Natalia L. Kononenko

Clathrin/AP-2 Mediate Synaptic Vesicle Reformation from Endosome-like Vacuoles but Are Not Essential for Membrane Retrieval at Central Synapses

Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

Molecular Mechanisms of Presynaptic Membrane Retrieval and Synaptic Vesicle Reformation

The function of the nervous system depends on the exocytotic release of neurotransmitter from synaptic vesicles (SVs). To sustain neurotransmission, SV membranes need to be retrieved, and SVs have to be reformed locally within presynaptic nerve terminals. In spite of more than 40 years of research, the mechanisms underlying presynaptic membrane retrieval and SV recycling remain controversial. Here, we review the current state of knowledge in the field, focusing on the molecular mechanism involved in presynaptic membrane retrieval and SV reformation. We discuss the challenges associated with studying these pathways and present perspectives for future research.

Tyramine as an independent transmitter and a precursor of octopamine in the locust central nervous system: An immunocytochemical study

Octopamine and its precursor tyramine are biogenic amines that are found ubiquitously in insects, playing independent but opposite neuromodulatory roles in a wide spectrum of behaviors, ranging from locomotion and aggression to learning and memory. We used recently available antibodies to octopamine and tyramine to label the distribution of immunoreactive profiles in the brain and ventral nerve cord of the locust. In the brain and all ventral cord ganglia all known octopaminergic neurons were labeled with both the tyramine and octopamine antisera. In the brain the subesophageal ganglion and all fused abdominal ganglia we found somata that were only labeled by the tyramine antibody. Some prominent architectural features of the brain, like the protocerebral bridge, the central body, and associated neuropils, also contain intensely labeled tyramine‐immunoreactive fibers. In addition, tyraminergic fibers occur in all ganglia of the ventral cord. For known octopaminergic neurons of the thoracic ganglia, octopamine‐immunoreactivity was confined to the cell body and to the varicosities or boutons, whereas fiber processes always expressed tyramine‐immunoreactivity. The distribution of the tyramine and octopamine content within these neurons turned out to be dependent on how the animal was handled before fixation for immunocytochemistry. We conclude that tyramine is an independent transmitter in locusts, and that in octopaminergic neurons the ratio between octopamine and its precursor tyramine is highly dynamic. J. Comp. Neurol. 512:433–452, 2009.

Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2

Significance Brain function depends on neurotransmission, and alterations in this process are linked to neuropsychiatric disorders. Neurotransmitter release requires the rapid recycling of synaptic vesicles (SVs) by endocytosis. How synapses can rapidly regenerate SVs, yet preserve their molecular composition, is poorly understood. We demonstrate that mice lacking the endocytic protein stonin 2 (Stn2) show changes in exploratory behavior and defects in SV composition, whereas the speed at which SVs are regenerated is increased. As Stn2 is implicated in schizophrenia and autism in humans, our findings bear implications for neuropsychiatric disorders. Neurotransmission depends on the exocytic fusion of synaptic vesicles (SVs) and their subsequent reformation either by clathrin-mediated endocytosis or budding from bulk endosomes. How synapses are able to rapidly recycle SVs to maintain SV pool size, yet preserve their compositional identity, is poorly understood. We demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) in mice compromises the fidelity of SV protein sorting, whereas the apparent speed of SV retrieval is increased. Loss of Stn2 leads to selective missorting of synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated SV protein endocytosis. The latter phenotype is mimicked by overexpression of endocytosis-defective variants of synaptotagmin 1. Increased speed of SV protein retrieval in the absence of Stn2 correlates with an up-regulation of SV reformation from bulk endosomes. Our results are consistent with a model whereby Stn2 is required to preserve SV protein composition but is dispensable for maintaining the speed of SV recycling.

Fast neurotransmitter release regulated by the endocytic scaffold intersectin

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.

