Axl receptor tyrosine kinase is a regulator of apolipoprotein E.

Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.

Co-expression of ß Subunits with the Voltage-Gated Sodium Channel NaV1.7: the Importance of Subunit Association and Phosphorylation and Their Effects on Channel Pharmacology and Biophysics.

The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary ß2 and ß3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either ß2 or ß3 subunits in HEK-293 cells. The ß2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the ß2 subunits did not change the biophysical properties of the channel. In contrast, ß3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated ß3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the ß3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the ß3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of ß subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with ß2. The ß3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.

Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway.

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

A stop codon mutation in SCN9A causes lack of pain sensation.

The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.