RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal.

Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus.

RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons.

In human cell lines, the caspase 2 adaptor RAIDD interacts selectively with caspase 2 through its caspase recruitment domain (CARD) and leads to caspase 2-dependent death. Whether RAIDD induces such effects in neuronal cells is unknown. We have previously shown that caspase 2 is essential for apoptosis of trophic factor-deprived PC12 cells and rat sympathetic neurons. We report here that rat RAIDD, cloned from PC12 cells, interacts with rat caspase 2 CARD. RAIDD overexpression induced caspase 2 CARD- and caspase 9-dependent apoptosis of PC12 cells and sympathetic neurons. Apoptosis correlated with the formation of discrete perinuclear aggregates. Both death and aggregates required the expression of full-length RAIDD. Such aggregates may enable more effective activation of caspase 2 through close proximity. Following trophic deprivation, RAIDD overexpression increased death and aggregate formation. Therefore, RAIDD aggregation is important for its death-promoting effects and may play a role in trophic factor withdrawal-induced neuronal apoptosis.

Caspase-2 redux.

It has been difficult to assign caspase-2 to the effector or initiator caspase groups. It bears sequence homology to initiators (caspase-9 and CED-3), but its cleavage specificity is closer to the effectors (caspase-3 and -7). Interest in caspase-2 was dampened by the lack of a dramatic phenotype in the caspase-2 null mouse. Studies have been inhibited by the lack of knowledge about its mechanism of activation and the lack of specific methods to assay its activity. Molecular studies have defined a unique role for caspase-2 in apoptosis initiated by beta-amyloid toxicity or by trophic factor deprivation. Recently, a role for caspase-2 as an upstream initiator of mitochondrial permeabilization has been proposed. Thus, while much remains to be deciphered about caspase-2, most critically the mode of activation, it is clear that caspase-2 plays critical and singular roles in the control of programmed cell death.

Death in the balance: alternative participation of the caspase-2 and -9 pathways in neuronal death induced by nerve growth factor deprivation.

The data presented here demonstrate that sympathetic neurons have the potential to activate two alternative caspase-dependent pathways either of which is capable of mediating death induced by NGF deprivation and that these neurons have the potential to switch from one pathway to the other. The presence of these two alternative pathways to trophic factor deprivation-induced death may have implications for ensuring the correct development of the nervous system. In wild-type neurons, a caspase-2-dependent pathway is required for death, and a caspase-9-dependent pathway appears to be suppressed by endogenous inhibitors of apoptosis proteins (IAPs). In contrast, for caspase-2-null neurons, death is dependent on the caspase-9 pathway. The mechanism underlying the shift is the result of a threefold compensatory elevation of caspase-9 expression and a doubling of levels of direct IAP binding protein with low pI/(DIABLO)/second mitochondria-derived activator of caspase (Smac), an IAP inhibitor, both at the mRNA and protein levels [corrected]. These findings resolve seemingly discrepant findings regarding the roles of various caspases after NGF deprivation and raise a cautionary note regarding the interpretation of findings with caspase-null animals. The choice of the death-mediating caspase pathway in the sympathetic neurons is thus dependent on the regulated relative expression of components of the pathways including those of caspases, IAPs, and IAP inhibitors.

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's 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 A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, 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 A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta 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 A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-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 A beta and hence identify it as a potential therapeutic target in AD.

Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.

CEP-1347 (KT7515), an inhibitor of JNK activation, rescues sympathetic neurons and neuronally differentiated PC12 cells from death evoked by three distinct insults.

The c-Jun N-terminal kinase signaling cascade appears to play a role in some cases of cell death, including neuronal apoptosis. CEP-1347 (KT7515), an indolocarbazole of the K252a family, blocks this stress signaling cascade and promotes survival. Here, we used CEP-1347 to probe whether neuronal death pathways activated by distinct insults also possess elements in common. Cultured rat sympathetic neurons and neuronally differentiated PC12 cells were induced to die by withdrawal of nerve growth factor, exposure to ultraviolet irradiation, or subjection to oxidative stress. In each case, death was prevented by 100-200 nM CEP-1347. Moreover, in each of these death paradigms, c-Jun N-terminal kinase 1 activity in neuronally differentiated PC12 cells was elevated by two- or threefold, and this increase was totally blocked by CEP-1347 at concentrations that promoted survival. In contrast, 200 nM CEP-1347 did not block death due to serum withdrawal from undifferentiated PC12 cells or to activation of Fas in Jurkat T cell cultures, even though in each case c-Jun N-terminal kinase 1 activation occurred and was inhibited by CEP-1347. These observations suggest that some but not all death pathways triggered by different insults can include a common mechanistic component, a likely candidate for which is activation of the c-Jun N-terminal kinase signaling cascade.

Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)-like activity.

We have previously shown that caspase-2 (Nedd-2) is required for apoptosis induced by withdrawal of trophic support from PC12 cells and sympathetic neurons. Here, we examine the relationship of caspase-2 processing and cell death to induction of caspase-3 (CPP32)-like activity in PC12 cells. Caspase-2 processing, at a site tentatively identified as D333, led to the formation of an N-terminal 37 kDa product. This processing correlated temporally with induction of caspase-3-like activity. Agents previously shown to inhibit caspase-3-like activation, such as bcl-2 and the Cdk inhibitor flavopiridol, also acted upstream of caspase-2 processing. The general caspase inhibitors BAF and zVAD-FMK inhibited N-terminal caspase-2 processing. In contrast, the more selective caspase inhibitor DEVD-FMK inhibited the induction of caspase-3-like activity but did not affect caspase-2 processing or significantly suppress death in PC12 cells or sympathetic neurons. This indicates that caspase-3-like activity is not required for either caspase-2 processing or apoptosis in this paradigm. An antisense oligonucleotide to caspase-2 inhibited cell death but did not affect caspase-3-like activity, indicating that caspase-2 is not upstream of this activity and that activation of caspase-3-like caspases is not sufficient for death. Thus, in our paradigm, caspase-2 processing and caspase-3-like activity are induced independently of each other. Moreover, although death requires caspase-2, caspase-3-like activity is neither necessary nor sufficient for death.

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

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-1beta-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 x 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 x fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICEinh and Z-VAD x 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.

Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress.

Here, we compare the pathways by which DNA-damaging agents, NGF deprivation, and superoxide dismutase 1 (SOD1) depletion evoke apoptosis of sympathetic neurons. Previous work raised the hypothesis that cell cycle signaling plays a required role in neuronal apoptosis elicited by NGF deprivation and the DNA-damaging agent camptothecin. To test this hypothesis, we extended our investigation of DNA-damaging agents to cytosine arabinoside (AraC) and UV irradiation. As with NGF deprivation and camptothecin treatment, the cyclin-dependent kinase inhibitors flavopiridol and olomoucine protected neurons from apoptosis induced by AraC and UV treatment. These observations support the model that camptothecin, AraC, and UV treatment cause DNA damage, which leads to apoptosis by a mechanism that, as in the case of NGF deprivation, includes activation of cell cycle components. Flavopiridol and olomoucine, however, had no effect on death induced by SOD1 depletion, suggesting that CDKs do not play a role in this paradigm of neuronal death. To compare further the mechanisms of death evoked by NGF withdrawal, SOD1 depletion, and DNA-damaging agents, we investigated their responses to inhibitors of cysteine aspartases, elements of apoptotic pathways. The V-ICEinh and BAF, two peptide inhibitors of cysteine aspartases, protected neurons in all three death paradigms. In contrast, the cysteine aspartase inhibitory peptide zVAD-fmk conferred protection from NGF withdrawal and SOD1 depletion, but not DNA-damaging agents, whereas acYVAD-cmk protected only from SOD1 depletion. Taken together, these findings indicate that three different apoptotic stimuli activate separate pathways of death in the same neuron type.

Inhibitors of trypsin-like serine proteases inhibit processing of the caspase Nedd-2 and protect PC12 cells and sympathetic neurons from death evoked by withdrawal of trophic support.

