In Vitro Neurotoxicity Resulting from Exposure of Cultured Neural Cells to Several Types of Nanoparticles.

Laboratory and industrial production of various nanoparticles, single-walled nanotubes (SWNTs), fullerene (C60), cadmium selenide (CdSe) quantum dots, carbon black (CB), and dye-doped silica nanospheres (NSs), has greatly increased in the past 15 years. However, little research has been done to analyze the toxicity of these materials. With recent studies showing that nano-substances can cross the blood-brain barrier, we examined the neurotoxicity of these manufactured nanoparticles. By employing the rat PC-12 neuronal-like cell line as the basis for our studies, we were able to evaluate the toxicity caused by these five nanoparticles. The level of toxicity was measured by testing for cell viability using the lactate dehydrogenase (LDH) cell viability assay, morphological analysis of changes in cellular structures, and Western blot analyses of αII-spectrin breakdown products (SBDP) as cell death indicators. Our results showed cytotoxicity in nondifferentiated PC-12 cells exposed to CB (10-100 µg/mL), SWNTs (10-100 µg/mL), C60 (100 µg/mL), CdSe (10 µg/mL), CB (500 µg/mL), and dye-doped silicon NSs (10 µg/mL). Exposure to higher concentrations (100 µg/mL) of SWNTs, CB, and C60 increased the formation of SBDP150/145, as well as cell membrane contraction and the formation of cytosolic vacuoles. The incorporations of the nanoparticles into cell cytoplasm were observed using the fluorescent dye-doped NSs in both nondifferentiated and nerve growth factor (NGF)-differentiated PC-12 cells. When PC-12 cells are differentiated, they appeared to be even more sensitive to cytotoxicity of nanoparticles such as CB 10 nm (10-100 µg/mL), CB 100 nm (10-100 µg/mL), and CdSe (1-10 µg/mL).

Assessment of Serum UCH-L1 and GFAP in Acute Stroke Patients.

A rapid and reliable diagnostic test to distinguish ischemic from hemorrhagic stroke in patients presenting with stroke-like symptoms is essential to optimize management and triage for thrombolytic therapy. The present study measured serum concentrations of ubiquitin C-terminal hydrolase (UCH-L1) and glial fibrillary astrocytic protein (GFAP) in acute stroke patients and healthy controls and investigated their relation to stroke severity and patient characteristics. We also assessed the diagnostic performance of these markers for the differentiation of intracerebral hemorrhage (ICH) from ischemic stroke (IS). Both UCH-L1 and GFAP concentrations were significantly greater in ICH patients than in controls (p < 0.0001). However, exclusively GFAP differed in ICH compared with IS (p < 0.0001). GFAP yielded an AUC of 0.86 for differentiating between ICH and IS within 4.5hrs of symptom onset with a sensitivity of 61% and a specificity of 96% using a cut-off of 0.34ng/ml. Higher GFAP levels were associated with stroke severity and history of prior stroke. Our results demonstrate that blood UCH-L1 and GFAP are increased early after stroke and distinct biomarker-specific release profiles are associated with stroke characteristics and type. We also confirmed the potential of GFAP as a tool for early rule-in of ICH, while UCH-L1 was not clinically useful.

A neuroproteomic and systems biology analysis of rat brain post intracerebral hemorrhagic stroke.

Intracerebral hemorrhage (ICH) is a devastating form of stroke leading to a high rate of death and disability worldwide. Although it has been hypothesized that much of the IHC insult occurs in the subacute period mediated via a series of complex pathophysiological cascades, the molecular mechanisms involved in ICH have not been systematically characterized. Among the best approaches to understand the underlying mechanisms of injury and recovery, protein dynamics assessment via proteomics/systems biology platforms represent one of the cardinal techniques optimized for mechanisms investigation and biomarker identification. A proteomics approach may provide a biomarker focused framework from which to identify candidate biomarkers of pathophysiological processes involved in brain injury after stroke. In this work, a neuroproteomic approach (LC-MS/MS) was applied to investigate altered expression of proteins that are induced in brain tissue 3 h after injury in a rat model of ICH. Data from sham and focal ischemic models were also obtained and used for comparison. Based on the differentially expressed protein profile, systems biology analysis was conducted to identify associated cellular processes and related interaction maps. After LC-MS/MS analysis of the 3 h brain lysates, 86 proteins were differentially expressed between hemorrhagic and sham tissues. Furthermore, 38 proteins were differentially expressed between ischemic and sham tissues. On the level of global pathway analysis, hemorrhagic stroke proteins were shown to be involved in autophagy, ischemia, necrosis, apoptosis, calpain activation, and cytokine secretion. Moreover, ischemic stroke proteins were related to cell death, ischemia, inflammation, oxidative stress, caspase activation and apoptotic injury. In conclusion, the proteomic responses identified in this study provide key information about target proteins involved in specific pathological pathways.

Different expression of ubiquitin C-terminal hydrolase-L1 and αII-spectrin in ischemic and hemorrhagic stroke: Potential biomarkers in diagnosis.

