Synergistic regulation of fusion pore opening and dilation by SNARE and synaptotagmin-1.

Fusion pore opening is a transient intermediate state of synaptic vesicle exocytosis, which is highly dynamic and precisely regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and synaptotagmin-1 (Syt1). Yet, the regulatory mechanism is not fully understood. In this work, using single-channel membrane fusion electrophysiology, we determined that SNAREpins are important for driving fusion pore opening and dilation but incapable of regulating the dynamics. When Syt1 was added, the closing frequency of fusion pores significantly increased, while the radius of fusion pores mildly decreased. In response to Ca2+, SNARE/Syt1 greatly increased the radius of fusion pores and reduced their closing frequency. Moreover, the residue F349 in the C2B domain of Syt1, which mediates Syt1 oligomerization, was required for clamping fusion pore opening in the absence of Ca2+, probably by extending the distance between the two membranes. Finally, in Ca2+-triggered fusion, the primary interface between SNARE and Syt1 plays a critical role in stabilizing and dilating the fusion pore, while the polybasic region of Syt1 C2B domain has a mild effect on increasing the radius of the fusion pore. In summary, our results suggest that Syt1, SNARE, and the anionic membrane synergically orchestrate the dynamics of fusion pore opening in synaptic vesicle exocytosis.

A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS-CoV-2 Spike-Mediated Viral Invasion.

The receptor-binding domain (RBD) of spike recognizing the receptor angiotensin-converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell-vesicle content-mixing assay, it is found that the cleavage at the S2' site by thrombin (Thr) protease strongly accelerates membrane fusion, compared to that of cleavage at the S1/S2 site by PreScission (3C) protease. Moreover, mutations at the RBD_ACE2 interface resulted in a positive correlation between binding affinity and fusion probability. In both the cell-vesicle and cell-cell fusion assays, by crosslinking two membranes via the neutravidin (NTV)_biotin interaction or complementary DNA strands, it is found that spike drives membrane fusion in the absence of ACE2, and a suitable distance between two membranes is critical for spike-mediated membrane fusion. Finally, unsuitable membrane crosslinkers significantly inhibited the fusion probability in the presence of ACE2. Taken together, the results suggest that the RBD_ACE2 complex may act as a crosslinker to bridge the viral and cell membranes at a suitable distance, which is critical, but also substitutable for spike-mediated SARS-CoV-2 entry.

Vesicle trafficking and vesicle fusion: mechanisms, biological functions, and their implications for potential disease therapy.

Intracellular vesicle trafficking is the fundamental process to maintain the homeostasis of membrane-enclosed organelles in eukaryotic cells. These organelles transport cargo from the donor membrane to the target membrane through the cargo containing vesicles. Vesicle trafficking pathway includes vesicle formation from the donor membrane, vesicle transport, and vesicle fusion with the target membrane. Coat protein mediated vesicle formation is a delicate membrane budding process for cargo molecules selection and package into vesicle carriers. Vesicle transport is a dynamic and specific process for the cargo containing vesicles translocation from the donor membrane to the target membrane. This process requires a group of conserved proteins such as Rab GTPases, motor adaptors, and motor proteins to ensure vesicle transport along cytoskeletal track. Soluble N-ethyl-maleimide-sensitive factor (NSF) attachment protein receptors (SNARE)-mediated vesicle fusion is the final process for vesicle unloading the cargo molecules at the target membrane. To ensure vesicle fusion occurring at a defined position and time pattern in eukaryotic cell, multiple fusogenic proteins, such as synaptotagmin (Syt), complexin (Cpx), Munc13, Munc18 and other tethering factors, cooperate together to precisely regulate the process of vesicle fusion. Dysfunctions of the fusogenic proteins in SNARE-mediated vesicle fusion are closely related to many diseases. Recent studies have suggested that stimulated membrane fusion can be manipulated pharmacologically via disruption the interface between the SNARE complex and Ca2+ sensor protein. Here, we summarize recent insights into the molecular mechanisms of vesicle trafficking, and implications for the development of new therapeutics based on the manipulation of vesicle fusion.

Impaired white matter microstructure associated with severe depressive symptoms in patients with PD.

