Nutrient Deprivation Induces Neuronal Autophagy and Implicates Reduced Insulin Signaling in Neuroprotective Autophagy Activation

Although autophagy maintains normal neural function by degrading misfolded proteins, little is known about how neurons activate this integral response. Furthermore, classical methods of autophagy induction used with nonneural cells, such as starvation, simply result in neuron death. To study neuronal autophagy, we cultured primary cortical neurons from transgenic mice that ubiquitously express green fluorescent protein-tagged LC3 and monitored LC3-I to LC3-II conversion by immunohistochemistry and immunoblotting. Evaluation of different culture media led us to discover that culturing primary neurons in Dulbecco''s modified Eagle''s medium without B27 supplementation robustly activates autophagy. We validated this nutrient-limited media approach for inducing autophagy by showing that 3-methyl-adenine treatment and Atg5 RNA interference knockdown each inhibits LC3-I to LC3-II conversion. Evaluation of B27 supplement components yielded insulin as the factor whose absence induced autophagy in primary neurons, and this activation was mammalian target of rapamycin-dependent. When we tested if nutrient-limited media could protect neurons expressing polyglutamine-expanded proteins against cell death, we observed a strong protective effect, probably due to autophagy activation. Our results indicate that nutrient deprivation can be used to understand the regulatory basis of neuronal autophagy and implicate diminished insulin signaling in the activation of neuronal autophagy.Most neurodegenerative disorders are characterized by the accumulation of misfolded proteins that coalesce into “inclusions” and become visible at the light microscope level in the brains and spinal cords of affected patients (1, 2). These inclusions manifest themselves pathologically in Alzheimer disease as extracellular plaques and neurofibrillary tangles, in Parkinson disease as Lewy bodies, and in poly(Q) repeat diseases as cytosolic and nuclear aggregates. A fundamental advance in our understanding of neurodegeneration has been the realization that protein misfolding is a common theme in many important neurological disorders, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, prion diseases, and poly(Q) diseases. The mechanistic underpinning of this “proteinopathy” hypothesis stems from the exquisite susceptibility of postmitotic cells in the central nervous system to misfolded protein stress, since neural cells are not continually replenished by cell division, unlike most of their nonneural counterparts.The ubiquitin-proteasome system is the main intracellular degradation pathway to remove short lived proteins and to eliminate peptides that exit from the protein-folding machinery of the endoplasmic reticulum with an aberrant conformation. However, many aggregate-prone proteins, such as poly(Q) proteins, are inefficiently degraded by the proteasome (3–5). Failure of adequate degradation of aggregate-prone proteins activates alternative protein turnover pathways in the cell, including macroautophagy (hereafter referred to as autophagy). Autophagy is a degradative process that begins with engulfment of cytosolic materials and/or organelles and progresses through a series of steps involving production of a double membrane bound structure, culminating in the delivery of the engulfed material to lysosomes (6). In the central nervous system, basal levels of autophagy are required for the continued health and normal function of neurons, since conditional inactivation of the autophagy pathway in neural cells in mice yields neuronal dysfunction and neurodegeneration characterized by the accumulation of proteinaceous material (7, 8). Furthermore, the presence of aggregate-prone proteins, not degraded by the proteasome, induces autophagy above basal levels, and activation of autophagy appears capable of clearing misfolded proteins, decreasing cytotoxicity, and preventing neurodegeneration in Drosophila and mouse models of misfolded protein stress (9–11).Although numerous reports have documented the protective effects of inducing autophagy in different areas of the diseased brain in model organisms (reviewed in Ref. 12), little is known about how neurons activate this integral response. Indeed, classical methods of autophagy induction used with cultured nonneural cells, such as starvation, simply result in the death of cultured primary neurons. Furthermore, starvation elicits quite different effects in neurons and nonneural cells, both in vitro and in vivo (13, 14). To directly study neuronal autophagy, we devised a primary neuron culture system where we can induce autophagy activation by withdrawal of a key supplement from the culture media. After independently validating the activation of autophagy in our system through pharmacological and genetic inhibition, we identified insulin as the factor responsible for autophagy induction in primary cortical neurons grown in nutrient-limited media. Further characterization of autophagy induction in primary neurons subjected to nutrient deprivation indicated that such autophagy activation is mammalian target of rapamycin (mTOR)²-dependent. We then tested if the autophagy response induced by nutrient deprivation could counter misfolded protein stress by expressing a poly(Q)-expanded protein in primary neurons and found that nutrient limitation prevented neuron cell death caused by misfolded protein stress.     

