IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-β receptor (LT-βR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-βR death pathway. The expression of LIGHT and LT-βR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.     

Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression

Although much is known about the global effects of insulin-like growth factor 1 receptor (IGF1R)-mediated signaling on fetal growth and the clinical manifestations resulting from IGF/IGF1R deficiencies, we have an incomplete understanding of the cellular actions of this essential pathway during vertebrate embryogenesis. In this study, we inhibited IGF1R signaling during zebrafish embryogenesis using antisense morpholino oligonucleotides or a dominant-negative IGF1R fusion protein. IGF1R inhibition resulted in reduced embryonic growth, arrested development and increased lethality. IGF1R-deficient embryos had significant defects in the retina, inner ear, motoneurons and heart. No patterning abnormalities, however, were found in the brain or other embryonic tissues. At the cellular level, IGF1R inhibition increased caspase 3 activity and induced neuronal apoptosis. Coinjection of antiapoptotic bcl2-like mRNA attenuated the elevated apoptosis and rescued the retinal and motoneuron defects, but not the developmental arrest. Subsequent cell cycle analysis indicated an increased percentage of cells in G1 and a decreased percentage in S phase in IGF1R-deficient embryos independent of apoptosis. These results provide novel insight into the cellular basis of IGF1R function and show that IGF1R signaling does not function as an anteriorizing signal but regulates embryonic growth and development by promoting cell survival and cell cycle progression.     

Necdin protects embryonic motoneurons from programmed cell death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.     

Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.     

Regulation of neural progenitor cell state by ephrin-B

Maintaining a balance between self-renewal and differentiation in neural progenitor cells during development is important to ensure that correct numbers of neural cells are generated. We report that the ephrin-B-PDZ-RGS3 signaling pathway functions to regulate this balance in the developing mammalian cerebral cortex. During cortical neurogenesis, expression of ephrin-B1 and PDZ-RGS3 is specifically seen in progenitor cells and is turned off at the onset of neuronal differentiation. Persistent expression of ephrin-B1 and PDZ-RGS3 prevents differentiation of neural progenitor cells. Blocking RGS-mediated ephrin-B1 signaling in progenitor cells through RNA interference or expression of dominant-negative mutants results in differentiation. Genetic knockout of ephrin-B1 causes early cell cycle exit and leads to a concomitant loss of neural progenitor cells. Our results indicate that ephrin-B function is critical for the maintenance of the neural progenitor cell state and that this role of ephrin-B is mediated by PDZ-RGS3, likely via interacting with the noncanonical G protein signaling pathway, which is essential in neural progenitor asymmetrical cell division.     

Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn-superoxide dismutase

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor function and eventual death as a result of degeneration of motor neurons in the spinal cord and brain. The discovery of mutations in SOD1, the gene encoding the antioxidant enzyme Cu/Zn-superoxide dismutase (CuZnSOD), in a subset of ALS patients has led to new insight into the pathophysiology of ALS. Utilizing a novel adenovirus gene delivery system, our laboratory has developed a human cell culture model using chemically differentiated neuroblastoma cells to investigate how mutations in SOD1 lead to neuronal death. Expression of mutant SOD1 (G37R) resulted in a time and dose-related death of differentiated neuroblastoma cells. This cell death was inhibited by overexpression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). These observations support the hypothesis that mutant SOD1-associated neuronal death is associated with alterations in oxidative stress, and since MnSOD is a mitochondrial enzyme, suggest that mitochondria play a key role in disease pathogenesis. Our findings in this model of inhibition of mutant SOD1-associated death by MnSOD represent an unique approach to explore the underlying mechanisms of mutant SOD1 cytotoxicity and can be used to identify potential therapeutic agents for further testing.     

Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.     

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc-interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G(0)/G(1) cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell.     

G protein hyperactivation of the Caenorhabditis elegans adenylyl cyclase SGS-1 induces neuronal degeneration.

