12 days in serum-free or fetal calf serum supplemented medium, respectively. The neuronal subpopulations expressing substance P or calbindin D-28k displayed similar morphological properties in both media and a higher resistance to culture conditions than the whole neuronal cell population, especially in serum-free medium. It is therefore concluded that a defined synthetic medium offers reproducible conditions to culture dorsal root ganglion cells for at least 5 days, stimulates the expression of substance P and enriches preferentially neuronal phenotypes expressing substance P or calbindin D-28k, for a longer period of culture.     

Stimulation of non-neuronal cell proliferation in vitro by mitogenic factors present in highly purified sympathetic neurons.

Sympathetic neurons have been demonstrated to contain one or more mitogens which are active on highly purified non-neuronal cells cultured in medium containing an optimal concentration of fetal calf serum. Neurons and homologous non-neuronal cells were separated by a method recently developed in this laboratory. The highly purified neurons were either sonicated or homogenized prior to addition to nonneuronal cultures. The presence of neuronal sonicate (1) greatly stimulated [3H]thymidine incorporation into acid-precipitable macromolecules without altering the soluble [3H]thymidine pool, (2) increased both the fraction of non-neuronal cells which took up [3H]thymidine and the density of labeling as observed by autoradiography, and (3) increased the number of cells present in treated cultures after 40 h. The enhancement of [3H]thymidine incorporation was dose-dependent and did not involve cyclic AMP. Addition of neuronal sonicate also caused marked non-neuronal cell elongation which resulted in the elaboration of very long cell processes. The active factor(s) in the neuronal sonicate were partially heat-labile. Norepinephrine was ruled out as a possible mitogenic factor.     

A procedure for purifying neuron-like cells in cultures from central nervous tissue with a defined medium

A serum-free medium (N1) containing the supplements insulin, transferrin, progesterone, putrescine and selenium was used to culture cells from a variety of embryonic chick central nervous system tissues, namely, optic lobe, neural retina, spinal cord and telencephalon. The N1 medium supported the survival of fiber-bearing cells (features typical of cultured neurons) as well as or better than horse serum, while permitting no, or almost no flat cells. Survival and growth of chick flat cells required fetal calf, but not horse serum. Cultivation of newborn mouse telencephalon under these conditions yielded similar results, except that either fetal calf or horse serum supported flat cells.     

Age-dependent responses of ciliary ganglion neurons to conditioned media on cells at different stages of embryonic development

Neurons from ciliary ganglia (CG) from 8 to 14 day-old chick embryos were cultured in presence of conditioned media (CM) by eye tissue cells (ETC) on nonneuronal cells from ciliary ganglia (NFGC). These conditioning cells were obtained from 8 and 14 day-old embryos. Two parameters, surviving neurons and neurons displaying neurites, were determined after 48 h of culture. For neuronal survival, CMs did not show an effect on CG14 neurons. In the other neuronal ages ETC-CMs maintained a similar neuronal survival, whereas NFGC-CMs were more effective on older neurons. CM14 media were more effective maintaining neuronal survival than CM8 media respectives. The number of neurons displaying neurites decreased with neuronal ages in presence of all CMs. ETC8-CM was the better promoting neurite extension in all neuronal ages tested.     

Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor.

We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1-7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30-40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18-20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occurred in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrate that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.     

Factors influencing neuronal growth in primary cultures derived from 3-day-old chick embryos

We compared neuronal growth patterns in primary cultures prepared by dissociating 3-day-old chick embryos, either whole embryo (E3WE) or head only (E3H) and plating the dispersed cells onto Petri dishes coated with either poly-L-lysine, collagen or laminin. The culture medium was Dulbecco's Modified Eagle's Medium (DMEM), supplemented with either 5 or 10% fetal bovine calf serum (FCS). As we have previously described, in E3WE cultures on poly-L-lysine the neuronal primary growth patterns were aggregation with neuritic fasciculation, presence of growth cones with microspikes and very few flat cells. In contrast with cultures grown on poly-L-lysine, in cultures grown on collagen or laminin the distinct growth pattern was extensive networks of isolated and differentiated neurons lying on acquired monolayers of flat cells. When 5% FCS was used, as compared to 10% FCS, neuronal aggregates were fewer and smaller on poly-L-lysine; on collagen or laminin a tendency to aggregate was observed. Several differences were observed in the E3H cultures when compared to E3WE: (a) aggregates were less numerous with the prevailing pattern being a web-like, self-contained aggregate; (b) aggregates connected with other aggregates or flat cells were rare and the aggregate adhesivity was minimized; (c) neurons on collagen or laminin formed networks with the exception of a few, small aggregates displaying no fasciculation; (d) flat cells did not form a monolayer but islets which hosted the neuronal meshy networks. We attribute these differences in the growth patterns between the various types of cultures to be the combined result of a variety of environmental signals, derived from the provided substrata, the serum and the nonneuronal cell factors and cell surface, all primarily regulating neuronal adhesivity.     

