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...

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) has an important function in cell regulation both as a precursor of second messenger molecules and by means of its direct interactions with cytosolic and membrane proteins. Biochemical studies have suggested a role for PtdIns(4,5)P2 in clathrin coat dynamics, and defects in its dephosphorylation at the synapse produce an accumulation of coated endocytic intermediates. However, the involvement of PtdIns(4,5)P2 in synaptic vesicle exocytosis remains unclear. Here, we show that decreased levels of PtdIns(4,5)P2 in the brain and an impairment of its depolarization-dependent synthesis in nerve terminals lead to early postnatal lethality and synaptic defects in mice. These include decreased frequency of miniature currents, enhanced synaptic depression, a smaller readily releasable pool of vesicles, delayed endocytosis and slower recycling kinetics. Our results demonstrate a critical role for PtdIns(4,5)P2 synthesis in the regulation of multiple steps of the synaptic vesicle cycle.

Synaptic dysfunction caused by oligomeric assemblies of amyloid-β peptide (Aβ) has been linked to cognitive deficits in Alzheimer's disease. Here we found that incubation of primary cortical neurons with oligomeric Aβ decreases the level of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ), a phospholipid that regulates key aspects of neuronal function. The destabilizing effect of Aβ on PtdIns (4,5)P 2 metabolism was Ca 2+ -dependent and was not observed in neurons that were derived from mice that are haploinsufficient for Synj1. This gene encodes synaptojanin 1, the main PtdIns(4,5) P 2 phosphatase in the brain and at the synapses. We also found that the inhibitory effect of Aβ on hippocampal long-term potentiation was strongly suppressed in slices from Synj1 +/− mice, suggesting that Aβ-induced synaptic dysfunction can be ameliorated by treatments that maintain the normal PtdIns(4,5)P 2 balance in the brain.