The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally different mechanisms and thus different treatment options. These two stroke categories were applied to rat models to identify potential biomarkers that can distinguish between them. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain hemispheres and biofluids were collected at two time points: 3 and 6h after stroke. Known molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF) and Banyan's proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after 6h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were elevated after 6h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels were increased after 3 and 6h of ischemia detected by ELISA. However, levels of those proteins in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. These molecules behaved differently in the two stroke models and thus may be capable of being differentiated.

Systems biology and theranostic approach to drug discovery and development to treat traumatic brain injury.

Traumatic brain injury is a significant disease affecting 1.4 to 2 million patients every year in the USA. Currently, there are no FDA-approved therapeutic remedies to treat TBI despite the fact that there have been over 200 clinical drug trials, all which have failed. These drugs used the traditional single drug-to-target approach of drug discovery and development. An alternative based upon the advances in genomics, proteomics, bioinformatic tools, and systems biology software has enabled us to use a Systems Biology-based approach to drug discovery and development for TBI. It focuses on disease-relevant converging pathways as potential therapeutic intervention points and is accompanied by downstream biomarkers that allow for the tracking of drug targeting and appears to correlate with disease mitigation. When realized, one is able to envision that a companion diagnostic will be codeveloped along the therapeutic compound. This "theranostic" approach is perfectly positioned to align with the emerging trend toward "personalized medicine".

Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells.

Neuronal cell death after traumatic brain injury, Alzheimer's disease and ischemic stroke may in part be mediated through endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78 and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145 levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120 was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7 substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site D(341) detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2 and caspase-3/7.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).

Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype.

BACKGROUND: Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA). RESULTS: NMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment. CONCLUSIONS: Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.

Multiple alphaII-spectrin breakdown products distinguish calpain and caspase dominated necrotic and apoptotic cell death pathways.

Apoptosis and oncotic necrosis in neuronal and glial cells have been documented in many neurological diseases. Distinguishing between these two major types of cell death in different neurological diseases is needed in order to better reveal the injury mechanisms so as to open up opportunities for therapy development. Accumulating evidence suggests apoptosis and oncosis epitomize the extreme ends of a broad spectrum of morphological and biochemical events. Biochemical markers that can distinguish between the calpain and caspase dominated types of cell death would help in this process. In this study, three chemical agents, maitotoxin (MTX), staurosporine (STS) and thylenediaminetetraacetic acid (EDTA), were used to induce different types of cell death in PC12 neuronal-like cells. MTX-induced necrosis, as determined by the increased levels of calpain-specific cleaved fragments of spectrin by antibodies specific to the calpain-cleaved 150 kDa alphaII-spectrin breakdown product (SBDP150) and 145 kDa alphaII-spectrin breakdown product (SBDP145). In this paradigm, there were no detectable SBDP150i and SBDP120 fragments as determined by antibodies specific to the caspase-cleaved specific fragments similar to those seen in the EDTA-mediated apoptotic PC-12 cells. In contrast to the calpain specific MTX necrosis treatment and the caspase EDTA apoptotic treatment is the STS treatment which induced both proteases as shown by the increase in all the SBDP fragments. Furthermore, compared to SBDP150, SBDP145 appears to be a more specific and sensitive biomarker for calpain activation. Taken together, our results suggested calpains and caspases which dominate the two major types of cell death could be independently discriminated by specifically examining the multiple alphaII-spectrin cleavage breakdown products.

Biomarkers of blast-induced neurotrauma: profiling molecular and cellular mechanisms of blast brain injury.

The nature of warfare in the 21st century has led to a significant increase in primary blast or over-pressurization injuries to the whole body and head, which manifest as a complex of neuro-somatic damage, including traumatic brain injury (TBI). Identifying relevant pathogenic pathways in reproducible experimental models of primary blast wave exposure is therefore vital to the development of biomarkers for diagnostics of blast brain injury. Comparative analysis of mechanisms and putative biomarkers of blast brain injury is complicated by a deficiency of experimental studies. In this article, we present an overview of current TBI biomarkers, as well as outline experimental strategies to investigate molecular signatures of blast neurotrauma and to develop a pathway network map for novel biomarker discovery. These biomarkers will be effective for triaging and managing both combat and civilian casualities.

Neuroproteomics and systems biology-based discovery of protein biomarkers for traumatic brain injury and clinical validation.

The rapidly growing field of neuroproteomics has expanded to track global proteomic changes underlying various neurological conditions such as traumatic brain injury (TBI), stroke, and Alzheimer's disease. TBI remains a major health problem with approximately 2 million incidents occurring annually in the United States, yet no affective treatment is available despite several clinical trials. The absence of brain injury diagnostic biomarkers was identified as a significant road-block to therapeutic development for brain injury. Recently, the field of neuroproteomics has undertaken major advances in the area of neurotrauma research, where several candidate markers have been identified and are being evaluated for their efficacy as biological biomarkers in the field of TBI. One scope of this review is to evaluate the current status of TBI biomarker discovery using neuroproteomics techniques, and at what stage we are at in their clinical validation. In addition, we will discuss the need for strengthening the role of systems biology and its application to the field of neuroproteomics due to its integral role in establishing a comprehensive understanding of specific brain disorder and brain function in general. Finally, to achieve true clinical input of these neuroproteomic findings, these putative biomarkers should be validated using preclinical and clinical samples and linked to clinical diagnostic assays including ELISA or other high-throughput assays.

