Leonidas Stefanis

α-Synuclein Overexpression in PC12 and Chromaffin Cells Impairs Catecholamine Release by Interfering with a Late Step in Exocytosis

α-Synuclein (α-syn), a protein implicated in Parkinsons disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how α-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous α-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant α-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically “docked” vesicles was apparent in the α-syn-overexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) α-syn, as well as chromaffin cells from control and α-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant α-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P α-syn-overexpressing cells. The α-syn-overexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that α-syn overexpression inhibits a vesicle “priming” step, after secretory vesicle trafficking to “docking” sites but before calcium-dependent vesicle membrane fusion.

α-Synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells

α-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegenerative diseases. Although the function of α-synuclein is not thoroughly elucidated, we found that α-synuclein regulates dopamine synthesis by binding to and inhibiting tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis. Understanding α-synuclein function in dopaminergic cells should add to our knowledge of this key protein, which is implicated in Parkinsons disease and other disorders. Herein, we report a mechanism by which α-synuclein diminishes tyrosine hydroxylase phosphorylation and activity in stably transfected dopaminergic cells. Short-term regulation of tyrosine hydroxylase depends on the phosphorylation of key seryl residues in the amino-terminal regulatory domain of the protein. Of these, Ser40 contributes significantly to tyrosine hydroxylase activation and dopamine synthesis. We observed that α-synuclein overexpression caused reduced Ser40 phosphorylation in MN9D cells and inducible PC12 cells. Ser40 is phosphorylated chiefly by the cyclic AMP-dependent protein kinase PKA and dephosphorylated almost exclusively by the protein phosphatase, PP2A. Therefore, we measured the impact of α-synuclein overexpression on levels and activity of PKA and PP2A in our cells. PKA was unaffected by α-synuclein. PP2A protein levels also were unchanged, however, the activity of PP2A increased in parallel with α-synuclein expression. Inhibition of PP2A dramatically increased Ser40 phosphorylation only in α-synuclein overexpressors in which α-synuclein was also found to co-immunoprecipitate with PP2A. Together the data reveal a functional interaction between α-synuclein and PP2A that leads to PP2A activation and underscores a key role for α-synuclein in protein phosphorylation.

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.

Proteasomal Inhibition-Induced Inclusion Formation and Death in Cortical Neurons Require Transcription and Ubiquitination

Increasing evidence suggests that proteasomal dysfunction plays a role in the pathogenesis of Lewy body diseases. We have used pharmacological inhibitors of the proteasome to model proteasomal dysfunction in cultured rat cortical neurons. Proteasomal inhibition induced apoptotic death and formation of cytoplasmic ubiquitinated inclusions, which were present only in viable neurons. Actinomycin D, but not a caspase inhibitor, prevented inclusion formation, whereas both agents inhibited cell death. alpha-Synuclein and thioflavin S staining were found within the inclusions. alpha-Synuclein, however, did not appear to be ubiquitinated or aggregated. A dominant-negative mutant of an E2 ubiquitin-conjugating enzyme, cdc34, prevented inclusion formation and attenuated cell death. Our results suggest that in cortical neurons: (a) proteasomal dysfunction plays a role in formation of ubiquitin/alpha-synuclein-positive inclusions, (b) inclusion formation is an active cell process requiring transcription, and (c) ubiquitination of certain proteins is required for inclusion formation and may participate in neuronal death.

Activation of Caspase-3 in Developmental Models of Programmed Cell Death in Neurons of the Substantia Nigra

Abstract: Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase‐3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase‐3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6‐hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase‐3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The sub‐cellular distribution of activated caspase‐3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.

Involvement of caspase 3 in apoptotic death of cortical neurons evoked by DNA damage

Previous reports have shown that DNA-damage-evoked death of embryonic cortical neurons is delayed by general caspase inhibitors and is accompanied by an increase in DEVD-AFC cleavage activity. We show here that this cleavage activity is lacking in camptothecin-treated caspase 3-deficient neurons. Moreover, we report that death of camptothecin-treated caspase 3-deficient neurons cultured from E16 embryos is delayed and that no significant increase in survival is observed with cotreatment with the general caspase inhibitor BAF. These results indicate that caspase-dependent death of camptothecin-treated cortical neurons requires caspase 3 activity. The delay in death is accompanied by impairment of DNA fragmentation. However, Bax-dependent cytochrome c release still occurs in camptothecin-treated caspase 3-deficient cortical neurons. Accordingly, we hypothesize that the delayed death which occurs in the absence of caspase 3 activity may be due to mitochondrial dysfunction. Finally, we show that the delay in death observed with E16 caspase 3-deficient neurons does not occur in neurons cultured from E19 embryos. This suggests that the requirement for caspase 3 in death of neurons evoked by DNA damage may differ depending upon the developmental state of the cell.