Depression is a common occurrence in patients with Parkinson's disease (PD); however, its pathophysiology is still unclear. This study assessed the association between the integrity of white matter and depressive symptoms in patients with PD. 67 patients with PD were divided into a non-depressed PD group (ndPD, n = 30) and a depressed PD group (dPD, n = 37). The dPD group was further subdivided into a mild-moderately depressed PD (mdPD, n = 22) and a severely depressed PD group (sdPD, n = 15). Tract-Based Spatial Statistics was used to compare fractional anisotropy (FA) between groups. Region-of-interest analysis was used to explore changes in diffusivity indices in the regions showing FA abnormalities. The sdPD patients exhibited significantly reduced FA in the left superior longitudinal fasciculus, uncinate fasciculus, anterior corona radiata, corticospinal tract, and bilateral inferior fronto-occipital fasciculus when compared with the ndPD patients, but the decreased FA was within a smaller area when compared with the mdPD patients. No significant difference in FA was found between the mdPD and ndPD groups. Among the dPD patients, FA values in the left superior longitudinal fasciculus negatively correlated with BDI scores. Impaired white matter integrity in the prefronto-limbic/temporal circuitry, mainly in the left hemisphere, is associated with severe, but not mild-moderate depressive symptoms in patients with PD.

Distinct Changes in Global Brain Synchronization in Early-Onset vs. Late-Onset Parkinson Disease.

Early- and late-onset Parkinson's disease (EOPD and LOPD, respectively) have different risk factors, clinical features, and disease course; however, the functional outcome of these differences have not been well characterized. This study investigated differences in global brain synchronization changes and their clinical significance in EOPD and LOPD patients. Patients with idiopathic PD including 25 EOPD and 24 LOPD patients, and age- and sex-matched healthy control (HC) subjects including 27 younger and 26 older controls (YCs and OCs, respectively) were enrolled. Voxel-based degree centrality (DC) was calculated as a measure of global synchronization and compared between PD patients and HC groups matched in terms of disease onset and severity. DC was decreased in bilateral Rolandic operculum and left insula and increased in the left superior frontal gyrus (SFG) and precuneus of EOPD patients compared to YCs. DC was decreased in the right putamen, mid-cingulate cortex, bilateral Rolandic operculum, and left insula and increased in the right cerebellum-crus1 of LOPD patients compared to OCs. Correlation analyses showed that DC in the right cerebellum-crus1 was inversely associated with the Hamilton Depression Scale (HDS) score in LOPD patients. Thus, EOPD and LOPD patients show distinct alterations in global synchronization relative to HCs. Furthermore, our results suggest that the left SFG and right cerebellum-crus1 play important roles in the compensation for corticostriatal-thalamocortical loop injury in EOPD and LOPD patients, whereas the cerebellum is a key hub in the neural mechanisms underlying LOPD with depression. These findings provide new insight into the clinical heterogeneity of the two PD subtypes.

Changed Resting-State Brain Signal in Parkinson's Patients With Mild Depression.

Background: Depression is reported to occur 5-10 years early than the onset of motor symptoms in Parkinson (PD) patients. However, markers for early diagnosis of PD in individuals with sub-clinical depression still remain to be identified. Purpose: This study utilized Regional Homogeneity (ReHo) to investigate the alterations in resting state brain activities in Parkinson (PD) patients with different degrees of depression. Methods: Twenty non-depressed PD patients, twenty mild to moderately depressed PD patients, and thirteen severely depressed PD patients were recruited. Hamilton Depression Scale (HDS) and the Beck Depression Inventory (BDI) were assessed depression. Resting-state functional magnetic resonance imaging (rs-MRI) was analyzed with ReHo. Results: PD patients with mild to moderate depression had decreased ReHo in the left dorsal anterior cingulate cortex when compared with PD patients without depression. PD patients with severe depression exhibited increased ReHo in the left inferior prefrontal gyrus and right orbitofrontal area when compared with PD patients with mild to moderate depression. ReHo values in the bilateral supplementary motor area (SMA) in PD patients with severe depression was also increased when compared with PD patients without depression. Conclusions: This study suggests that rs-MRI with ReHo analysis can detect early changes in brain function that associate with depression in PD patients, which could be biomarkers for early diagnosis and treatment of PD related depression.