p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells.

This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons', oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro.     

Mature neurons: equipped for survival

Neurons completely transform how they regulate cell death over the course of their lifetimes. Developing neurons freely activate cell death pathways to fine-tune the number of neurons that are needed during the precise formation of neural networks. However, the regulatory balance between life and death shifts as neurons mature beyond early development. Mature neurons promote survival at all costs by employing multiple, often redundant, strategies to prevent cell death by apoptosis. This dramatic shift from permitting cell death to ensuring cellular survival is critical, as these post-mitotic cells must provide neuronal circuitry for an organism''s entire lifetime. Importantly, as many neurodegenerative diseases afflict adult neuronal populations, the survival mechanisms in mature neurons are likely to be either reversed or circumvented during neurodegeneration. Examining the adaptations for inhibiting apoptosis during neuronal maturation is key to comprehending not just how neurons survive long term, but may also provide insight for understanding how neuronal toxicity in various neurodegenerative diseases may ultimately lead to cell death.     

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

Neuronal life and death: an essential role for the p53 family

Recent evidence indicates that the p53 tumor suppressor protein, and its related family member, p73, play an essential role in regulating neuronal apoptosis in both the developing and injured, mature nervous system. In the developing nervous system, they do so by regulating naturally-occurring cell death in neural progenitor cells and in postmitotic neurons, acting to ensure the apoptosis of cells that either do not appropriately undergo the progenitor to postmitotic neuron transition, or that fail to compete for sufficient quantities of trophic support. Somewhat surprisingly, in developing postmitotic neurons, p53 plays a proapoptotic role, while a naturally-occurring, truncated form of p73, DeltaNp73, antagonizes p53 and plays an anti-apoptotic role. In the mature nervous system, numerous studies indicate that p53 is essential for the neuronal death in response to a variety of insults, including DNA damage, ischemia and excitotoxicity. It is likely that all of these insults culminate in DNA damage, which may well be a common trigger for neuronal apoptosis. In this regard, the signaling pathways that are responsible for triggering p53-dependent neuronal apoptosis are starting to be elucidated, and involve cell cycle deregulation and activation of the JNK pathway. Finally, accumulating evidence indicates that p53 is perturbed in the CNS in a number of neurodegenerative disorders, leading to the hypothesis that longterm oxidative damage and/or excitotoxicity ultimately trigger p53-dependent apoptosis in the chronically degenerating nervous system.     

Role of RB and RB2/P130 genes in marrow stromal stem cells plasticity

Marrow stromal cells (MSCs) are stem-like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC-independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC-dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination.     

Insulin-like growth factor-1 inhibits STS-induced cell death and increases functional recovery of in vitro differentiated neurons

NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-xL content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and down-regulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17,1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude without giving rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to 100% after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.     

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits

Neurogenesis takes place in the adult mammalian brain in three areas:Subgranular zone of the dentate gyrus(DG);subventricular zone of the lateral ventricle;olfactory bulb.Different molecular markers can be used to characterizethe cells involved in adult neurogenesis.It has been recently suggested that a population of bone marrow(BM)progenitor cells may migrate to the brain and differentiate into neuronal lineage.To explore this hypothesis,we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells.Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells,then after several months in mature neurons and microglial cells,and thus without central nervous system(CNS)lesion.Most of transgene-expressing cells expressed NeuN,a marker of mature neurons.Thus,BM-derived cells may function as progenitors of CNS cells in adult animals.The mechanism by which the cells from the BM come to be neurons remains to be determined.Although the observed gradual increase in transgene-expressing neurons over 16mo suggests that the pathway involved differentiation of BM-resident cells into neurons,cell fusion as the principal route cannot be totally ruled out.Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons.Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector.In addition to cells expressing markers of mature neurons,transgene-positive cells were also positive for nestin and doublecortin,molecules expressed by developing neuronal cells.These cells were actively proliferating,as shown by short term BrdU incorporation studies.Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40vectors migrating to the hippocampus,and these cells were seen at earlier time points in the DG.We show that the cell membrane chemokine receptor,CCR5,and its ligands,enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity.SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells,suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS,both in the basal state and in response to injury.Furthermore,reduction in CCR5 expression incirculating cells provides profound neuroprotection from excitotoxic neuronal injury,reduces neuroinflammation,and increases neuronal regeneration following this type of insult.These results suggest that BM-derived,transgeneexpressing,cells can migrate to the brain and that they become neurons,at least in part,by differentiating into neuron precursors and subsequently developing into mature neurons.     

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.     

Small stress protein Hsp27 accumulation during dopamine-mediated differentiation of rat olfactory neurons counteracts apoptosis

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.     

Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons

Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1-2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Neuroleukin inhibition sensitises neuronal cells to caspase-dependent apoptosis

Neuroleukin (NLK) is a multifunctional protein, involved in neuronal growth, glucose metabolism, cell motility, and differentiation. Expressed in the brain, it supports the growth of embryonic spinal, skeletal motor, and sensory neurons. We have previously demonstrated that NLK is up-regulated in the brain during Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats. In order to study the biological role of NLK, we have generated an inducible rat pheochromocytoma PC12 cell line in which the expression of NLK is selectively down-regulated by antisense strategy. We show here that the block of NLK commits PC12 cells to caspase-dependent apoptosis. This priming effect elicited by NLK inhibition is independent from the differentiation state of the neuronal cells. These results suggest a general protective role of NLK in the control of cell death in neuronal cells.     

FGF-2 Inhibits Apoptosis in Human Teratocarcinoma Cells during Differentiation on Collagen Substratum

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24–48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop.In vivo,such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.     

Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC.     

Apoptotic cell death in neuronal differentiation of P19 EC cells: Cell death follows reentry into S phase

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation. J. Cell. Physiol. 172:25–35, 1997. © 1997 Wiley-Liss, Inc.     

Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.