Presubiculum Layer III Conveys Retrosplenial Input to the Medial Entorhinal Cortex

Navigation is mediated by a network of brain areas, and research has focused on the head‐direction system in the presubiculum (PrS), the grid cell containing medial entorhinal cortex (EC) (MEC) and place cells in the hippocampus. Less research addressed the interactions of the retrosplenial cortex (RSC) and the navigational system, although it is well established that damage to the RSC leads to navigational deficits. We previously showed that RSC provides a dense input to deep layers of MEC and to superficial layers of PrS. In this study we use confocal microscopical analysis and show that the dense projection from the caudal part of the ventral retrosplenial granular cortex targets neurons in Layer III of PrS, which provide input to superficial layers of MEC. Our high resolution anatomical data indicate that sparsely spiny pyramidal neurons in Layer III of PrS that originate projections to Layer III of MEC are the main target of these retrosplenial projections. Retrosplenial axonal boutons were found to equally contact spines and shafts of basal dendrites in Layer III, but contacts on shafts are more prominent close to the soma, indicating the potential for efficient synaptic transfer. These observations suggest that neurons in Layer III of PrS have an important role in mediating RSC contributions to navigation.

Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles

Significance Brain function depends on neurotransmission, and alterations in this process are linked to neurological disorders. Neurotransmitter release requires the rapid recycling of synaptic vesicles (SVs) by endocytosis. How synapses maintain the molecular composition of SVs during recycling is poorly understood. We demonstrate that overlapping functions of two completely distinct proteins, the vesicle protein SV2A/B and the adaptor stonin 2, mediate endocytic sorting of the vesicular calcium sensor synaptotagmin 1. As SV2A is the target of the commonly used antiepileptic drug levetiracetam and is linked to late onset Alzheimer’s disease, our findings bear implications for the treatment of neurological and neurodegenerative disorders. Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.

Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration

Autophagosomes primarily mediate turnover of cytoplasmic proteins or organelles to provide nutrients and eliminate damaged proteins. In neurons, autophagosomes form in distal axons and are trafficked retrogradely to fuse with lysosomes in the soma. Although defective neuronal autophagy is associated with neurodegeneration, the function of neuronal autophagosomes remains incompletely understood. We show that in neurons, autophagosomes promote neuronal complexity and prevent neurodegeneration in vivo via retrograde transport of brain-derived neurotrophic factor (BDNF)-activated TrkB receptors. p150Glued/dynactin-dependent transport of TrkB-containing autophagosomes requires their association with the endocytic adaptor AP-2, an essential protein complex previously thought to function exclusively in clathrin-mediated endocytosis. These data highlight a novel non-canonical function of AP-2 in retrograde transport of BDNF/TrkB-containing autophagosomes in neurons and reveal a causative link between autophagy and BDNF/TrkB signalling.

Dendritic projections of different types of octopaminergic unpaired median neurons in the locust metathoracic ganglion

Octopaminergic dorsal unpaired median (DUM) neurons of locust thoracic ganglia are important components of motor networks and are divided into various sub-populations. We have examined individually stained metathoracic DUM neurons, their dendritic projection patterns, and their relationship to specific architectural features of the metathoracic ganglion, such as longitudinal tracts, transverse commissures, and well-defined sensory neuropils. The detailed branching patterns of individually characterized DUM neurons of various types were analyzed in vibratome sections in which architectural features were revealed by using antibodies against tubulin and synapsin. Whereas DUM3,4,5 and DUM5 neurons (the group innervating leg and “non-wing-power” muscles) had many ventral and dorsal branches, DUM1 and DUM3,4 neurons (innervating “wing-power” muscles) branched extensively only in dorsal areas. The structure of DUM3 neurons differed markedly from that of the other DUM neurons examined in that they sent branches into dorsal areas and had differently structured side branches that mostly extended laterally. The differences between the branching patterns of these neurons were quantified by using currently available new reconstruction algorithms. These structural differences between the various classes of DUM neurons corresponded to differences in their function and biophysical properties.

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.