Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD). ApoE4 has sex-dependent effects, whereby the risk of developing AD is higher in apoE4-expressing females than males. However, the mechanism underlying the sex difference, in relation to apoE4, is unknown. Previous findings indicate that apoE4 causes age-dependent impairments of hilar GABAergic interneurons in female mice, leading to learning and memory deficits. Here, we investigate whether the detrimental effects of apoE4 on hilar GABAergic interneurons are sex-dependent using apoE knock-in (KI) mice across different ages. We found that in female apoE-KI mice, there was an age-dependent depletion of hilar GABAergic interneurons, whereby GAD67- or somatostatin-positive–but not NPY- or parvalbumin-positive–interneuron loss was exacerbated by apoE4. Loss of these neuronal populations was correlated with the severity of spatial learning deficits at 16 months of age in female apoE4-KI mice; however, this effect was not observed in female apoE3-KI mice. In contrast, we found an increase in the numbers of hilar GABAergic interneurons with advancing age in male apoE-KI mice, regardless of apoE genotype. Moreover, male apoE-KI mice showed a consistent ratio of hilar inhibitory GABAergic interneurons to excitatory mossy cells approximating 1.5 that is independent of apoE genotype and age, whereas female apoE-KI mice exhibited an age-dependent decrease in this ratio, which was exacerbated by apoE4. Interestingly, there are no apoE genotype effects on GABAergic interneurons in the CA1 and CA3 subregions of the hippocampus as well as the entorhinal and auditory cortexes. These findings suggest that the sex-dependent effects of apoE4 on developing AD is in part attributable to inherent sex-based differences in the numbers of hilar GABAergic interneurons, which is further modulated by apoE genotype.

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The ability to distinguish between similar experiences is a critical feature of episodic memory and is primarily regulated by the dentate gyrus (DG) region of the hippocampus. However, the molecular mechanisms underlying such pattern separation tasks are poorly understood. We report a novel role for the small GTPase ADP ribosylation factor 4 (Arf4) in controlling pattern separation by regulating dendritic spine development. Arf4+/− mice at 4–5 months of age display severe impairments in a pattern separation task, as well as significant dendritic spine loss and smaller miniature excitatory post-synaptic currents (mEPSCs) in granule cells of the DG. Arf4 knockdown also decreases spine density in primary neurons, whereas Arf4 overexpression promotes spine development. A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls. Arf4′s effects on spine development are regulated by ASAP1, a GTPase-activating protein that modulates Arf4 GTPase activity. ASAP1 overexpression decreases spine density, and this effect is partially rescued by concomitant overexpression of wildtype Arf4 or Arf4-Q71L. In addition, Arf4 overexpression rescues spine loss in primary neurons from an Alzheimer's disease-related apolipoprotein (apo) E4 mouse model. Our findings suggest that Arf4 is a critical modulator of DG-mediated pattern separation by regulating dendritic spine development.

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