␣-Synuclein (␣-syn), a protein implicated in Parkinson's disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how ␣-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous ␣-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant ␣-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 ␣-synoverexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) ␣-syn, as well as chromaffin cells from control and ␣-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 ␣-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 ␣-syn-overexpressing cells. The ␣-synoverexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that ␣-syn overexpression inhibits a vesicle "priming" step, after secretory vesicle trafficking to "docking" sites but before calcium-dependent vesicle membrane fusion.

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 Parkinson's 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 a-synucleinpositive 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.

Critical illness affects body composition profoundly, especially body cell mass (BCM). BCM loss reflects lean tissue wasting and could be a nutritional marker in critically ill patients. However, BCM assessment with usual isotopic or tracer methods is impractical in intensive care units (ICUs). We aimed to modelize the BCM of critically ill patients using variables available at bedside. Fat-free mass (FFM), bone mineral (Mo), and extracellular water (ECW) of 49 critically ill patients were measured prospectively by dual-energy X-ray absorptiometry and multifrequency bioimpedance. BCM was estimated according to the four-compartment cellular level: BCM ϭ FFM Ϫ (ECW/0.98) Ϫ (0.73 ϫ Mo). Variables that might influence the BCM were assessed, and multivariable analysis using fractional polynomials was conducted to determine the relations between BCM and these data. Bootstrap resampling was then used to estimate the most stable model predicting BCM. BCM was 22.7 Ϯ 5.4 kg. The most frequent model included height (cm), leg circumference (cm), weight shift (⌬) between ICU admission and body composition assessment (kg), and trunk length (cm) as a linear function: BCM (kg) ϭ 0.266 ϫ height ϩ 0.287 ϫ leg circumference ϩ 0.305 ϫ ⌬weight Ϫ 0.406 ϫ trunk length Ϫ 13.52. The fraction of variance explained by this model (adjusted r 2 ) was 46%. Including bioelectrical impedance analysis variables in the model did not improve BCM prediction. In summary, our results suggest that BCM can be estimated at bedside, with an error lower than Ϯ20% in 90% subjects, on the basis of static (height, trunk length), less stable (leg circumference), and dynamic biometric variables (⌬weight) for critically ill patients. critical illness; body composition; bioelectrical impedance; dualenergy X-ray absorptiometry OTHER THAN THE BASIC TWO-COMPARTMENT [fat and fat-free mass (FFM)] model, the composition of the human body can be described by multicompartment models from the atomic to the functional level (11). Body cell mass (BCM) is the metabolically active compartment of FFM that reflects the body's cellular components involved in oxygen consumption, carbon dioxide production, and resting metabolism (11,19,20). Thus, physiological modeling of body composition at the cellular level can be separated into different compartments associated with functions: energy store in fat mass (FM), energy expenditure and metabolism by BCM, exchanges in extracellular water (ECW), and support by bone mineral (Mo).In healthy adults, homeostasis maintains a stable body composition. Conversely, body composition is profoundly modified in acutely or chronically ill patients (28). BCM is altered by changes of nutritional status and the catabolic effects of disease (11,39,47). In this context, BCM could be a marker of malnutrition or prognosis of chronically or critically ill patients (14, 37). BCM is associated with resting energy expenditure (REE), and it has been shown that assessment of energy balance may have particular significance for the nutritional management of the...

a-Synuclein is a neuronal protein that is implicated in the control of synaptic vesicle function and in Parkinson's disease (PD). Consequently, alterations of a-synuclein levels may play a role in neurotransmission and in PD pathogenesis. However, the factors that regulate a-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 a-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 a-synuclein protein levels with maturation in culture. Application of basic Fibroblast growth factor (bFGF) promoted a-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. a-Synuclein levels were not altered by glial-derived neurotrophic factor (GDNF), or by apoptotic stimuli. We conclude that bFGF promotes a-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.

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