Hardy J. Rideout

Proteasomal inhibition leads to formation of ubiquitin/α-synuclein-immunoreactive inclusions in PC12 cells

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinsons disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O‐tBu]‐A‐leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose‐dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT‐cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein‐1, the rat homologue of human α‐synuclein. However the total level of synuclein‐1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.

Proteasome inhibition and Parkinson's disease modeling.

Impaired proteasome function is a potential mechanism for dopaminergic neuron degeneration. To model this molecular defect, we administered systemically the reversible lipophilic proteasome inhibitor, carbobenzoxy‐L‐isoleucyl‐γ‐t‐butyl‐L‐glutamyl‐L‐alanyl‐L‐leucinal (PSI), to rodents. In contrast to a previous report, this approach failed to cause any detectable behavioral or neuropathological abnormality in either rats or mice. Although theoretically appealing, this specific model of Parkinsons disease appears to exhibit poor reproducibility. Ann Neurol 2006;60:260–264

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.

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.

Neurobiology of α-synuclein

Abstractα-Synuclein is an abundant neuronal protein that has been linked both to normal synaptic function and to neurodegeneration. Most significantly, mutations in the gene encoding for α-synuclein are responsible for Parkinson’s disease (PD) in rare familial cases, and the aggregated protein is a major component of Lewy bodies found in sporadic PD. Here we review recent data regarding the structure, the regulation at the transcriptional and posttranslational level, and the physiologic and aberrant functions of α-synuclein. We focus in particular on the fibrilization potential of α-synuclein and on its link with defects in protein degradation.

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 7 days of nerve growth factor treatment. Synuclein‐1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1–2 days 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 cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c 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.

Lack of p53 delays apoptosis, but increases ubiquitinated inclusions, in proteasomal inhibitor-treated cultured cortical neurons

Proteasomal dysfunction may contribute to neurodegenerative diseases; however, its effects on primary neurons are largely unknown. We have previously reported that pharmacological proteasomal inhibition leads to apoptosis and cytoplasmic ubiquitinated inclusions in primary rat cortical neurons. In cell lines the transcription factor p53 is regulated by the proteasome and in some cases it mediates death following proteasomal inhibition. It is unclear, however, if this is the case in primary neurons. Here we show in proteasome inhibitor-treated cortical neurons an early increase of p53 levels, accompanied by nuclear translocation. At later time points p53 is found sequestered within ubiquitinated inclusions. Compared to controls, p53-deficient mouse neurons show delayed apoptosis, but increased numbers of inclusions, likely secondary to enhanced survival. We conclude that p53 plays a role in cortical neuron apoptosis induced by proteasomal inhibition and, despite the fact that it localizes to inclusions, it is not necessary for their formation.