The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

␣-Synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, ␣-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, ␣-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of ␣-synuclein with cell membranes, we find that ␣-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of ␣-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of ␣-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute ␣-synuclein away from synapses, indicating an important role for raft association in the normal function of ␣-synuclein and its role in the pathogenesis of PD.

␣-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.

β-Amyloid (Aβ) peptides are thought to be critically involved in the etiology of Alzheimer's disease (AD). The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aβ, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aβ40, Aβ42, and sAPPβ (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aβ40, Aβ42, and sAPPβ in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aβ pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

This study was undertaken to determine the prevalence and correlates of cognitive impairment (CI) and neuropsychiatric symptoms (NPS) in early, untreated patients with Parkinson’s disease (PD). Background Both CI and NPS are common in PD and impact disease course and quality of life. However, limited knowledge is available about cognitive abilities and NPS. Methods Parkinson’s Progression Markers Initiative (PPMI) is a multi-site study of early, untreated PD patients and healthy controls (HCs), the latter with normal cognition. At baseline, participants were assessed with a neuropsychological battery and for symptoms of depression, anxiety, impulse control disorders (ICDs), psychosis, and apathy. Results Baseline data of 423 PD patients and 196 HCs yielded no between-group differences in demographic characteristics. Twenty-two percent of PD patients met the PD-recommended screening cutoff for CI on the Montral Cognitive Assessment (MoCA), but only 9% met detailed neuropsychological testing criteria for mild cognitive impairment (MCI)-level impairment. The PD patients were more depressed than HCs (P < 0.001), with twice as many (14% vs. 7%) meeting criteria for clinically significant depressive symptoms. The PD patients also experienced more anxiety (P < 0.001) and apathy (P < 0.001) than HCs. Psychosis was uncommon in PD (3%), and no between-group difference was seen in ICD symptoms (P = 0.51). Conclusions Approximately 10% of PD patients in the early, untreated disease state met traditional criteria of CI, which is a lower frequency compared with previous studies. Multiple dopaminergic-dependent NPS are also more common in these patients compared with the general population, but others associated with dopamine replacement therapy are not or are rare. Future analyses of this cohort will examine biological predictors and the course of CI and NPS.

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Dysregulation of dopamine homeostasis and elevation of the cytosolic level of the transmitter have been suggested to underlie the vulnerability of catecholaminergic neurons in Parkinson's disease. Because several known mutations in ␣-synuclein or overexpression of the wild-type (WT) protein causes familial forms of Parkinson's disease, we investigated possible links between ␣-synuclein pathogenesis and dopamine homeostasis. Chromaffin cells isolated from transgenic mice that overexpress A30P ␣-synuclein displayed significantly increased cytosolic catecholamine levels as measured by intracellular patch electrochemistry, whereas cells overexpressing the WT protein and those from knock-out animals were not different from controls. Likewise, catechol concentrations were higher in L-DOPAtreated PC12 cells overexpressing A30P or A53T compared with those expressing WT ␣-synuclein, although the ability of cells to maintain a low cytosolic dopamine level after L-DOPA challenge was markedly inhibited by either protein. We also found that incubation with low-micromolar concentrations of WT, A30P, or A53T ␣-synuclein inhibited ATP-dependent maintenance of pH gradients in isolated chromaffin vesicles and that the WT protein was significantly less potent in inducing the proton leakage. In summary, we demonstrate that overexpression of different types of ␣-synuclein disrupts vesicular pH and leads to a marked increase in the levels of cytosolic catechol species, an effect that may in turn trigger cellular oxyradical damage. Although multiple molecular mechanisms may be responsible for the perturbation of cytosolic catecholamine homeostasis, this study provides critical evidence about how ␣-synuclein might exert its cytotoxicity and selectively damage catecholaminergic cells.

Mutations in leucine-rich repeat kinase 2 ( LRRK2 ) are the most common genetic risk factors for Parkinson’s disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.