Abstract: Neurotransmitter-containing synaptic vesicles (SVs) form tight clusters at synapses. These clusters act as a reservoir from which SVs are drawn for exocytosis during sustained activity. Several components associated with SVs that are likely to help form such clusters have been reported, including synapsin. Here we found that synapsin can form a distinct liquid phase in an aqueous environment. Other scaffolding proteins could coassemble into this condensate but were not necessary for its formation. Importantly, the synapsin phase could capture small lipid vesicles. The synapsin phase rapidly disassembled upon phosphorylation by calcium/calmodulin-dependent protein kinase II, mimicking the dispersion of synapsin 1 that occurs at presynaptic sites upon stimulation. Thus, principles of liquid-liquid phase separation may apply to the clustering of SVs at synapses.

Abstract: Close appositions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are a general feature of all cells and are abundant in neurons. A function of these appositions is lipid transport between the two adjacent bilayers via tethering proteins that also contain lipid transport modules. However, little is known about the properties and dynamics of these proteins in neurons. Here we focused on TMEM24/C2CD2L, an ER-localized SMP domain containing phospholipid transporter expressed at high levels in the brain, previously shown to be a component of ER–PM contacts in pancreatic β-cells. TMEM24 is enriched in neurons versus glial cells and its levels increase in parallel with neuronal differentiation. It populates ER–PM contacts in resting neurons, but elevations of cytosolic Ca 2+ mediated by experimental manipulations or spontaneous activity induce its transient redistribution throughout the entire ER. Dissociation of TMEM24 from the plasma membrane is mediated by phosphorylation of an array of sites in the C-terminal region of the protein. These sites are only partially conserved in C2CD2, the paralogue of TMEM24 primarily expressed in nonneuronal tissues, which correspondingly display a much lower sensitivity to Ca 2+ elevations. ER–PM contacts in neurons are also sites where Kv2 (the major delayed rectifier K + channels in brain) and other PM and ER ion channels are concentrated, raising the possibility of a regulatory feedback mechanism between neuronal excitability and lipid exchange between the ER and the PM.

Abstract: Numerous genes whose mutations cause, or increase the risk of, Parkinson’s disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1 RQ KI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a “synthetic” effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1 RQ KI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1 RQ KI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.

Abstract: VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson’s disease–linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER–endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo–electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.

Abstract: Pancreatic β cells operate with a high rate of membrane recycling for insulin secretion, yet endocytosis in these cells is not fully understood. We investigate this process in mature mouse β cells by genetically deleting dynamin GTPase, the membrane fission machinery essential for clathrin-mediated endocytosis. Unexpectedly, the mice lacking all three dynamin genes ( DNM1 , DNM2 , DNM3 ) in their β cells are viable, and their β cells still contain numerous insulin granules. Endocytosis in these β cells is severely impaired, resulting in abnormal endocytic intermediates on the plasma membrane. Although insulin granules are abundant, their release upon glucose stimulation is blunted in both the first and second phases, leading to hyperglycemia and glucose intolerance in mice. Dynamin triple deletion impairs insulin granule exocytosis and decreases intracellular Ca 2+ responses and granule docking. The docking defect is correlated with reduced expression of Munc13-1 and RIM1 and reorganization of cortical F-actin in β cells. Collectively, these findings uncover the role of dynamin in dense-core vesicle endocytosis and secretory capacity. Insulin secretion deficiency in the absence of dynamin-mediated endocytosis highlights the risk of impaired membrane trafficking in endocrine failure and diabetes pathogenesis.

Abstract: Chorea-acanthocytosis (ChAc) and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an endoplasmic reticulum (ER)-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER–PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK, and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.

Abstract: The origin of millet from Neolithic China has generally been accepted, but it remains unknown whether common millet ( Panicum miliaceum ) or foxtail millet ( Setaria italica ) was the first species domesticated. Nor do we know the timing of their domestication and their routes of dispersal. Here, we report the discovery of husk phytoliths and biomolecular components identifiable solely as common millet from newly excavated storage pits at the Neolithic Cishan site, China, dated to between ca. 10,300 and ca. 8,700 calibrated years before present (cal yr BP). After ca. 8,700 cal yr BP, the grain crops began to contain a small quantity of foxtail millet. Our research reveals that the common millet was the earliest dry farming crop in East Asia, which is probably attributed to its excellent resistance to drought.

Abstract: Zintl compounds are considered to be potential thermoelectric materials due to their “phonon glass electron crystal” (PGEC) structure. A promising Zintl-phase thermoelectric material, 2-1-2–type Eu 2 ZnSb 2 ( P 6 3 / mmc ), was prepared and investigated. The extremely low lattice thermal conductivity is attributed to the external Eu atomic layers inserted in the [Zn 2 Sb 2 ] 2- network in the structure of 1-2-2–type EuZn 2 Sb 2 ( P 3 ¯ m 1 ) , as well as the abundant inversion domain boundary. By regulating the Zn deficiency, the electrical properties are significantly enhanced, and the maximum ZT value reaches ∼1.0 at 823 K for Eu 2 Zn 0.98 Sb 2 . Our discovery provides a class of Zintl thermoelectric materials applicable in the medium-temperature range.

Abstract: Nuclear factor κB (NF-κB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo , rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-κB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induced time-dependent NF-κB nuclear translocation. The nuclear NF-κB protein after QA treatment was comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein were markedly increased at the time of QA-induced NF-κB nuclear translocation. Immunohistochemical analysis showed that c-Myc and p53 induction occurred in the excitotoxin-sensitive medium-sized striatal neurons. NF-κB nuclear translocation was blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-κB SN50, but not by the NF-κB SN50 control peptide. NF-κB SN50 significantly inhibited the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-κB SN50, but not the control peptide, also substantially reduced the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-κB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating an unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis.