Neuronal macroautophagy: from development to degeneration

Macroautophagy, a lysosomal pathway responsible for the turnover of organelles and long-lived proteins, has been regarded mainly as an inducible process in neurons, which is mobilized in states of stress and injury. New studies show, however, that macroautophagy is also constitutively active in healthy neurons and is vital to cell survival. Neurons in the brain, unlike cells in the periphery, are protected from large-scale autophagy induction because they can use several different energy sources optimally, receive additional nutrients and neurotrophin support from glial cells, and benefit from hypothalamic regulation of peripheral nutrient supplies. Due to its exceptional efficiency, constitutive autophagy in healthy neurons proceeds in the absence of easily detectable autophagic vacuole intermediates. These intermediates can accumulate rapidly, however, when late steps in the autophagic process are blocked. Autophagic vacuoles also accumulate abnormally in affected neurons of several major neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, where they have been linked to various aspects of disease pathogenesis including neuronal cell death. The build-up of autophagic vacuoles in these neurological disorders and others may reflect either heightened autophagy induction, impairment in later digestive steps in the autophagy pathway, or both. Determining the basis for AV accumulation is critical for understanding the pathogenic significance of autophagy in a given pathologic state and for designing possible therapies based on modulating autophagy. In this review, we discuss the special features of autophagy regulation in the brain, its suspected roles in neurodevelopment and plasticity, and recent progress toward understanding how dysfunctional autophagy contributes to neurodegenerative disease.     

Organization of the autophagy pathway in neurons

Macroautophagy (hereafter referred to as autophagy) is an essential quality-control pathway in neurons, which face unique functional and morphological challenges in maintaining the integrity of organelles and the proteome. To overcome these challenges, neurons have developed compartment-specific pathways for autophagy. In this review, we discuss the organization of the autophagy pathway, from autophagosome biogenesis, trafficking, to clearance, in the neuron. We dissect the compartment-specific mechanisms and functions of autophagy in axons, dendrites, and the soma. Furthermore, we highlight examples of how steps along the autophagy pathway are impaired in the context of aging and neurodegenerative disease, which underscore the critical importance of autophagy in maintaining neuronal function and survival.     

Phagocytic Removal of Neuronal Debris by Olfactory Ensheathing Cells Enhances Neuronal Survival and Neurite Outgrowth via p38MAPK Activity

Compelling evidence from animal models and clinical studies suggest that transplantation of olfactory ensheathing cells (OECs), specialized glia in the olfactory system, combined with specific training may be therapeutically useful in the central nervous system (CNS) injuries and neurodegenerative diseases. The unique function of OECs could mainly attribute to both production of cell adhesion molecules and secretion of growth factors in OECs, which support neuron survival and neurite outgrowth. However, little is known about whether engulfment of neuronal degenerative debris by OECs also equally contributes to neuronal survival and neurite outgrowth. Furthermore, the molecular mechanisms responsible for neuronal degenerative corpses' removal remain elusive. Here, we used an in vitro model of primary culture of spinal cord neurons to investigate the effect of engulfment of degenerative neuron debris by OECs on neuronal survival and neurite outgrowth and the possible molecular mechanisms. Our results showed that OECs can engulf an amount of degenerated neuron debris, and this phagocytosis can make a substantial contribution to neuron growth, as demonstrated by increased number of neurons with longer neurite length and richer neurite branches when compared with the combination of neuron debris and OEC conditioned medium (OECCM). Moreover, p38 mitogen-activated protein kinase (p38MAPK) signaling pathway may mediate the OEC engulfment of debris because the p38MAPK-specific inhibitor, SB203580, can abrogate all the positive effects of OECs, including clearance of degenerated neuron debris and generation of bioactive molecules, indicating that p38MAPK is required for the process of phagocytosis of the neuron debris. In addition, the OEC phagocytic activity had no influence on its generation of bioactive molecules. Therefore, these findings provide new insight into further investigations on the OEC role in the repair of traumatic CNS injury and neurodegenerative diseases.     

Autophagy in neurons: it is not all about food

The primary function of autophagy in most cell types is adaptation to starvation. Because neurons are protected from this type of stress, the physiological role of autophagy in normal functioning neurons was, until now, poorly understood. Using genetic manipulations to block autophagy in neurons selectively, Mizushima's and Tanaka's groups have recently presented conclusive evidence for an essential role of constitutive autophagy in neuronal survival. These studies provide new insights into the relationship between autophagy malfunctioning and neurodegenerative disorders.     

