Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation

Mechanisms Controlling the Expression and Secretion of BDNF

Brain-derived nerve factor (BDNF), through TrkB receptor activation, is an important modulator for many different physiological and pathological functions in the nervous system. Among them, BDNF plays a crucial role in the development and correct maintenance of brain circuits and synaptic plasticity as well as in neurodegenerative diseases. The proper functioning of the central nervous system depends on the available BDNF concentrations, which are tightly regulated at transcriptional and translational levels but also by its regulated secretion. In this review we summarize the new advances regarding the molecular players involved in BDNF release. In addition, we will address how changes of their levels or function in these proteins have a great impact in those functions modulated by BDNF under physiological and pathological conditions.Research in Juan Carlos Arévalo’s and Rubén Deogracias’ laboratories was funded by MCIN/AEI/10.13039/501100011033, grant numbers PID2020-113130RB-100 to JCA, RYC2018-025215- I and PID2020-113086RB-100 to RD

Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis

Phosphorylation of Synaptotagmin 4 captures transiting dense core vesicles at active synapses

Synaptic modulation requires fast recruitment of neuronal dense core vesicles (DCVs) containing various neuropeptides and neurotrophins at nerve terminals. DCVs undergo long-range trafficking in axons to deliver cargoes at release sites. However, the question of whether and how specific sites capture these transiting vesicles upon neuronal activity is open. In this study, we have used a Synaptotagmin (Syt) isoform, Syt4, as a DCV marker to investigate trafficking and activity-dependent capture of DCVs in hippocampal neurons. We found that Syt4-harboring vesicles are highly mobile on microtubules and switch directions only at the distal end of axons in hippocampal neurons. We examined the effects of phosphorylation of Syt4 at S135 on trafficking, capture and fusion of DCVs in mature neurons. We found that phosphomimetic Syt4 vesicles traffic less and are more concentrated at synapses. Conversely, phosphodeficient Syt4 vesicles had the most processivity and were least localized at synapses. We also found that disrupting actin, which is enriched at pre-synaptic sites, enhances the mobility of phosphomimetic vesicles. We found that the motor protein Kif1A is associated with Syt4 vesicles but phosphomimetic vesicles had less interaction with Kif1A. Over-expression of Kif1A rescued the trafficking of phosphomimetic Syt4 vesicles. In addition, we found that c-Jun N-terminal kinase (JNK) phosphorylates Syt4 at S135 specifically causing decreased motility of transiting DCVs. Furthermore, increased neuronal activity promoted capture of transiting vesicles at synapses via a JNK phosphorylation dependent mechanism. Phosphorylation of Syt4 did not affect the fusion of vesicles at synaptic and non-synaptic sites in hippocampal neurons. Together, this study reveals a JNK-dependent phosphorylation mechanism involved in trafficking and capture of Syt4 harboring DCVs in hippocampal neurons. We propose a mechanism whereby JNK at active synapses phosphorylates Syt4 at S135 on transiting DCVs, promoting destabilization of Syt4-Kif1A binding and allowing capture of DCVs at synapses by actin. This mechanism would potentially allow fast recruitment of dense core vesicles to active synapses, ensuring the efficient delivery of neuropeptides and neurotrophins to specific sites in hippocampal neurons whenever needed

Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B

An epilepsy-associated mutation of salt-inducible kinase 1 increases the susceptibility to epileptic seizures and interferes with adrenocorticotropic hormone therapy for infantile spasms in mice（Salt-induced kinase 1遺伝子のてんかん関連変異はてんかん発作の感受性を高めるとともに、マウスの点頭てんかんに対するACTHの効果を減弱させる。）

信州大学(Shinshu university)博士（医学）次の雑誌に発表。 /International Journal of Molecular Sciences 23(14) :7927(2022); doi:10.3390/ijms23147927 © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).ThesisPANG BO. An epilepsy-associated mutation of salt-inducible kinase 1 increases the susceptibility to epileptic seizures and interferes with adrenocorticotropic hormone therapy for infantile spasms in mice（Salt-induced kinase 1遺伝子のてんかん関連変異はてんかん発作の感受性を高めるとともに、マウスの点頭てんかんに対するACTHの効果を減弱させる。）. 信州大学, 2021, 博士論文. 博士（医学）, 甲第1301号, 令和03年09月30日授与.doctoral thesi

The role of HIG1/MYB51 in the regulation of indolic glucosinolate biosynthesis

Glucosinolates are amino-acid derived plant secondary metabolites found mainly in Brassicaceae, including the model plant Arabidopsis thaliana. Due to their role in plant defence and their cancer-preventive properties in human nutrition, they have gained increasing interest over the last years. This study presents the characterisation of the activation-tagging mutant HIG1-1D, which displays a high indolic glucosinolate phenotype, caused by an activation of the R2R3-type MYB transcription factor HIG1/MYB51. A positive correlation between HIG1/MYB51 transcription and the accumulation of indolic glucosinolates could be confirmed in gain and loss-of-function mutants. HIG1/MYB51 expression overlaps with sites of indolic glucosinolate biosynthesis and the expression of biosynthesis genes, which are activated by HIG1/MYB51 in trans. Unlike previously characterised mutants affected in indolic glucosinolate biosynthesis, HIG1-1D displays only minor effects on auxin biosynthesis. However, a role of HIG1/MYB51 in the biotic stress response of A. thaliana appears likely, due to the mechano-sensitive expression of HIG1/MYB51 along with an increased resistance of HIG1-1D plants against a generalist herbivore. Yeast-two-hybrid screening allowed identifying the interaction of HIG1/MYB51 with ATR2/bHLH05, a putative regulator of tryptophan and indolic glucosinolate biosynthesis. Therefore, HIG1/MYB51 appears to be part of a complex network controlling indolic glucosinolate biosynthesis