Mutational analysis of N-Bak reveals different structural requirements for antiapoptotic activity in neurons and proapoptotic activity in nonneuronal cells

N-Bak, a neuron-specific BH3-only splice variant of Bak, is proapoptotic when overexpressed in nonneuronal cells, but antiapoptotic in NGF-deprived sympathetic neurons. We generated mutants of N-Bak and compared their activities in COS-7 or Neuro2A cells to those in NGF-deprived sympathetic neurons. A C-terminal deletion shortly after the BH3 domain of N-Bak compromised its neuroprotective activity but had little effect on its cytotoxic activity in nonneuronal cells. Amino acid changes in the BH3 domain of N-Bak differently affected its function in nonneuronal cells and in neurons. The same changes in the BH3 domain of longer Bak isoform affected its function similarly in nonneuronal cells and neurons. C-terminally truncated Bax, a structural analogue of N-Bak, was also neuroprotective, whereas Blk, a different BH3-only protein was apoptotic in neurons. Thus, neuron-specific antiapoptotic interactions require a "N-Bak-type" conformation, not just a BH3 domain, whereas the presence of a BH3 domain in the Bak protein is sufficient to kill nonneuronal cells.     

Not all brains are made the same: new views on brain scaling in evolution

Evolution has generated mammalian brains that vary by a factor of over 100,000 in mass. Despite such tremendous diversity, brain scaling in mammalian evolution has tacitly been considered a homogeneous phenomenon in terms of numbers of neurons, neuronal density, and the ratio between glial and neuronal cells, with brains of different sizes viewed as similarly scaled-up or scaled-down versions of a shared basic plan. According to this traditional view, larger brains would have more neurons, smaller neuronal densities (and, hence, larger neurons), and larger glia/neuron ratios than smaller brains. Larger brains would also have a cerebellum that maintains its relative size constant and a cerebral cortex that becomes relatively larger to the point that brain evolution is often equated with cerebral cortical expansion. Here I review our recent data on the numbers of neuronal and nonneuronal cells that compose the brains of 28 mammalian species belonging to 3 large clades (Eulipotyphla, Glires, and Primata, plus the related Scandentia) and show that, contrary to the traditional notion of shared brain scaling, both the cerebral cortex and the cerebellum scale in size as clade-specific functions of their numbers of neurons. As a consequence, neuronal density and the glia/neuron ratio do not scale universally with structure mass and, most importantly, mammalian brains of a similar size can hold very different numbers of neurons. Remarkably, the increased relative size of the cerebral cortex in larger brains does not reflect an increased relative concentration of neurons in the structure. Instead, the cerebral cortex and cerebellum appear to gain neurons coordinately across mammalian species. Brain scaling in evolution, hence, should no longer be equated with an increasing dominance of the cerebral cortex but rather with the concerted addition of neurons to both the cerebral cortex and the cerebellum. Strikingly, all brains appear to gain nonneuronal cells in a similar fashion, with relatively constant nonneuronal cell densities. As a result, while brain size can no longer be considered a proxy for the number of brain neurons across mammalian brains in general, it is actually a very good proxy for the number of nonneuronal cells in the brain. Together, these data point to developmental mechanisms that underlie evolutionary changes in brain size in mammals: while the rules that determine how neurons are added to the brain during development have been largely free to vary in mammalian evolution across clades, the rules that determine how other cells are added in development have been mostly constrained and to this day remain largely similar both across brain structures and across mammalian groups.     

