Carol M. Troy

Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway

We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier “vector” peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide.

β‐Amyloid‐induced neuronal apoptosis requires c‐Jun N‐terminal kinase activation

β‐Amyloid (Aβ) has been strongly implicated in the pathophysiology of Alzheimers disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c‐Jun N‐terminal kinases (JNKs) and of their substrate, c‐Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated Aβ. The activities of JNK family members increased in neuronal PC12 cells within 2 h of Aβ treatment and reached 3–4‐fold elevation by 6 h. To test the role of these changes in death caused by Aβ, we examined the effects of CEP‐1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP‐1347 (100–300 nm) in the culture medium effectively blocked the increases in cellular JNK activity caused by Aβ and, at similar concentrations, protected both PC12 cells and sympathetic neurons from Aβ‐evoked‐death. Effective protection required addition of CEP‐1347 within 2 h of Aβ treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant‐negative c‐Jun construct also conferred protection from Aβ‐evoked death, supporting a model in which JNK activation contributes to death via activation of c‐Jun. Finally, CEP‐1347 blocked Aβ‐stimulated activation of caspase‐2 and ‐3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by Aβ and hence identify it as a potential therapeutic target in AD.

Highly Efficient Small Interfering RNA Delivery to Primary Mammalian Neurons Induces MicroRNA-Like Effects before mRNA Degradation

The study of protein function in neurons has been hindered by the lack of highly efficient, nontoxic methods of inducing RNA interference in such cells. Here we show that application of synthetic small interfering RNA (siRNA) linked to the vector peptide Penetratin1 results in rapid, highly efficient uptake of siRNA by entire populations of cultured primary mammalian hippocampal and sympathetic neurons. This treatment leads to specific knock-down of targeted proteins within hours without the toxicity associated with transfection. In contrast to current methods, our technique permits study of protein function across entire populations with minimal disturbance of complex cellular networks. Using this technique, we found that protein knock-down (evident after 6 hr) precedes any decrease in targeted message (evident after 24 hr), suggesting an early, translational repression by perfectly targeted siRNAs.

Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

Abstract: Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N‐methyl‐d‐aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin‐1β‐converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA‐induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V‐ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z‐VAD·fmk) that inhibits caspase activity. Pretreatment with either V‐ICEinh or Z‐VAD·fmk protects cerebrocortical neurons from NMDA‐induced apoptosis, suggesting a role for caspases in NMDA‐induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA‐induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V‐ICEinh or Z‐VAD·fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V‐ICEinh and Z‐VAD·fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.

Ontogeny of the neuronal intermediate filament protein, peripherin, in the mouse embryo

The expression of peripherin, a type III neuron-specific intermediate filament protein, and the middle neurofilament subunit were studied in the mouse embryo using immunofluorescence staining. The earliest staining for both proteins is seen at embryonic day 9 in the myelencephalon, initially as fiber staining followed by cell body staining in the developing facial and acoustic nuclei. As the embryo develops, there is rostral as well as caudal extension of peripherin and staining is seen in the trigeminal ganglia, nerve fibers and in the enteric nervous system. As the spinal cord forms there is anti-peripherin staining in developing motoneurons of the anterior horns while little cell body staining is seen for the middle neurofilament subunit. Both antibodies stain the developing dorsal root and its entry zone, but peripherin is found in the secondary sensory and commissural fibers while the middle neurofilament subunit is not. While both proteins are found in the neurons of the dorsal root ganglia, their distribution varies. The larger peripheral cells of the ganglia contain both proteins while the smaller more central cells, constituting over 60% of the cells in the ganglia, contain only peripherin. A similar picture is found in the sympathetic ganglia where there are cells which contain peripherin. middle neurofilament subunit or both, but where the majority of the neurons have only peripherin in their cell bodies. Peripherin is not found in the developing retina or in the adrenal medulla. Peripherin is also completely absent from cell bodies in the cerebral and cerebellar cortices. These results indicate that peripherin is found in development only in regions in which it is found in the adult. It can either co-exist with neurofilaments in the same neuron or the two may be independently expressed.

Endogenous amyloid-β is necessary for hippocampal synaptic plasticity and memory

The goal of this study was to investigate the role of endogenous amyloid‐β peptide (Aβ) in healthy brain.

