Marta M. Lipinski

Diversity in the Mechanisms of Neuronal Cell Death

Neurons may die as a normal physiological process during development or as a pathological process in diseases. The best-understood mechanism of neuronal cell death is apoptosis, which is regulated by an evolutionarily conserved cellular pathway that consists of the caspase family, the Bcl-2 family, and the adaptor protein Apaf-1. Apoptosis, however, may not be the only cellular mechanism that regulates neuronal cell death. Neuronal cell death may exhibit morphological features of autophagy or necrosis, which differ from that of the canonical apoptosis. This review evaluates the evidence supporting the existence of alternative mechanisms of neuronal cell death and proposes the possible existence of an evolutionarily conserved pathway of necrosis.

The retinoblastoma gene family in differentiation and development.

The retinoblastoma (Rb) tumor suppressor gene and its close relatives p107 and p130 are best known for their function in the control of cell cycle progression. In recent years, however, a new role for these proteins has been emerging as they have been linked with regulation of terminal differentiation of many tissues and cell types. In fact, Rb and its family members have been shown to be involved in multiple stages of the differentiation process including irreversible exit from the cell cycle, protection from apoptosis, induction of cell type specific gene expression and maintenance of the post-mitotic state. They also play a critical role in assuring the orderly progression through all these stages of differentiation.

Cell death assays for drug discovery

Cell death has an important role in many human diseases, and strategies aimed at modulating the associated pathways have been successfully applied to treat various disorders. Indeed, several clinically promising cytotoxic and cytoprotective agents with potential applications in cancer, ischaemic and neurodegenerative diseases have recently been identified by high-throughput screening (HTS), based on appropriate cell death assays. Given that different cell death modalities may be dysregulated in different diseases, it is becoming increasingly clear that such assays need to not only quantify the extent of cell death, but they must also be able to distinguish between the various pathways. Here, we systematically describe approaches to accurately quantify distinct cell death pathways, discuss their advantages and pitfalls, and focus on those techniques that are amenable to HTS.

Genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer's disease

Dysregulation of autophagy, a cellular catabolic mechanism essential for degradation of misfolded proteins, has been implicated in multiple neurodegenerative diseases. However, the mechanisms that lead to the autophagy dysfunction are still not clear. Based on the results of a genome-wide screen, we show that reactive oxygen species (ROS) serve as common mediators upstream of the activation of the type III PI3 kinase, which is critical for the initiation of autophagy. Furthermore, ROS play an essential function in the induction of the type III PI3 kinase and autophagy in response to amyloid β peptide, the main pathogenic mediator of Alzheimers disease (AD). However, lysosomal blockage also caused by Aβ is independent of ROS. In addition, we demonstrate that autophagy is transcriptionally down-regulated during normal aging in the human brain. Strikingly, in contrast to normal aging, we observe transcriptional up-regulation of autophagy in the brains of AD patients, suggesting that there might be a compensatory regulation of autophagy. Interestingly, we show that an AD drug and an AD drug candidate have inhibitory effects on autophagy, raising the possibility that decreasing input into the lysosomal system may help to reduce cellular stress in AD. Finally, we provide a list of candidate drug targets that can be used to safely modulate levels of autophagy without causing cell death.

A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions

Autophagy is a cellular catabolic mechanism that plays an essential function in protecting multicellular eukaryotes from neurodegeneration, cancer, and other diseases. However, we still know very little about mechanisms regulating autophagy under normal homeostatic conditions when nutrients are not limiting. In a genome-wide human siRNA screen, we demonstrate that under normal nutrient conditions upregulation of autophagy requires the type III PI3 kinase, but not inhibition of mTORC1, the essential negative regulator of starvation-induced autophagy. We show that a group of growth factors and cytokines inhibit the type III PI3 kinase through multiple pathways, including the MAPK-ERK1/2, Stat3, Akt/Foxo3, and CXCR4/GPCR, which are all known to positively regulate cell growth and proliferation. Our study suggests that the type III PI3 kinase integrates diverse signals to regulate cellular levels of autophagy, and that autophagy and cell proliferation may represent two alternative cell fates that are regulated in a mutually exclusive manner.

Autophagy Limits Listeria monocytogenes Intracellular Growth in the Early Phase of Primary Infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a delta actA mutant strain, the latter being impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria co-localize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a delta plcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.

Negative Regulation of Vps34 by Cdk Mediated Phosphorylation

Vacuolar protein sorting 34 (Vps34) complexes, the class III PtdIns3 kinase, specifically phosphorylate the D3 position of PtdIns to produce PtdIns3P. Vps34 is involved in the control of multiple key intracellular membrane trafficking pathways including endocytic sorting and autophagy. In mammalian cells, Vps34 interacts with Beclin 1, an ortholog of Atg6 in yeast, to regulate the production of PtdIns3P and autophagy. We show that Vps34 is phosphorylated on Thr159 by Cdk1, which negatively regulates its interaction with Beclin 1 during mitosis. Cdk5/p25, a neuronal Cdk shown to play a role in Alzheimers disease, can also phosphorylate Thr159 of Vps34. Phosphorylation of Vps34 on Thr159 inhibits its interaction with Beclin 1. We propose that phosphorylation of Thr159 in Vps34 is a key regulatory mechanism that controls the class III PtdIns3 kinase activity in cell-cycle progression, development, and human diseases including neurodegeneration and cancers.

Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system

The retinoblastoma tumor suppressor (RB) plays an important role in the regulation of cell cycle progression and terminal differentiation of many cell types. Rb−/− mouse embryos die at midgestation with defects in cell cycle regulation, control of apoptosis and terminal differentiation. However, chimeric mice composed of wild‐type and Rb‐deficient cells are viable and show minor abnormalities. To determine the role of Rb in development more precisely, we analyzed chimeric embryos and adults made with marked Rb−/− cells. Like their germline Rb−/− counterparts, brains of midgestation chimeric embryos exhibited extensive ectopic S‐phase entry. In Rb‐mutants, this is accompanied by widespread apoptosis. However, in chimeras, the majority of Rb‐deficient cells survived and differentiated into neuronal fates. Rescue of Rb−/− neurons in the presence of wild‐type cells occurred after induction of the p53 pathway and led to accumulation of cells with 4n DNA content. Therefore, the role of Rb during development can be divided into a cell‐autonomous function in exit from the cell cycle and a non‐cell‐autonomous role in the suppression of apoptosis and induction of differentiation.

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.

A novel tracing algorithm for high throughput imaging Screening of neuron-based assays.

High throughput neuron image processing is an important method for drug screening and quantitative neurobiological studies. The method usually includes detection of neurite structures, feature extraction, quantification, and statistical analysis. In this paper, we present a new algorithm for fast and automatic extraction of neurite structures in microscopy neuron images. The algorithm is based on novel methods for soma segmentation, seed point detection, recursive center-line detection, and 2D curve smoothing. The algorithm is fully automatic without any human interaction, and robust enough for usage on images with poor quality, such as those with low contrast or low signal-to-noise ratio. It is able to completely and accurately extract neurite segments in neuron images with highly complicated neurite structures. Robustness comes from the use of 2D smoothening techniques and the idea of center-line extraction by estimating the surrounding edges. Efficiency is achieved by processing only pixels that are close enough to the line structures, and by carefully chosen stopping conditions. These make the proposed approach suitable for demanding image processing tasks in high throughput screening of neuron-based assays. Detailed results on experimental validation of the proposed method and on its comparative performance with other proposed schemes are included.