Dopaminergic neurons in rat ventral midbrain cultures undergo selective apoptosis and form inclusions, but do not up‐regulate iHSP70, following proteasomal inhibition

Dysfunction of the ubiquitin‐dependent protein degradation system, either at the level of the proteasome itself, or at the level of ubiquitination, may play a role in the pathogenesis of Parkinsons disease (PD) and other related neurodegenerative disorders. We have employed a cellular model of this dysfunction in which lactacystin or epoxomicin, selective pharmacological inhibitors of the proteasome, are applied to primary cultures of embryonic rat ventral midbrain. Proteasomal inhibition with either agent led to apoptotic death specifically within phenotypically defined tyrosine hydroxylase (TH)‐positive dopaminergic neurons, with little or no apoptotic death induced in GABAergic neurons. Inhibition of the proteasome also led to the formation of ubiquitin and α‐synuclein‐positive cytoplasmic inclusions in TH‐positive and TH‐negative neurons. Inclusions were observed in viable as well as apoptotic neurons, and required new or ongoing transcription. Tyrosine hydroxylase immunolabeling was often present within the inclusions. Such mislocalization may lead to dysfunction of dopamine biosynthesis. Interestingly, dopaminergic neurons, unlike other neurons within these cultures or cultured cortical neurons, failed to induce the chaperone Hsp70 in response to proteasomal inhibition. This failure may explain in part the increased sensitivity of these neurons to proteasomal inhibitors.

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells

Mutations in the α‐synuclein gene have recently been identified in families with inherited Parkinsons disease and the protein product of this gene is a component of Lewy bodies, indicating that α‐synuclein is involved in Parkinsons disease pathogenesis. A role for normal α‐synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein‐1, the rat homolog of human α‐synuclein, is highly and selectively up‐regulated at the mRNA and protein levels after 7u2003days of nerve growth factor treatment. Synuclein‐1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1–2u2003days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein‐1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor‐treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein‐1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein‐1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for α‐synuclein in plasticity‐maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.

Application of proteasomal inhibitors to mouse sympathetic neurons activates the intrinsic apoptotic pathway

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinsons disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival‐promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochromeu2003c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochromeu2003c release and caspase 3 activation, and also provided complete protection against proteasomal inhibition‐induced death. Bcl‐2 overexpression achieved a similar survival‐promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3‐only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl‐2 family members and the mitochondria.

Regulation of α‐synuclein by bFGF in cultured ventral midbrain dopaminergic neurons

α‐Synuclein is a neuronal protein that is implicated in the control of synaptic vesicle function and in Parkinsons disease (PD). Consequently, alterations of α‐synuclein levels may play a role in neurotransmission and in PD pathogenesis. However, the factors that regulate α‐synuclein levels are unknown. Growth factors mediate neurotrophic and plasticity effects in CNS neurons, and may play a role in disease states. Here we examine the regulation of α‐synuclein levels in primary CNS neurons, with particular emphasis on dopaminergic neurons. E18 rat cortical neurons and dopaminergic neurons of E14 rat ventral midbrain showed an induction of α‐synuclein protein levels with maturation in culture. Application of basic Fibroblast growth factor (bFGF) promoted α‐synuclein expression selectively within dopaminergic, and not GABAergic or cortical neurons. This induction was blocked by actinomycin D, but not by inhibition of bFGF‐induced glial proliferation. α‐Synuclein levels were not altered by glial‐derived neurotrophic factor (GDNF), or by apoptotic stimuli. We conclude that bFGF promotes α‐synuclein expression in cultured ventral midbrain dopaminergic neurons through a direct transcriptional effect. These results suggest that distinct growth factors may thus mediate plasticity responses or influence disease states in ventral midbrain dopaminergic neurons.