Expression of a constitutively activated version of the heterotrimeric G protein alpha-subunit Galphas results in the swelling and vacuolization of a specific subset of ventral nerve cord motoneurons of Caenorhabditis elegans. A second site modifier (sgs-1) that completely suppresses this neuronal degeneration has been isolated. sgs-1 was cloned and was shown to encode an adenylyl cyclase which is most similar to mammalian adenylyl cyclase type IX. Mutations in sgs-1 change residues that are conserved among different adenylyl cyclases. These mutations are located in the two catalytic domains and in the first multiple transmembrane spanning region of the predicted protein. An sgs-1 reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Galphas-induced cell death. A second C.elegans adenylyl cyclase gene (acy-2) was analyzed as well. In contrast to sgs-1, acy-2 shows a restricted expression pattern and loss of acy-2 function results in early larval lethality. These results suggest that SGS-1 is a target of Galphas signaling in motoneurons, whereas an interaction of Galphas with ACY-2, probably in the canal-associated neurons, is required for viability.     

Mitochondrial Function in Apoptotic Neuronal Cell Death

Apoptosis can be defined as the regulated death of a cell and is conducted by conserved pathways. Apoptosis of neurons after injury or disease differs from programed cell death, in the sense that neurons in an adult brain are not "meant" to die and results in a loss of function. Thus apoptosis is an honorable process by a neuron, a cell with limited potential to replace itself, choosing instead to commit suicide to save neighboring cells from release of cellular components that cause injury directly or trigger secondary injury resulting from inflammatory reactions. The excess of apoptosis of neuronal cells underlies the progressive loss of neuronal populations in neurodegenerative disorders and thus is harmful. Mitochondria are the primary source for energy in neurons but are also poised, through the "mitochondrial apoptosis pathway," to signal the demise of cells. This duplicity of mitochondria is discussed, with particular attention given to the specialized case of pathological neuronal cell death.     

Resistance of cell lines to prion toxicity aided by phospho-ERK expression

Prion diseases are fatal neurodegenerative disorders. They are characterised by neuronal loss and the accumulation of an abnormal protein in the CNS. Cell lines exist that express the toxic form of the prion protein (PrP) with little evidence of cell death. Other cell based models studying the mechanism by which cell death occurs employ exogenous application of peptides or fragments of PrP. In this study, we demonstrated that full-length recombinant PrP binding manganese was toxic to PrP-expressing cell lines and primary neuronal cultures but not to PrP-knockout neurones. This toxic form of PrP was also toxic to cell lines equivalently regardless of whether they were infected with scrapie or not. Both scrapie-infected cells and cells resistant to the toxicity of PrP showed increased levels of phosphorylated ERK protein. Scrapie-infected cells also showed elevated levels of caspase 12. Inhibition of phospho-ERK resulted in increased cell death suggesting the increased levels of phospho-ERK served a protective effect. These results suggest that scrapie-infected cell lines resist the toxicity of the prions they generate because they produce only low levels of abnormal protein and have increased resistance to apoptotic signs because of heightened activity of the MAP kinase pathway.     

Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins,including nitrated proteins

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.     

SIRT1 silencing confers neuroprotection through IGF‐1 pathway activation

The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF‐1 protein expression and secretion and of IGF‐1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF‐1 and IGF‐1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF‐1 and IGF‐1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF‐1 survival pathway. J. Cell. Physiol. 228: 1754–1761, 2013. © 2013 Wiley Periodicals, Inc.     

S-nitrosylated GAPDH mediates neuronal apoptosis induced by amyotrophic lateral sclerosis-associated mutant SOD1G93A

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with the selective loss of motor neurons in the brain, brain stem, and spinal cord. A number of the mutants of the human gene for superoxide dismutase 1 (SOD1) have been shown to cause familial ALS as a result of gain-of-function toxicity by an unknown mechanism. In this study, we show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a critical mediator of the apoptotic cell death signaling cascade induced by the ALS-associated G93A mutant of human SOD1 [SOD1(G93A)]. We observed that SOD1(G93A) induces S-nitrosylation of GAPDH and the subsequent binding of GAPDH and Siah1 in NSC34 motor neuron-like cells. Furthermore, SOD1(G93A) promoted nuclear translocation of S-nitrosylated GAPDH in the cells. In addition, SOD1(G93A)-induced apoptotic cell death was inhibited by deprenyl, a chemical inhibitor of GAPDH S-nitrosylation, in NSC34 cells. Taken together, our findings suggest that S-nitrosylation of GAPDH plays a critical role in SOD1(G93A)-induced neuronal apoptosis.     

