Live Cell Fluorescence Imaging Shows Neurotransmitter Activation Promotes Aggregation of the Intracellular Domain of Amyloid Precursor Protein.

Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCß signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (N&B) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCß1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCß reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.

Activation of Gαq sequesters specific transcripts into Ago2 particles.

The Gαq/phospholipase Cß1 (PLCß1) signaling system mediates calcium responses from hormones and neurotransmitters. While PLCß1 functions on the plasma membrane, there is an atypical cytosolic population that binds Argonaute 2 (Ago2) and other proteins associated with stress granules preventing their aggregation. Activation of Gαq relocalizes cytosolic PLCß1 to the membrane, releasing bound proteins, promoting the formation of stress granules. Here, we have characterized Ago2 stress granules associated with Gαq activation in differentiated PC12 cells, which have a robust Gαq/PLCß1 signaling system. Characterization of Ago2-associated stress granules shows shifts in protein composition when cells are stimulated with a Gαq agonist, or subjected to heat shock or osmotic stress, consistent with the idea that different stresses result in unique stress granules. Purified Ago2 stress granules from control cells do not contain RNA, while those from heat shock contain many different mRNAs and miRs. Surprisingly, Ago2 particles from cells where Gαq was stimulated show only two transcripts, chromogranin B, which is involved in secretory function, and ATP synthase 5f1b, which is required for ATP synthesis. RT-PCR, western blotting and other studies support the idea that Gαq-activation protects these transcripts. Taken together, these studies show a novel pathway where Gαq/PLCß regulates the translation of specific proteins.

Stimulation of phospholipase Cß1 by Gαq promotes the assembly of stress granule proteins.

During adverse conditions, mammalian cells suppress protein production by sequestering the translational machinery in membrane-less organelles known as stress granules. Here, we found that activation of the G protein subunit Gαq promoted the formation of particles that contained stress granule proteins through a mechanism linked to a cytosolic fraction of phospholipase Cß1 (PLCß1). In experiments with PC12 and A10 cells, we showed that under basal conditions, cytosolic PLCß1 bound to stress granule­associated proteins, including PABPC1, eIF5A, and Ago2. Knockdown of cytosolic PLCß1 with siRNA or promoting its relocalization to the plasma membrane by activating Gαq resulted in the formation of particles containing these stress granule­associated proteins. Our studies showed that the composition of these particles resembled those formed under osmotic stress and were distinct from those formed in response to other types of stress. Our results fit a simple thermodynamic model in which cytosolic PLCß1 solubilizes stress granule proteins such that its movement to activated Gαq releases these proteins to enable the formation of stress granules. Together, our data suggest a link between Gαq-coupled signals and protein translation through stress granule formation.

Stimulation of the Gαq/phospholipase Cß1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cß1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCß1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCß1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK-N-SH cells to an undifferentiated state. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down-regulated. This return to a stem-like state is accompanied by shifts in multiple miR populations. Surprisingly, de-differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real-time hydrolysis of a fluorescent-tagged miR in cells where PLCß1 or C3PO were down-regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCß1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cß/G protein pathway.

Some proteins can serve multiple functions depending on different cellular conditions. An example of a bifunctional protein is inositide-specific mammalian phospholipase Cß (PLCß). PLCß is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium. Recently, alternate cellular function(s) of PLCß have become uncovered. However, the conditions that allow these different functions to be operative are unclear. Like many mammalian proteins, PLCß has a conserved catalytic core along with several regulatory domains. These domains modulate the intensity and duration of calcium signals in response to external sensory information, and allow this enzyme to inhibit protein translation in a noncatalytic manner. In this review, we first describe PLCß's cellular functions and regulation of the switching between these functions, and then discuss the thermodynamic considerations that offer insight into how cells manage multiple and competitive associations allowing them to rapidly shift between functional states.

Dopaminergic regulation of orexin neurons.

Orexin/hypocretin neurons in the lateral hypothalamus and adjacent perifornical area (LH/PFA) innervate midbrain dopamine (DA) neurons that project to corticolimbic sites and subserve psychostimulant-induced locomotor activity. However, it is not known whether dopamine neurons in turn regulate the activity of orexin cells. We examined the ability of dopamine agonists to activate orexin neurons in the rat, as reflected by induction of Fos. The mixed dopamine agonist apomorphine increased Fos expression in orexin cells, with a greater effect on orexin neurons located medial to the fornix. Both the selective D1-like agonist, A-77636, and the D2-like agonist, quinpirole, also induced Fos in orexin cells, suggesting that stimulation of either receptor subtype is sufficient to activate orexin neurons. Consistent with this finding, combined SCH 23390 (D1 antagonist)-haloperidol (D2 antagonist) pretreatment blocked apomorphine-induced activation of medial as well as lateral orexin neurons; in contrast, pretreatment with either the D1-like or D2-like antagonists alone did not attenuate apomorphine-induced activation of medial orexin cells. In situ hybridization histochemistry revealed that LH/PFA cells rarely express mRNAs encoding dopamine receptors, suggesting that orexin cells are transsynaptically activated by apomorphine. We therefore lesioned the nucleus accumbens, a site known to regulate orexin cells, but this treatment did not alter apomorphine-elicited activation of medial or lateral orexin neurons. Interestingly, apomorphine failed to activate orexin cells in isoflurane-anaesthetized animals. These data suggest that apomorphine-induced arousal but not accumbens-mediated hyperactivity is required for dopamine to transsynaptically activate orexin neurons.