alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.

MicroRNAs (miRNAs) are evolutionarily conserved, 18-25 nucleotide non-protein coding transcripts that play an important function in post-transcriptional regulation of gene expression during development (1-4). However, the significance of miRNAs in postmitotic cells, such as neurons in the mammalian CNS, is less well characterized. Here we investigate the role of miRNAs in the terminal differentiation, function, and survival of mammalian midbrain dopaminergic neurons (DNs). We identify a miRNA, miR-133b, that is specifically expressed in midbrain DNs and deficient in Parkinson's disease midbrain tissue that has lost midbrain DNs. MiR-133b regulates the maturation and function of midbrain DNs within a negative feedback circuit that includes the paired-like homeodomain transcription factor Pitx3. KeywordsmiRNA; miR-133b; midbrain dopamine neurons; Parkinson's disease; Dicer miRNAs are derived from long primary transcripts through sequential processing by the Drosha ribonuclease (5) and the Dicer enzyme (1,6). In the context of an RNA-induced silencing complex (RISC), miRNAs guide the cleavage of target mRNAs and/or inhibit their translation (2). miRNAs were first characterized in invertebrates, where they function to regulate developmental cell fate decisions in the nervous system (7,8) and elsewhere (9).Midbrain dopamine neurons (DNs) play a central role in complex behaviors such as reward and addiction, and these cells are lost in Parkinson's disease. Furthermore, a number of transcription factors have been identified that regulate midbrain DN development, function, and survival (16). However, the role of post-transcriptional mechanisms in these processes is uncharacterized. We sought to establish a role for miRNAs in mammalian dopamine neuron differentiation, function, and survival. To facilitate a kinetic analysis, we first used an in vitro model system: the differentiation of murine ES cells into DNs (17,18). An ES cell line was obtained that expresses Dicer enzyme containing LoxP recombinase sites that flank both chromosomal copies of the Dicer gene (floxed Dicer alleles)(19). Introduction of Cre recombinase into these cells by lentiviral transduction leads to the deletion of Dicer in nearly 100% of cells (Supplementary Figure 1A). Floxed Dicer ES cultures were differentiated to a midbrain DN phenotype using the embryoid body protocol (EB; Supplementary Figure 1B) (18). Briefly, cells were initially grown in non-adherent conditions in the context of defined media, including growth factors, to generate neuronal precursors (stage 2); subsequently, neuronal precursors were expanded in the presence of basic fibroblast growth factor (bFGF; stages 3 and 4); and finally, the bFGF was withdrawn to obtain mature DNs (stage 5), which constitute 10-25% of the cells in these cultures (18). Cre-mediated deletion of the floxed Dicer alleles at stage 4, when postmitotic dopamine neurons first arise, led to a nearly complete loss of dopamine neuron accumulation at stage 5, as quantified by the expression of markers i...

Mutations in LRRK2 underlie an autosomal-dominant, inherited form of Parkinson's disease (PD) that mimics the clinical features of the common "sporadic" form of PD. The LRRK2 protein includes putative GTPase, protein kinase, WD40 repeat, and leucine-rich repeat (LRR) domains of unknown function. Here we show that PD-associated LRRK2 mutations display disinhibited kinase activity and induce a progressive reduction in neurite length and branching both in primary neuronal cultures and in the intact rodent CNS. In contrast, LRRK2 deficiency leads to increased neurite length and branching. Neurons that express PD-associated LRRK2 mutations additionally harbor prominent phospho-tau-positive inclusions with lysosomal characteristics and ultimately undergo apoptosis.

SUMMARY Recent genome-wide association studies have linked common variants in the human genome to Parkinson’s disease (PD) risk. Here we show that the consequences of variants at 2 such loci, PARK16 and LRRK2, are highly interrelated, both in terms of their broad impacts on human brain transcriptomes of unaffected carriers, and in terms of their associations with PD risk. Deficiency of the PARK16 locus gene RAB7L1 in primary rodent neurons, or of a RAB7L1 orthologue in Drosophila dopamine neurons, recapitulated degeneration observed with expression of a familial PD mutant form of LRRK2, whereas RAB7L1 overexpression rescued the LRRK2 mutant phenotypes. PD-associated defects in RAB7L1 or LRRK2 led to endolysosomal and Golgi apparatus sorting defects and deficiency of the VPS35 component of the retromer complex. Expression of wild-type VPS35, but not a familial PD-associated mutant form, rescued these defects. Taken together, these studies implicate retromer and lysosomal pathway alterations in PD risk.