Defective mitophagy in human Niemann-Pick Type C1 neurons is due to abnormal autophagy activation

Although traditionally regarded as a cellular adaptive process triggered by nutrient deprivation, autophagy in neurons appears to provide an important neuroprotective mechanism. Neurons in the brain are protected from starvation, and neuronal autophagy serves a critical role in the turnover of abnormal proteins and damaged organelles. As post-mitotic, highly polarized cells with active protein trafficking, neurons rely heavily on an efficient autophagic pathway. Using human embryonic stem cell-derived neurons engineered to mimic the cholesterol lysosomal storage disease Niemann Pick type C1 (NPC1), we have shown that excessive activation and impaired progression of the autophagic pathway conspire to cause abnormal mitochondrial clearance. Defective mitophagy is exceptionally severe in human NPC1 neurons, as compared with patient fibroblasts, and may explain the selective neuronal failure observed in NPC1 and related neurodegenerative disorders.     

Defective mitophagy in human Niemann-Pick Type C1 neurons is due to abnormal autophagy activation

Although traditionally regarded as a cellular adaptive process triggered by nutrient deprivation, autophagy in neurons appears to provide an important neuroprotective mechanism. Neurons in the brain are protected from starvation, and neuronal autophagy serves a critical role in the turnover of abnormal proteins and damaged organelles. As post-mitotic, highly polarized cells with active protein trafficking, neurons rely heavily on an efficient autophagic pathway. Using human embryonic stem cell-derived neurons engineered to mimic the cholesterol lysosomal storage disease Niemann Pick type C1 (NPC1), we have shown that excessive activation and impaired progression of the autophagic pathway conspire to cause abnormal mitochondrial clearance. Defective mitophagy is exceptionally severe in human NPC1 neurons, as compared with patient fibroblasts, and may explain the selective neuronal failure observed in NPC1 and related neurodegenerative disorders.     

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1^G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1^G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1^G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.     

Disruption of neuronal autophagy by infected microglia results in neurodegeneration

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.     

Cellular model of neuronal atrophy induced by DYNC1I1 deficiency reveals protective roles of RAS-RAF-MEK signaling

Neuronal atrophy is a common pathological feature occurred in aging and neurodegenerative diseases. A variety of abnormalities including motor protein malfunction and mitochondrial dysfunction contribute to the loss of neuronal architecture; however, less is known about the intracellular signaling pathways that can protect against or delay this pathogenic process. Here, we show that the DYNC1I1 deficiency, a neuron-specific dynein intermediate chain, causes neuronal atrophy in primary hippocampal neurons. With this cellular model, we are able to find that activation of RAS-RAF-MEK signaling protects against neuronal atrophy induced by DYNC1I1 deficiency, which relies on MEK-dependent autophagy in neuron. Moreover, we further reveal that BRAF also protects against neuronal atrophy induced by mitochondrial impairment. These findings demonstrate protective roles of the RAS-RAF-MEK axis against neuronal atrophy, and imply a new therapeutic target for clinical intervention.     

Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons

The aim was to define a primary culture system enriched in neurons using a defined culture medium, and characterize the model system as to cellular morphology and neuronal phenotypes. We found that these primary neuron enriched cultures from either newborn rat cerebral cortex or hippocampus contain small GABAergic and large glutamatergic neurons as well as astrocytes and microglia. Astrocytes in these cultures are morphologically differentiated with long, slender processes and interact with soluble factors responsible for induction and expression of the glutamate transporter GLT-1. The cultures achieve the highest expression of the vesicular glutamate transporter 1 (VGLUT1) and GLT-1 after 20 days in vitro. Conditioned media from these neuron enriched cultures also induced GLT-1 expression in primary astrocytic cultures, which were free from neurons. The amount of glutamatergic neurons guides the morphological maturation of astrocytes and GLT-1 expression both in the neuron enriched cultures and in the conditioned media supplemented astrocytic cultures. Interestingly, these cultures were not influenced or activated by the inflammatory stimulus lipopolysaccharide. This suggests that soluble factors from neurons protect microglia and astrocytes to become inflammatory reactive. In conclusion we have developed a well characterized culture model system enriched in neurons, taken from newborn rats and cultured in defined media. The neurons express different neuronal phenotypes. Such a model system is valuable when studying interactions between neurons and glial cells.     