Rat pheochromocytoma (PC12) cells and sympathetic neurons undergo apoptotic cell death upon withdrawal of trophic support. We have shown previously that selective cysteine aspartase (caspase) inhibitors protect PC12 cells and sympathetic neurons from such death, and that the caspase Nedd-2 is required for this type of death to occur. We now show that 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and N(alpha)-p-tosyl-L-lysine chloromethyl ketone (TLCK), agents that inhibit another class of proteases, the trypsin-like serine proteases, also suppress cell death in this paradigm. The site of action of these agents is upstream of the caspases, because the CPP32-like and Nedd-2-cleaving activities that are induced upon withdrawal of trophic support in PC12 cells are inhibited when AEBSF and TLCK are applied to the cells. Both agents inhibit thymidine incorporation in PC12 cells at concentrations similar to those that promote survival, raising the possibility that they may promote survival in neuronal cells through inhibition of aberrant activation of cell cycle components.

Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells.

Activation of cysteine aspartases (caspases) seems to be a required element of apoptotic death in many paradigms. We have shown previously that general inhibitors of cysteine aspartases block apoptosis of PC12 cells and sympathetic neurons evoked by either trophic factor (nerve growth factor and/or serum) deprivation or superoxide dismutase (SOD1) downregulation. Moreover, activation of a caspase family member similar or equivalent to the interleukin-1beta-converting enzyme (ICE) was implicated for death caused by SOD1 downregulation, but not withdrawal of trophic support. The experiments presented here demonstrate that diminished expression of the cysteine aspartase Nedd2 in PC12 cells and sympathetic neurons induced by an appropriate vector peptide-linked antisense oligonucleotide rescues them from death caused by trophic factor deprivation without inhibiting apoptosis in the same cell types evoked by SOD1 downregulation. Neither the level (as revealed by Western immunoblotting) nor the cellular distribution (as revealed immunohistochemically) of Nedd2 was altered demonstrably by trophic factor deprivation. However, evidence for proteolytic processing of Nedd2 (consistent with commencement of activation) was observed in PC12 cells after withdrawal of trophic support. These findings indicate that neuronal death triggered by different initial causes may be mediated by distinct members of the cysteine aspartase family.

Induction of CPP32-like activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis.

Inhibitors of interleukin-1beta 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.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

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.

Down-regulation of copper/zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells.

The discovery of missense mutations leading to reduced enzymatic activity in the copper/zinc superoxide dismutase (SOD1) in human familial amyotrophic lateral sclerosis has heightened interest in the role of free radicals in neurodegenerations but left the mechanisms by which they may cause neuronal death unexplained. We have approached this problem by specifically inhibiting the synthesis of SOD1 in cultured PC12 cells with antisense oligonucleotides. Cell survival in both untreated and nerve growth factor (NGF)-treated PC12 cells was inhibited by down-regulation of SOD1, with NGF-treated cells dying at lower levels of inhibition than untreated cells. Dying cells showed DNA degradation characteristic of apoptosis and could be rescued by the antioxidant vitamin E, with a combination of vitamin E and NGF being most efficacious. These results suggest that the induction of cell death by inhibition of SOD1 is due to free radical induction of apoptosis and that growth factor therapy for free-radical-mediated disease may require antioxidants in order to be effective.

Neurite outgrowth in peripherin-depleted PC12 cells.

Peripherin is the major neuronal intermediate filament (IF) protein in PC12 cells and both its synthesis and amount increase during nerve growth factor (NGF) promoted neuronal differentiation. To address the question of the biological function of peripherin in neurite initiation we have used an antisense oligonucleotide complementary to the 5' region of peripherin mRNA to specifically inhibit its transcription. The oligonucleotide blocks both the synthesis of peripherin and its increase in response to NGF. Peripherin was found to be a stable protein with a cellular half-life of approximately 7 d. 6 wk of incubation with the oligonucleotide decreases peripherin to 11% of the level in naive control cells and to 3% of that in NGF-treated control cells. Despite the depletion, NGF elicits apparently normal neurite outgrowth from the oligonucleotide-treated cells. As evaluated by EM, there are few IFs in these cells, either in the cell bodies or neurites. There is no compensatory increase in NF-M, NF-L, or vimentin levels as a result of the inhibition of peripherin synthesis. These findings suggest that peripherin is not required for neurite formation, but is necessary for the formation of a cellular IF network which could be involved in process stability. They also demonstrate the utility of antisense oligonucleotides for the study of proteins with long half-lives.

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.

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.