Induction of CPP32-like Activity in PC12 Cells by Withdrawal of Trophic Support DISSOCIATION FROM APOPTOSIS

Inhibitors of interleukin-1β converting enzyme (ICE) and a related group of cysteine aspartases of the ICE/ced-3 family inhibit cell death in a variety of settings, including in PC12 cells and sympathetic neurons following withdrawal of trophic support. To assess the particular member(s) of the ICE/ced-3 family that are relevant to cell death and to position their activation within the apoptotic pathway, we have used specific substrates to measure ICE-like and CPP32-like enzymatic activity in naive and neuronally differentiated PC12 cells that had been deprived of trophic support (nerve growth factor and/or serum). Rapid induction of CPP32-like, but not ICE-like, activity was observed. c-Jun kinase activation and the action of bcl-2 and other survival agents, such as cell cycle blockers, a NO generator, N-acetylcysteine, aurintricarboxylic acid, and actinomycin D occurred at a point further upstream in the apoptotic pathway compared with the aspartase activation. In living cells, zVAD-FMK, a pseudosubstrate aspartase inhibitor, blocked the activity/activation of the aspartase at concentrations about one order of magnitude lower than those required to promote survival, raising the possibility that the CPP32-like aspartase is not the main death effector in this model.

Regulation of peripherin and neurofilament expression in regenerating rat motor neurons

Northern blotting, in situ hybridization and immunocytochemistry were used to study the changes in levels of mRNA coding for peripherin and in immunoreactivity of peripherin, a type III neuronal intermediate filament, in rat spinal motor neurons following axotomy of the sciatic nerve. For comparison, parallel studies examined the biology of neurofilament (NF) proteins in this model. The sciatic nerve was crushed at the junction of the L4-L5 spinal nerves. Levels of messenger RNA (mRNA) coding for peripherin in the motor neurons doubled by 4 days postaxotomy and remained elevated for a period of 6 weeks. Within 4-7 days of injury peripherin immunoreactivity increased significantly in cell bodies of motor neurons and remained elevated through 6 weeks. In contrast, no changes were detected in NF-M immunoreactivity over the same time period. By 8 weeks postaxotomy, levels of peripherin mRNA and protein returned to control values. The increases in the expression of peripherin parallel those of beta-tubulin and actin, and these changes are quite different from the alterations in neurofilament mRNA that decrease after axotomy. The contrasting responses of peripherin and NF to nerve injury indicates that each of these intermediate filaments may play distinct roles in nerve growth and regeneration.

The Parkinson Disease Protein Leucine-Rich Repeat Kinase 2 Transduces Death Signals via Fas-Associated Protein with Death Domain and Caspase-8 in a Cellular Model of Neurodegeneration

Neurodegenerative illnesses such as Parkinson and Alzheimer disease are an increasingly prevalent problem in aging societies, yet no therapies exist that retard or prevent neurodegeneration. Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD), but the mechanisms by which mutant forms of LRRK2 disrupt neuronal function and cause cell death remain poorly understood. We report that LRRK2 interacts with the death adaptor Fas-associated protein with death domain (FADD), and that in primary neuronal culture LRRK2-mediated neurodegeneration is prevented by the functional inhibition of FADD or depletion of caspase-8, two key elements of the extrinsic cell death pathway. This pathway is activated by disease-triggering mutations, which enhance the LRRK2-FADD association and the consequent recruitment and activation of caspase-8. These results establish a direct molecular link between a mutant PD gene and the activation of programmed cell death signaling, and suggest that FADD/caspase-8 signaling contributes to LRRK2-induced neuronal death.

Caspases on the brain

The basic mechanisms that underlie neurodegenerative diseases are unknown. Loss of function of specific regions of the brain is due to incapacitation of cells that constitute those regions. Cells can simply stop functioning normally (neurons may cease to transmit signals), or they may die. There is now evidence that the pathology of several neurodegenerative diseases is due to inappropriate apoptosis. This being the case, an understanding of the mediators of apoptosis, their identities, and their role in orchestrating death would be a vital step toward remedying the diseases. The central components of apoptotic pathways, proteases of the caspase family, are present in latent forms in all nucleated cells. Their activity is balanced by specific activation and inactivation events, and the molecular and biochemical controls have been well established in vitro and in model transformed cell lines. In this Mini‐Review, we consider the current status of the basic control mechanisms and how these may be subverted during neurodegeneration.