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.

Decades of research have correlated increased levels of amyloid β-peptide (Aβ) with neuropathological progression in Alzheimer’s disease (AD) patients and transgenic models. Aβ precipitates synaptic and neuronal anomaliesby perturbing intracellular signaling which, in turn, may underlie cognitive impairment. Aβ also alters lipid metabolism, notably causinga deficiency of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phospholipid that regulates critical neuronal functions. Haploinsufficiency of the gene encoding synaptojanin 1 (Synj1), a major PI(4,5)P2 phosphatase in the brain, provided protection against PI(4,5)P2 breakdown and electrophysiological deficits attributable to Aβ. Based on these data, we tested whether reduction of Synj1 could rescue cognitive deficits and Aβ-induced morphological alterations of synapses. We found that hemizygous deletion of Synj1 in the context of a mouse model expressing the Swedish mutant of amyloid precursor protein rescues deficits in learning and memory without affecting amyloid load. Synj1 heterozygosity also rescued PI(4,5)P2 deficiency in a synaptosome-enriched fraction from the brain of Tg2576 mice. Genetic disruption of Synj1 attenuated Aβ oligomer-induced changes in dendritic spines of cultured hippocampal neurons, sparing maturespine classes, which corroborates the protective role for Synj1 reduction against Aβ insult at the synapse. These results indicate that Synj1 reduction ameliorates AD-associated behavioral and synaptic deficits, providing evidence that Synj1 and, more generally, phosphoinositide metabolism, may be promising therapeutic targets. Our work expands on recent studies identifying lipid metabolism and lipid modifying enzymes as targets of AD-associated synaptic and behavioral impairment.

SUMMARY Little is known about the cellular events promoting metastasis. We show that knockout of phospholipase D2 (PLD2), which generates the signaling lipid phosphatidic acid (PA), inhibits lung metastases in the mammary tumor virus (MMTV)-Neu transgenic mouse breast cancer model. PLD2 promotes local invasion through the regulation of the plasma membrane targeting of MT1-MMP and its associated invadopodia. A liposome pulldown screen identifies KIF5B, the heavy chain of the motor protein kinesin-1, as a new PA-binding protein. In vitro assays reveal that PA specifically and directly binds to the C-terminus of KIF5B. The binding between PLD2-generated PA and KIF5B is required for the vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia, and invasion, in cancer cells. Taken together, these results identify a role of PLD2-generated PA in the regulation of kinesin-1 motor functions and breast cancer metastasis, and suggest PLD2 as a potential therapeutic target for metastatic breast cancer.

Alzheimer’s disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aβ. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

Apolipoprotein E ε4 (APOE4) is the primary genetic risk factor for the late-onset form of Alzheimer’s disease (AD). Although the reason for this association is not completely understood, researchers have uncovered numerous effects of APOE4 expression on AD-relevant brain processes, including amyloid beta (Aβ) accumulation, lipid metabolism, endosomal-lysosomal trafficking, and bioenergetics. In this study, we aimed to determine the effect of APOE4 allelic dosage on regional brain lipid composition in aged mice, as well as in cultured neurons. We performed a targeted lipidomic analysis on an AD-vulnerable brain region (entorhinal cortex; EC) and an AD-resistant brain region (primary visual cortex; PVC) from 14–15 month-old APOE3/3, APOE3/4, and APOE4/4 targeted replacement mice, as well as on neurons cultured with conditioned media from APOE3/3 or APOE4/4 astrocytes. Our results reveal that the EC possesses increased susceptibility to APOE4-associated lipid alterations compared to the PVC. In the EC, APOE4 expression showed a dominant effect in decreasing diacylglycerol (DAG) levels, and a semi-dominant, additive effect in the upregulation of multiple ceramide, glycosylated sphingolipid, and bis(monoacylglycerol)phosphate (BMP) species, lipids known to accumulate as a result of endosomal-lysosomal dysfunction. Neurons treated with conditioned media from APOE4/4 vs. APOE3/3 astrocytes showed similar alterations of DAG and BMP species to those observed in the mouse EC. Our results suggest that APOE4 expression differentially modulates regional neuronal lipid signatures, which may underlie the increased susceptibility of EC-localized neurons to AD pathology.

SUMMARY Given the complex nature of Alzheimer’s disease (AD), a cell-based model that recapitulates physiological properties of the target neuronal population carries significant value in discovering improved drug candidates and chemical probes for uncovering disease mechanisms. We established phenotypic neuronal assays for biogenesis and synaptic action of amyloid β-peptide (Aβ) based on embryonic stem (ES) cell-derived neurons (ESNs). ESNs enriched with pyramidal neurons were robust, scalable and amenable to a small molecule screening assay, overcoming apparent limitations of neuronal models derived from human pluripotent cells. Small molecule screening of clinical compounds identified four compounds capable of reducing Aβ levels in ESNs derived from the Tg2576 mouse model of AD. Our approach is therefore highly suitable for phenotypic screening in AD drug discovery and has the potential to identify therapeutic candidates with improved efficacy and safety potential.

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Elevation of amyloid β-peptide (Aβ) is critically associated with Alzheimer’s disease (AD) pathogenesis. Aβ-induced synaptic abnormalities, including altered receptor trafficking and synapse loss, have been linked to cognitive deficits in AD. Recent work implicates a lipid critical for neuronal function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], in Aβ-induced synaptic and behavioral impairments. Synaptojanin 1 (Synj1), a lipid phosphatase mediating the breakdown of PI(4,5)P2, has been shown to play a role in synaptic vesicle recycling and receptor trafficking in neurons. Heterozygous deletion of Synj1 protected neurons from Aβ-induced synaptic loss and restored learning and memory in a mouse model of AD. Thus, inhibition of Synj1 may ameliorate Aβ-associated impairments, suggesting Synj1 as a potential therapeutic target. To this end, we developed a screening assay for Synj1 based on detection of inorganic phosphate liberation from a water-soluble, short chain PI(4,5)P2. The assay displayed saturable kinetics and detected Synj1’s substrate preference for PI(4,5)P2 over PI(3,4,5)P3. The assay will enable identification of novel Synj1 inhibitors which have potential utility as chemical probes to dissect the cellular role of Synj1 as well as potential to prevent or reverse AD-associated synaptic abnormalities.