Differentiation and maturation of zebrafish dorsal root and sympathetic ganglion neurons

The trunk neural crest of vertebrate embryos gives rise to dorsal root ganglion (DRG) sensory neurons and autonomic sympathetic neurons, among other derivatives. We have examined the development of DRG and sympathetic neurons during development in the zebrafish. We found that sensory neurons differentiate rapidly and that their overt neuronal differentiation significantly precedes that of sympathetic neurons in the trunk. Sympathetic neurons in different regions differentiate at different times. The most rostral population, which we call the cervical ganglion, differentiates several days before trunk sympathetic neurons. After undergoing overt neuronal differentiation, sympathetic neurons subsequently express the adrenergic differentiation markers dopamine beta-hydroxylase and tyrosine hydroxylase. A second population of adrenergic nonneuronal cells initially localized with cervical sympathetic neurons appears to represent adrenal chromaffin cells. In more mature fish, these cells were present in clusters within the kidneys. Individual DRG and sympathetic ganglia initially contain few neurons. However, the number of neurons in DRG and sympathetic ganglia increases continuously at least up to 4 weeks of age. Analysis of phosphohistone H3 expression and bromodeoxyuridine incorporation studies suggests that the increases in DRG and sympathetic ganglion neuronal cell number are due wholly or in part to the division of neuronal cells within the ganglia.     

Replication-Deficient Adenovirus Vector Transfer ofgfpReporter Gene into Supraoptic Nucleus and Subfornical Organ Neurons

The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 × 10⁶pfu) and sacrificed 1–7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successfulin vitrotransfection of cultured cells from the SON and SFO was also achieved with Ad (2 × 10⁶to 2 × 10⁸pfu). The expression of GFP inin vitrotransfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO–SON axis.     

Basic fibroblast growth factor in neuronal cultures of human fetal brain

The presence of basic fibroblast growth factor (bFGF) was investigated in neuronal cells derived from 12 and 18 week-old human fetal brain cultures. To this purpose, the ability of bFGF to stimulate plasminogen activator (PA) production in fetal bovine aortic endothelial GM 7373 cells was used as an assay for this molecule in neuronal cell extracts. The identity of the PA-stimulating activity of neuronal cell extract with bFGF was confirmed by its high affinity for heparin and by its cross-reactivity with polyclonal antibodies to human placental bFGF. These antibodies recognized a Mr 18,000 cell-associated protein both in Western blot and in immuno-precipitation experiments. All the neurons showed bFGF immunoreactivity, as demonstrated by immunocytochemical staining, while nonneuronal cells were unstained. The data demonstrate for the first time that cultured human fetal brain neurons contain and synthesize bFGF.     

Telomerase and neuronal marker status of differentiated NT2 and SK-N-SH human neuronal cells and primary human neurons

Upon treatment with retinoic acid, NTera-2 (NT2) human teratocarcinoma and SK-N-SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT-2 cells is partially dependent on the time of differentiation (24-55 days). SK-N-SH cells differentiate into a mixed population of neuronal and epithelium-like cells. Here we report modified protocols that increase the number of differentiated NT-2 and SK-N-SH cells and that establish an enriched neuronal SK-N-SH-derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton-associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down-regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK-N-SH cells may represent an excellent source for studies investigating the role of telomerase or other survival-promoting activities in protecting human neuronal cells from cell death-mediating stresses associated with neurodegenerative diseases.     

Ultrastructural Changes of Sympathetic Neurons Following Neurotrophin 3 Antiserum Treatment in Young Rat

We have previously demonstrated that neurotrophin-3 antiserum administration to rats during the first 2 postnatal weeks results in a massive reduction of neurons in the superior cervical ganglion. In the present study, an ultrastructural analysis was undertaken to elucidate the mechanism by which neurotrophin-3 deprivation causes neuronal death. Newborn and 4-week-old rats were injected with either neurotrophin-3 antiserum or normal rabbit serum or used without injection. Superior cervical ganglia from each animal were examined by routine electron microscopy. Most neurons in the ganglia from untreated rats had a large and round nucleus with one or two nucleoli. Chromatin within the nucleus was evenly distributed. A double-layer nuclear membrane could be distinguished and the cytoplasm contained abundant organelles. Treatment with neurotrophin-3 antiserum for 24 h in neonates resulted in chromatin clumping in the nucleus of many neurons. The nuclear membrane became rough and occasionally folded. In the cytoplasm, the Golgi apparatus was disrupted. Three days after treatment, these changes became more obvious. The chromatin in the nucleus was often aggregated and marginalized. Vacuolation was present in many membranous organelles throughout the cytoplasm. Although neurotrophin-3 antiserum given to 4-week-old rats had little effect on overall neuronal numbers (Tafreshi, Zhou, and Rush, unpublished), a few neurons, undergoing either apoptotic or cytolytic cell death, were identified 7 days later. Most affected neurons were located near small blood vessels or capillaries and were associated with numerous nonneuronal cells. The debris of degenerating neurons were surrounded by the processes of glia cells. These findings support the view that loss of endogenous neurotrophin-3 following neutralization with specific antibody leads to activation of apoptotic pathways within the affected neurons. However, the presence of neurons dying as a result of cytolysis suggests that other mechanisms may also be involved.     

