Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA.
The cnm gene coding for the glycosylated collagen- and laminin-binding surface adhesin Cnm is found in the genome of approximately 20% of Streptococcus mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans such as P1 and WapA have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the β-sheet rich N-terminal collagen-binding domain (CBD) of Cnm has propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids either in its native glycosylated or recombinant non-glycosylated forms and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro , ex vivo and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, amyloid formation by Cnm might act as a negative regulatory mechanism to modulate collagen-binding activity within S. mutans biofilms and warrants further investigation. IMPORTANCE Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor S. mutans . Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body including collagen-rich surfaces such as dentin and heart valves. Here, we suggest that Cnm function might be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm could self-assemble into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light into the role of functional amyloids in S. mutans pathobiology and ecology.
Several studies examining vesicle fusion have been reported in last decades and have established a number of factors favoring the process of vesicle fusion. To determine whether variations to the physicochemical properties of the membrane affect the process of vesicle fusion, we worked with binary and ternary mixtures of large unilamellar vesicles (LUVs). The selected binary models were dioleoyl phosphocholine-cholesterol (DOPC-chol) and disteraroyl phosphocholine-cholesterol (DSPC-chol), and the tertiary mixtures were phosphatidylcholine-phsophatidylethanolamine-cholesterol (PC-PE-Chol); phosphatidylcholine-sphingomyelincholesterol (PC-SM-Chol); and phosphatidylcholine-phosphatidylserine-cholesterol (PC-PS-Chol). For all these models, the effect of cholesterol content on the lamella physicochemical properties was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy, generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan), and DPH fluorescence lifetime. To determine whether fusion of these vesicles varied according to lipid composition, the % mixing content and the % leakage were determined. Examining membrane incorporation using fluorescence steady-state and time-resolved probe assays in the models indicated that cholesterol content affected packing order and lamellar hydration. In most of the models, nonmonotonic variations were observed for these parameters, and these variations could be interpreted as increases in the proportion of ordered microdomains. When the proportion of these domains is higher, the packing order increases, and the lamellar water decrease. Similarly, the % mixing, which was assessed as a fusion parameter, also exhibited nonmonotonic behavior, indicating that the fusion process is enhanced at these concentrations of cholesterol. However, DSPC vesicles do not merge, so more than the presence of microdomains is required to stabilize fusion.
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