HHV-6A infection dysregulates autophagy/UPR interplay increasing beta amyloid production and tau phosphorylation in astrocytoma cells as well as in primary neurons,possible molecular mechanisms linking viral infection to Alzheimer's disease

HHV-6A and HHV-6B are neurotropic viruses able to dysregulate autophagy and activate ER stress/UPR in several cell types. The appropriate functioning of these processes is required for cell homeostasis, particularly in post-mitotic cells such as neuronal cells. Interestingly, neurodegenerative diseases such as Alzheimer's disease (AD) are often accompanied by autophagy dysregulation and abnormal UPR activation. This study demonstrated for the first time that HHV-6A infection of astrocytoma cells and primary neurons reduces autophagy, increases Aβ production and activates ER stress/UPR promoting tau protein hyper-phosphorylation. Our results support previous studies suggesting that HHV-6A infection may play a role in AD and unveil the possible underlying molecular mechanisms involved.     

Autophagy Is Involved in the Sevoflurane Anesthesia-Induced Cognitive Dysfunction of Aged Rats

Autophagy is associated with regulation of both the survival and death of neurons, and has been linked to many neurodegenerative diseases. Postoperative cognitive dysfunction is commonly observed in elderly patients following anesthesia, but the pathophysiological mechanisms are largely unexplored. Similar effects have been found in aged rats under sevoflurane anesthesia; however, the role of autophagy in sevoflurane anesthesia-induced hippocampal neuron apoptosis of older rats remains elusive. The present study was designed to investigate the effects of autophagy on the sevoflurane-induced cognitive dysfunction in aged rats, and to identify the role of autophagy in sevoflurane-induced neuron apoptosis. We used 20-month-old rats under sevoflurane anesthesia to study memory performance, neuron apoptosis, and autophagy. The results demonstrated that sevoflurane anesthesia significantly impaired memory performance and induced hippocampal neuron apoptosis. Interestingly, treatment of rapamycin, an autophagy inducer, improved the cognitive deficit observed in the aged rats under sevoflurane anesthesia by improving autophagic flux. Rapamycin treatment led to the rapid accumulation of autophagic bodies and autophagy lysosomes, decreased p62 protein levels, and increased the ratio of microtubule-associated protein light chain 3 II (LC3-II) to LC3-I in hippocampal neurons through the mTOR signaling pathway. However, administration of an autophagy inhibitor (chloroquine) attenuated the autophagic flux and increased the severity of sevoflurane anesthesia-induced neuronal apoptosis and memory impairment. These findings suggest that impaired autophagy in the hippocampal neurons of aged rats after sevoflurane anesthesia may contribute to cognitive impairment. Therefore, our findings represent a potential novel target for pro-autophagy treatments in patients with sevoflurane anesthesia-induced neurodegeneration.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

Role of LKB1-SAD/MARK pathway in neuronal polarization

The formation of axon/dendrite polarity is critical for the neuron to perform its signaling function in the brain. Recent advance in our understanding of cellular and molecular mechanisms underlying the development and maintenance of neuronal polarity has been greatly facilitated by the use of the culture system of dissociated hippocampal neurons. Among many polarization-related proteins, we here focus on the mammalian LKB1, the counterpart of the C. elegans Par-4, which is an upstream regulator among six Par (partitioning-defective) genes that act as master regulators of cell polarity in different cell types across evolutionary distant species. Recent studies have identified LKB1 and its downstream targets SAD/MARK kinases (mammalian homologs of Par-1) as key regulators of neuronal polarization and axon development in cultured neurons and in developing cortical neurons in vivo. We will review the properties of and interactions among proteins in this LKB1-SAD/MARK pathway, drawing upon information obtained from both neuronal and non-neuronal systems. Due to central role of the protein kinase A-dependent phosphorylation of LKB1 in the activation of this pathway, we will review recent findings on how cAMP and cGMP signaling may serve as antagonistic second messengers for axon/dendrite development, and how these cyclic nucleotides may mediate the action of extracellular polarizing factors by modulating the activity of the LKB1-SAD/MARK pathway.     

Light-addressed single-neuron stimulation in dissociated neuronal cultures with sparse expression of ChR2

Individual neurons are heterogeneous and have profound impact on population activity in a complex cortical network. Precise experimental control of the firing of multiple neurons would be therefore beneficial to advance our understanding of cell-network interactions. Except for direct intracellular stimulation, however, it is difficult to gain precise control of targeted neurons without inducing antidromic activation of untargeted neurons. To overcome this problem, we attempt to create a sparse group of photosensitized neurons via transfection of Channelrhodopsin-2 (ChR2) in primary dissociated cultures and then deliver light-addressed stimulation exclusively to these target neurons. We first show that liposome transfection was able to express ChR2 in 0.3-1.9% of cells plated depending on cell density. This spatially sparse but robust expression in our neuronal cultures offered the capability of single cell activation by illuminating a spot of light. We then demonstrated that delivering a pulsed train to photo-activate a single neuron had a substantial effect on the activity level of an entire neuronal culture. Furthermore, the activity level was controllable by altering the frequency of light illumination when 4 neurons were recruited as stimulation targets. These results suggest that organized activation of a very small population of neurons can provide better control over global activity of neuronal circuits than can single-neuron activities by themselves.     