Abstract: In summary, our results show the existence of mechanisms that prevent the rapid depletion of synaptic vesicles at release sites when the supply of such vesicles is drastically reduced by the absence of both dynamin 1 and dynamin 3. They suggest that, when synaptic vesicle components are stranded within clathrin-coated pits (which happens in the absence of dynamin 1 and 3) with a resulting dramatic accumulation of membrane at the cell surface, a feedback mechanism exists that affects the probability of release. This finding reveals an interesting example of the coupling between endocytosis and exocytosis, a process that is increasingly recognized as playing an important role in short-term synaptic plasticity and thus in the proper functioning of neuronal networks. Overnight silencing of the DKO cultures with tetrodotoxin, which blocks the sodium ion channels responsible for the propagation of electrical impulses along axons, decreased both the synaptic facilitation and the activation of CaMKII. This finding indicates that the effects observed in DKO cultures are dependent on neuronal activity and most likely result (at least to a great extent) from the trapping of recycling vesicle membranes in endocytic intermediates. Inhibition of CaMKII also reversed the facilitation observed at DKO synapses, suggesting that this enzyme plays a role in relaying the status of endocytic traffic to the exocytic machinery. We also examined the physiological parameters of DKO synapses that could account for these observations. The size of the readily releasable pool of vesicles, which was assessed by two independent methods, was greatly decreased at DKO synapses, although not as strongly as was the amplitude decrease of the EPSCs. Likewise, EM showed that docked vesicles at active zones (i.e., the vesicles thought to reflect the readily releasable pool) were less abundant in DKO cultures. Why then did DKO synapses not quickly run out of vesicles, resulting in transmission failure? Additional analysis revealed a reduction in release probability ( p ). A lower p (reluctance to release the available vesicles) explains the greater effect of the loss of dynamin 1 and 3 on EPSCs than on the readily releasable pool. It also, therefore, explains the enhanced preservation of a secretory response from this pool during a stimulus train. We additionally found an increased activation state of a calcium/calmodulin-dependent protein kinase (CaMKII) in DKO cultures, consistent with the previously reported increase in the state of phosphorylation of synapsin 1 (a synaptic vesicle associate protein that is a substrate of this kinase) ( 1 ). Surprisingly, instead of a faster depression, we have now found robust synaptic facilitation on repeated stimulation at synapses of cultured neurons derived from DKO newborn mice. Although the average amplitude of the first excitatory postsynaptic current (EPSC) in a stimulus train was dramatically reduced in DKO cultures, subsequent EPSCs in the train were higher than the first response. They did, however, remain much smaller in absolute values than in control neurons ( Fig. P1 B , Upper ). After this initial facilitation phase, the response underwent depression, but the average normalized EPSC curves revealed overall lower depression in DKO cultures relative to controls throughout the train ( Fig. P1 B ). Here, we have investigated the impact of the combined absence of dynamin 1 and 3 [double KO (DKO)] on short-term synaptic plasticity (i.e., the changes in the strength of synaptic transmission that occur in response to repetitive stimulation). In cortical neuron cultures, most synapses exhibit a progressively smalle r postsynaptic response, known as depression, in response to a train of electrical pulses or action potentials ( Fig. P1 B ). This response is caused by a progressive depletion of vesicles available for release ( 4 ). As shown by studies in mice and other species, such depression is typically enhanced at the synapses of neurons, where the function of endocytic proteins has been impaired by either gene disruption or other manipulations, reflecting the less-efficient resupply of synaptic vesicles and thus, the smaller pools of available vesicles. For example, using precisely the same conditions used here for studies of dynamin DKO neurons, enhanced depression was observed at synapses of endophilin triple KO mouse neurons ( 5 ). Three dynamin genes are present in mammals and encode three very similar proteins, dynamins 1, 2, and 3. Although dynamin 2 performs functions in all cells, dynamin 1 and 3 are expressed primarily in the nervous system, where they are present at very high levels (particularly dynamin 1) and contribute to most of the dynamin levels in neurons. Recent studies of neurons from newborn mice in which the genes that encode dynamin 1 and 3 had been knocked out have shown a major exo-/endocytosis imbalance; synaptic vesicles are greatly reduced in number, and much of the synaptic vesicle membrane material is sequestered in endocytic clathrin-coated pits ( 1 – 3 ). However, synaptic transmission, albeit greatly reduced, is not abolished ( 1 – 3 ). Most likely, the low levels of dynamin 2 in such neurons are sufficient to support low level of vesicle endocytosis, although a dynamin-independent endocytosis cannot be excluded. At synapses—the sites where nerve cells pass signals to each other—the fusion of small membrane-bound packages known as synaptic vesicles with the plasma membrane (i.e., exocytosis) is rapidly followed by the reinternalization (i.e., endocytosis) of their membranes to regenerate new vesicles. Much of this endocytic recycling occurs by clathrin-mediated endocytosis, and the enzyme dynamin plays a critical role in this process by mediating the fission of clathrin-coated pits to generated free vesicles. Here, we have investigated the impact of the absence of the bulk of neuronal dynamin on synaptic changes in response to repetitive stimulation. In contrast to the expectation that such absence would result in enhanced postsynaptic depression on repetitive stimulation, we found synaptic facilitation and reduced depression, and we further investigated the mechanism behind this effect.