Prevention of mutant SOD1 motoneuron degeneration by copper chelators in vitro

An animal model of familial amyotrophic lateral sclerosis (FALS) has been generated by overexpression of human CuZn superoxide dismutase (SOD1) containing a substitution of glycine to alanine at position 93 in transgenic G93A mice. The loss of motoneurons shown in this model has been attributed to a dominant gain of function of this mutated enzyme, which might be due to copper toxicity. This hypothesis was tested in purified spinal motoneurons cultures originating from G93A transgenic embryos. Spinal motoneurons were isolated from E13 embryos by several steps including density gradient centrifugation. The effect of copper chelators on survival and neurite growth of motoneurons was investigated. Survival of G93A motoneurons was decreased by 46% as compared to wild-type motoneurons. Moreover, G93A motoneurons showed reduced neurite outgrowth. Copper chelators strikingly increased viability of G93A motoneurons (by over 200%) but had no effect on wild-type cells. Presence of DDC in the medium increases the length of neurites from G93A motoneurons. The present results suggest the capacity of copper chelators to reduce the effect of reverse function of mutated SOD1 on motoneurons.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor impairs ER Ca(2+) buffering and causes neurodegeneration in primary cortical neurons

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3) R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3) R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3) R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3) R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3) R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+) ](i) ), increased [Ca(2+) ](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3) R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.     

Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes.

Treatment of chick embryos in ovo for 10-12 hr with inhibitors of protein and RNA synthesis during the peak time of normal cell death (Embryonic Day 8) for motoneurons and dorsal root ganglion cells markedly reduces the number of degenerating neurons in these populations. The massive neuronal death induced by the early absence of the limbs was also blocked almost completely by these agents. Further, the death of neurons following peripheral axotomy at the end of the normal cell death period (Embryonic Day 10) was reduced significantly by treatment with inhibitors of biosynthetic reactions. These results indicate that, in vivo, naturally occurring neuronal death, neuronal death induced by the absence of peripheral targets, and axotomy-induced neuronal death later in development all require active gene expression and protein and RNA synthesis. Therefore, neuronal death in a variety of situations may reflect the expression of a developmental fate that can normally only be overridden or suppressed by specific environmental signals (e.g., neurotrophic molecules).     

Identification of a key pathway required for the sterile inflammatory response triggered by dying cells

Dying cells stimulate inflammation, and this response is thought to contribute to the pathogenesis of many diseases. Very little has been known, however, about how cell death triggers inflammation. We found here that the acute neutrophilic inflammatory response to cell injury requires the signaling protein myeloid differentiation primary response gene 88 (Myd88). Analysis of the contribution of Myd88-dependent receptors to this response revealed only a minor reduction in mice doubly deficient in Toll-like receptor 2 (Tlr2) and Tlr4 and normal responses in mice lacking Tlr1, Tlr3, Tlr6, Tlr7, Tlr9, Tlr11 or the interleukin-18 receptor (IL-18R). However, mice lacking IL-1R showed a markedly reduced neutrophilic inflammatory response to dead cells and tissue injury in vivo as well as greatly decreased collateral damage from inflammation. This inflammatory response required IL-1alpha, and IL-1R function was required on non-bone-marrow-derived cells. Notably, the acute monocyte response to cell death, which is thought to be important for tissue repair, was much less dependent on the IL-1R-Myd88 pathway. Also, this pathway was not required for the neutrophil response to a microbial stimulus. These findings suggest that inhibiting the IL-1R-Myd88 pathway in vivo could block the damage from acute inflammation that occurs in response to sterile cell death, and do so in a way that might not compromise tissue repair or host defense against pathogens.     

Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death

Galectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis-inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1-induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.