Astrocytes protect neurons from neurotoxic injury by serum glutamate

Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be ∼0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30–100 μM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from ∼90 μM to less than 1 μM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED₅₀ values of > 100 μM (< 6 DIV) to ∼6 μM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED₅₀ for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state. GLIA 22:237–248, 1998. © 1998 Wiley-Liss, Inc.     

Sialin expression in the CNS implicates extralysosomal function in neurons

SLC17A5 encodes a lysosomal membrane protein, sialin, which transports sialic acid from lysosomes. Mutations in sialin result in neurodegenerative sialic acid storage disorders, Salla disease (SD) and infantile sialic acid storage disease (ISSD). Here we analyzed sialin in mouse central nervous system (CNS) and primary cortical and hippocampal neurons and glia. In the CNS, sialin was predominantly expressed in neurons, especially in the proliferative zone of the prospective neocortex and the hippocampus in developing brain. In nonneuronal cells and primary glial cell cultures, mouse sialin was localized into lysosomes but interestingly, in primary neuronal cultures sialin was not targeted into lysosomes but rather revealed a punctate staining along the neuronal processes and was also seen in the plasma membrane. These data demonstrate a nonlysosomal localization of sialin in neurons and would imply a role for sialin in the secretory processes of neuronal cells.     

Neurodegeneration in Excitotoxicity, Global Cerebral Ischemia, and Target Deprivation: A Perspective on the Contributions of Apoptosis and Necrosis

In the human brain and spinal cord, neurons degenerate after acute insults (e.g., stroke, cardiac arrest, trauma) and during progressive, adult-onset diseases [e.g., amyotrophic lateral sclerosis, Alzheimer’s disease]. Glutamate receptor-mediated excitotoxicity has been implicated in all of these neurological conditions. Nevertheless, effective approaches to prevent or limit neuronal damage in these disorders remain elusive, primarily because of an incomplete understanding of the mechanisms of neuronal death in in vivo settings. Therefore, animal models of neurodegeneration are crucial for improving our understanding of the mechanisms of neuronal death. In this review, we evaluate experimental data on the general characteristics of cell death and, in particular, neuronal death in the central nervous system (CNS) following injury. We focus on the ongoing controversy of the contributions of apoptosis and necrosis in neurodegeneration and summarize new data from this laboratory on the classification of neuronal death using a variety of animal models of neurodegeneration in the immature or adult brain following excitotoxic injury, global cerebral ischemia, and axotomy/target deprivation. In these different models of brain injury, we determined whether the process of neuronal death has uniformly similar morphological characteristics or whether the features of neurodegeneration induced by different insults are distinct. We classified neurodegeneration in each of these models with respect to whether it resembles apoptosis, necrosis, or an intermediate form of cell death falling along an apoptosis-necrosis continuum. We found that N-methyl-d-aspartate (NMDA) receptor- and non-NMDA receptor-mediated excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, in which neuronal death (appearing as apoptotic, necrotic, or intermediate between the two extremes) is influenced by the degree of brain maturity and the subtype of glutamate receptor that is stimulated. Global cerebral ischemia produces neuronal death that has commonalities with excitotoxicity and target deprivation. Degeneration of selectively vulnerable populations of neurons after ischemia is morphologically nonapoptotic and is indistinguishable from NMDA receptor-mediated excitotoxic death of mature neurons. However, prominent apoptotic cell death occurs following global ischemia in neuronal groups that are interconnected with selectively vulnerable populations of neurons and also in nonneuronal cells. This apoptotic neuronal death is similar to some forms of retrograde neuronal apoptosis that occur following target deprivation. We conclude that cell death in the CNS following injury can coexist as apoptosis, necrosis, and hybrid forms along an apoptosis–necrosis continuum. These different forms of cell death have varying contributions to the neuropathology resulting from excitotoxicity, cerebral ischemia, and target deprivation/axotomy. Degeneration of different populations of cells (neurons and nonneuronal cells) may be mediated by distinct or common causal mechanisms that can temporally overlap and perhaps differ mechanistically in the rate of progression of cell death.