Calpain and caspase proteolytic markers co-localize with rat cortical neurons after exposure to methamphetamine and MDMA.

Abuse of the club drugs Methamphetamine (Meth) and Ecstasy (MDMA) is an international problem. The seriousness of this problem is the result of what appears to be programmed cell death (PCD) occurring within the brain following their use. This follow up study focused on determining which cell types, neurons and/or glial cells, were affected in the brains of drug-injected rats. Two proteolytic enzyme families involved in PCD, calpains and caspases, were previously shown to be activated and to degrade the brain cytoskeletal associated protein alphaII-spectrin. Using methods employed and confirmed in traumatic brain injury (TBI) studies, rat brain tissues were examined, 24 and 48 h after Meth and MDMA exposure, for the activation of calpain-1 and caspase-3, and their subsequent alphaII-spectrin cleavage breakdown products (SBDPs), SBDP145, and SBDP120, respectively. Based upon our previous studies we know that activated calpain-1 and caspase-3 were up-regulated after drug use as were the levels of their cleaved SBDPs, SBDP145, and SBDP120, respectively, which is indicative of PCD. Here we show that activated calpain-1 and caspase-3 increases could be localized to neurons in the cortex where the products of their cleaved targets were found to be concentrated, particularly, to the axonal regions. These findings support the hypothesis that calpains and caspases mediate PCD in cortical neurons following club drug abuse and, more importantly, appear to contribute to the neuropathology suffered by abusers.

Calpain- and caspase-mediated alphaII-spectrin and tau proteolysis in rat cerebrocortical neuronal cultures after ecstasy or methamphetamine exposure.

Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

Neuroprotection targets after traumatic brain injury.

PURPOSE OF REVIEW: The scarcity of pharmacological neuroprotective treatments for traumatic brain injury is a concern being targeted on various fronts. This review examines the latest treatments under investigation. RECENT FINDINGS: In the last 12-18 months, no drug has completed phase III clinical trials as a clearly proven method to treat traumatic brain injury. While the drugs work in rodents, when they make it to clinical trial they have failed primarily due to negative side-effects. Those still in trial show promise, and even those rejected have undergone modifications and now show potential, e.g. second-generation N-methyl-D-aspartic acid and alpha-amino-3-hydroxy-methyl-4-isoxazolyl-propionic acid receptor antagonists, calpain inhibitors, and cyclosporine A analogues. Also, several drugs not previously given much attention, such as the antibiotic minocycline, estrogen and progesterone, and a drug already approved for other diseases, erythropoietin, are being examined. Finally, a treatment generating some controversy, but showing potential, is the application of hypothermia to the patients. SUMMARY: Clearly, finding treatments for traumatic brain injury is not going to be easy and is evidently going to require numerous trials. The good news is that we are closer to finding one or more methods for treating traumatic brain injury patients.

Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury.

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.

Direct Rho-associated kinase inhibition [correction of inhibiton] induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells.

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer's disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.

Unfolded protein response after neurotrauma.

The endoplasmic reticulum (ER) lumen, which actively monitors the synthesis, folding, and modification of newly synthesized transmembrane and secretory proteins as well as lipids, is quite sensitive to homeostatic perturbations. The biochemical, molecular, and physiological events that elevate cellular ER stress levels and disrupt Ca2+ homeostasis trigger secondary reactions. These reactions are factors in the ongoing neurological pathology contributing to the continual tissue loss. However, the cells are not without defensive systems. One of the reactive mechanisms, the unfolded protein response (UPR), when evoked, provides some measure of protection, unless the stress conditions become prolonged or overwhelming. UPR activation occurs when key ER membrane-bound sensor proteins detect the excess accumulation of misfolded or unfolded proteins within the ER lumen. The activation of these sensors leads to a general protein translation shut-down, transcriptional induction, and translation of select proteins to deal with the difficult and miscreant protein or to encourage their degradation so they will do no harm. If the stress is prolonged, caspase-12, along with other apoptotic proteins, are activated, triggering programmed cell death. UPR, once considered to be a rather simple response, can now be characterized as a multifaceted labyrinth of reactions that continues expanding as research intensifies. This review will examine what has been learned to date about how this highly efficient and specific signaling pathway copes with ER stress, by centering on the basic components, their roles, and the complex interactions engendered. Finally, the UPR impact in various central nervous system injuries is summarized.

Amino acid starvation induced autophagic cell death in PC-12 cells: evidence for activation of caspase-3 but not calpain-1.

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis.