Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] plays a fundamental role in clathrin-mediated endocytosis. However, precisely how PI(4,5)P2 metabolism is spatially and temporally regulated during membrane internalization and the functional consequences of endocytosis-coupled PI(4,5)P2 dephosphorylation remain to be explored. Using cell-free assays with liposomes of varying diameters, we show that the major synaptic phosphoinositide phosphatase, synaptojanin 1 (Synj1), acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P2 from curved membranes as opposed to relatively flat ones. Moreover, in vivo recruitment of Synj1's inositol 5-phosphatase domain to endophilin-induced membrane tubules results in fragmentation and condensation of these structures largely in a dynamin-dependent fashion. Our study raises the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P2 elimination during membrane internalization and suggests that the PI(4,5)P2-to-PI4P conversion achieved by Synj1 at sites of high curvature may cooperate with dynamin to achieve membrane fission.

Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate.

Ubiquitously expressed Mg(2+)-inhibitory cation (MIC) channels are permeable to Ca2+ and Mg2+ and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP2) recovered MIC current from cholesterol effect. As PIP2 is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP2. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP2-sensitive K+ channels: human Ether-a-go-go related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP2 in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP2 via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced gamma-(32)P incorporation into PIP2 by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP2. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP2.

Synaptojanin 1-linked phosphoinositide dyshomeostasis and cognitive deficits in mouse models of Down's syndrome.

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal PtdIns(4,5)P(2) balance is required for proper neurotransmission, the phosphoinositide phosphatase synaptojanin 1 is a key regulator of its metabolism. The underlying gene, SYNJ1, maps to human chromosome 21 and is thus a candidate for involvement in Down's syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that PtdIns(4,5)P(2) metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring Synj1 to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing Synj1 from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that PtdIns(4,5)P(2) dyshomeostasis caused by gene dosage imbalance for Synj1 may contribute to brain dysfunction and cognitive disabilities in DS.

Oligomeric amyloid-beta peptide disrupts phosphatidylinositol-4,5-bisphosphate metabolism.

Synaptic dysfunction caused by oligomeric assemblies of amyloid-beta peptide (Abeta) has been linked to cognitive deficits in Alzheimer's disease. Here we found that incubation of primary cortical neurons with oligomeric Abeta decreases the level of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), a phospholipid that regulates key aspects of neuronal function. The destabilizing effect of Abeta on PtdIns(4,5)P2 metabolism was Ca2+-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)P2 phosphatase in the brain and at the synapses. We also found that the inhibitory effect of Abeta on hippocampal long-term potentiation was strongly suppressed in slices from Synj1+/- mice, suggesting that Abeta-induced synaptic dysfunction can be ameliorated by treatments that maintain the normal PtdIns(4,5)P2 balance in the brain.

A MicroRNA feedback circuit in midbrain dopamine neurons.

MicroRNAs (miRNAs) are evolutionarily conserved, 18- to 25-nucleotide, non-protein coding transcripts that posttranscriptionally regulate gene expression during development. miRNAs also occur in postmitotic cells, such as neurons in the mammalian central nervous system, but their function is less well characterized. We investigated the role of miRNAs in mammalian midbrain dopaminergic neurons (DNs). We identified a miRNA, miR-133b, that is specifically expressed in midbrain DNs and is deficient in midbrain tissue from patients with Parkinson's disease. miR-133b regulates the maturation and function of midbrain DNs within a negative feedback circuit that includes the paired-like homeodomain transcription factor Pitx3. We propose a role for this feedback circuit in the fine-tuning of dopaminergic behaviors such as locomotion.

Presenilin mutations linked to familial Alzheimer's disease cause an imbalance in phosphatidylinositol 4,5-bisphosphate metabolism.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important cellular effector whose functions include the regulation of ion channels and membrane trafficking. Aberrant PIP2 metabolism has also been implicated in a variety of human disease states, e.g., cancer and diabetes. Here we report that familial Alzheimer's disease (FAD)-associated presenilin mutations cause an imbalance in PIP2 metabolism. We find that the transient receptor potential melastatin 7 (TRPM7)-associated Mg2+ -inhibited cation (MIC) channel underlies ion channel dysfunction in presenilin FAD mutant cells, and the observed channel deficits are restored by the addition of PIP2, a known regulator of the MIC/TRPM7 channel. Lipid analyses show that PIP2 turnover is selectively affected in FAD mutant presenilin cells. We also find that modulation of cellular PIP2 closely correlates with 42-residue amyloid beta-peptide (Abeta42) levels. Our data suggest that PIP2 imbalance may contribute to Alzheimer's disease pathogenesis by affecting multiple cellular pathways, such as the generation of toxic Abeta42 as well as the activity of the MIC/TRPM7 channel, which has been linked to other neurodegenerative conditions. Thus, our study suggests that brain-specific modulation of PIP2 may offer a therapeutic approach in Alzheimer's disease.