Autophagy in neurons: a review

Macroautophagy is a process of regulated turnover of cellular constituents that occurs during development and under conditions of stress such as starvation. Defects in autophagy have serious consequences, as they have been linked to neurodegenerative disease, cancer, and cardiomyopathy. This process, which exists in all eukaryotic cells, is tightly controlled, but in extreme cases results in the death of the cell. While major insights into the molecular and biochemical pathways involved have come from genetic studies in yeast, little is known about autophagic pathways in mammalian cells, particularly in neurons. Recently, research in neuronal culture models has begun to identify some characteristics of neuronal macroautophagy. The results suggest that macroautophagy in neurons may provide a neuroprotective mechanism. Here, we review the defining characteristics of autophagy with special attention to its role in neurodegenerative disorders, and recent efforts to delineate the pathway of autophagic protein degradation in neurons.     

Notch resolves mixed neural identities in the zebrafish epiphysis

Manipulation of Notch activity alters neuronal subtype identity in vertebrate neuronal lineages. Nonetheless, it remains controversial whether Notch activity diversifies cell fate by regulating the timing of neurogenesis or acts directly in neuronal subtype specification. Here, we address the role of Notch in the zebrafish epiphysis, a simple structure containing only two neural subtypes: projection neurons and photoreceptors. Reducing the activity of the Notch pathway results in an excess of projection neurons at the expense of photoreceptors, as well as an increase in cells retaining a mixed identity. However, although forced activation of the pathway inhibits the projection neuron fate, it does not promote photoreceptor identity. As birthdating experiments show that projection neurons and photoreceptors are born simultaneously, Notch acts directly during neuronal specification rather than by controlling the timing of neurogenesis. Finally, our data suggest that two distinct signals are required for photoreceptor fate specification: one for the induction of the photoreceptor fate and the other, involving Notch, for the inhibition of projection neuron traits. We propose a novel model in which Notch resolves mixed neural identities by repressing an undesired genetic program.     

Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons.

Apoptosis is a prominent mechanism of programmed cell death in lymphocytes and in cancer cells not previously found in neurons. We have identified apoptosis and internucleosomal DNA degradation in cultures of cerebellar granule neurons. 1-methyl-4-phenylpyridinium, a selective neurotoxin that destroys the dopaminergic nigrostriatal pathway and results in a parkinsonian syndrome, increases the rate of apoptosis and kills cerebellar granule cells in culture via induction of programmed cell death. Inhibition of gene expression in granule cells with cycloheximide prevents the MPP(+)-induced apoptosis and the DNA fragmentation. Our findings demonstrate a new pathway of neuron death and suggest the possibility that neurodegenerative diseases may result from the inappropriate activation of programmed cell death by apoptosis.     

DRAM Is Involved in Regulating Nucleoside Analog-Induced Neuronal Autophagy in a p53-Independent Manner

The widespread use of combined anti-retroviral therapy (cART) has not decreased the prevalence of HIV-1-associated neurocognitive disorder (HAND), a type of neurodegenerative disease, even though cART effectively inhibits virus colonization in the central nervous system. Therefore, anti-retroviral agents cannot be fully excluded from the pathogenesis of HAND. Our previous study reported that long-term nucleoside analogue (NA) exposure induced mitochondrial toxicity in the cortical neurons of HAND patients and mice, but the exact mechanism of NA-associated neurotoxicity has remained unclear. Alteration of autophagy can result in protein aggregation and the accumulation of dysfunctional organelles, which are hallmarks of some neurodegenerative diseases. In this study, we first found increased autophagy in cortical autopsy specimens of AIDS patients. We then found that a low dose of NAs could stimulate autophagy in primary cultured neurons, while a high dose of NAs could induce only neuronal apoptosis. The level of NA-induced Bcl-2 and Bax expressions determined whether neuronal autophagy or apoptosis occurred. Furthermore, the level of NA-induced neuronal apoptosis correlated with the dysfunction of cellular DNA polymerase gamma. Damage-regulated autophagy modulator (DRAM) overexpression was also involved in NA-induced neuronal autophagy. p53 played a role in the regulation of NA-induced neuronal apoptosis, but its role in NA-associated neuronal autophagy was uncertain. Our results suggest that DRAM is involved in the regulation of NA-induced neuronal autophagy in a p53-independent manner. Further research is needed to investigate the underlying mechanism.