Regulation of AMPA receptor trafficking by secreted protein factors.

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain. Regulation of AMPAR levels at synapses controls synaptic strength and underlies information storage and processing. Many proteins interact with the intracellular domain of AMPARs to regulate their trafficking and synaptic clustering. However, a growing number of extracellular factors important for glutamatergic synapse development, maturation and function have emerged that can also regulate synaptic AMPAR levels. This mini-review highlights extracellular protein factors that regulate AMPAR trafficking to control synapse development and plasticity. Some of these factors regulate AMPAR clustering and mobility by interacting with the extracellular N-terminal domain of AMPARs whereas others regulate AMPAR trafficking indirectly via their respective signaling receptors. While several of these factors are secreted from neurons, others are released from non-neuronal cells such as glia and muscle. Although it is apparent that secreted factors can act locally on neurons near their sites of release to coordinate individual synapses, it is less clear if they can diffuse over longer ranges to coordinate related synapses within a circuit or region of the brain. Given that there are hundreds of factors that can be secreted from neuronal and non-neuronal cells, it will not be surprising if more extracellular factors that modulate AMPARs and glutamatergic synapses are discovered. Many open questions remain including where and when the factors are expressed, what regulates their secretion from different cell types, what controls their diffusion, stability, and range of action, and how their cognate receptors influence intracellular signaling to control AMPAR trafficking.

Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.

Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment.

Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-ß-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.

2-Hydroxypropyl-ß-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking.

Cholesterol is an essential structural component of cellular membranes and precursor molecule for oxysterol, bile acid, and hormone synthesis. The study of intracellular cholesterol trafficking pathways has been limited in part due to a lack of suitable cholesterol analogues. Herein, we developed three novel diazirine alkyne cholesterol probes: LKM38, KK174, and KK175. We evaluated these probes as well as a previously described diazirine alkyne cholesterol analogue, trans-sterol, for their fidelity as cholesterol mimics and for study of cholesterol trafficking. LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.

Prolonged survival after intraperitoneal interleukin-2 immunotherapy for recurrent ovarian cancer.

•A case report of a 14 year remission of recurrent ovarian cancer with intraperitoneal aldesleukin (IL-2) is presented.•Intraperitoneal IL-2 was given with little toxicity.•Immunotherapy may have the potential for durable remissions in ovarian cancer.