Dual-Specificity Phosphatases in Neuroblastoma Cell Growth and Differentiation

Dual-specificity phosphatases (DUSPs) are important regulators of neuronal cell growth and differentiation by targeting proteins essential to neuronal survival in signaling pathways, among which the MAP kinases (MAPKs) stand out. DUSPs include the MAPK phosphatases (MKPs), a family of enzymes that directly dephosphorylate MAPKs, as well as the small-size atypical DUSPs, a group of low molecular-weight enzymes which display more heterogeneous substrate specificity. Neuroblastoma (NB) is a malignancy intimately associated with the course of neuronal and neuroendocrine cell differentiation, and constitutes the source of more common extracranial solid pediatric tumors. Here, we review the current knowledge on the involvement of MKPs and small-size atypical DUSPs in NB cell growth and differentiation, and discuss the potential of DUSPs as predictive biomarkers and therapeutic targets in human NB.This work was partially supported by the grants: BIO13/CI/001/BC from BIOEF (EITB maratoia), Basque Country, Spain; SAF2013-48812-R from the Ministerio de Educacion y Ciencia (to R.P.), and SAF2016-79847-R from the Ministerio de Economia y Competitividad (Spain and Fondo Europeo de Desarrollo Regional) (to R.P. and J.I.L.); and 239813 from the Research Council of Norway (to C.E.N-X.)

A Novel Communication Mechanism Between the Presynapse and Postsynapse Through Exosomes: A Dissertation

The minimal element of the nervous system, the synapse, is a plastic structure that has the ability to change in response to various internal and external factors. This property of the synapse underlies complex behaviors such as learning and memory. However, the exact molecular and cellular mechanisms involved in this process are not fully understood. To understand the mechanisms that regulate synapse development and plasticity I took advantage of a powerful model system, the Drosophila larval neuromuscular junction (NMJ). In this system, both anterograde and retrograde signaling pathways critical for coordinated synapse development and plasticity have been documented. An anterograde WNT/Wingless (Wg) signaling pathway plays a crucial role in both developmental and activity-dependent synaptic plasticity at the NMJ. Presynaptic motor neuron terminals secrete highly hydrophobic Wg, which travels to relatively distant postsynaptic sites where it activates a signal transduction pathway required for postsynaptic development. In the first half of my thesis I unraveled a previously unrecognized cellular mechanism by which Wg is shuttled to postsynaptic sites. In this mechanism Wg rides on secreted microvesicles or exosomes that contain a dedicated WNT secretion factor, the WNT-binding transmembrane protein, Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). To our knowledge, this was the first in vivo study demonstrating that neurons release exosomes, which are involved in trans-synaptic communication. Moreover, this was the first study showing that hydrophobic WNT signals are transported to the extracellular space on exosomes to reach WNT-receptor containing target cells. Retrograde signals are also critical during development and plasticity of synaptic connections. These signals function to adjust the activity of presynaptic cells according to postsynaptic cell outputs, to maintain synaptic function within a dynamic range. However, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are not clear. In the second half of my thesis, I provided evidence that a crucial component of retrograde signaling at the fly NMJ, Synaptotagmin-4 (Syt4), is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Drosophila Syt4 is known to reside on postsynaptic vesicles at the NMJ and function as a calcium sensor to release a retrograde signal upon synaptic activity. This event is required for coordinated maturation of the presynaptic terminal. We demonstrated that retrograde Syt4 function in postsynaptic muscle is required for activity-dependent presynaptic growth. However, surprisingly, Syt4 protein was not synthesized in postsynaptic muscles. Instead, Syt4 was produced in motorneurons and transferred to postsynaptic muscle cells via exosome secretion by presynaptic cells. The above study provided evidence for a presynaptic control of postsynaptic retrograde signaling through exosomal transfer of an essential retrograde signaling component. In summary, this body of work reveals a novel mechanism of trans-synaptic communication through exosomes. While intercellular communication through exosomes had been demonstrated during antigen presentation in the immune system, our studies were the first to substantiate this mode of communication in the nervous system. Thus, these studies provide a significantly deeper and novel understanding of the mechanisms underlying synapse development and plasticity

Cell-to-Cell Communication in Learning and Memory: From Neuro- and Glio-Transmission to Information Exchange Mediated by Extracellular Vesicles

Most aspects of nervous system development and function rely on the continuous crosstalk between neurons and the variegated universe of non-neuronal cells surrounding them. The most extraordinary property of this cellular community is its ability to undergo adaptive modifications in response to environmental cues originating from inside or outside the body. Such ability, known as neuronal plasticity, allows long-lasting modifications of the strength, composition and efficacy of the connections between neurons, which constitutes the biochemical base for learning and memory. Nerve cells communicate with each other through both wiring (synaptic) and volume transmission of signals. It is by now clear that glial cells, and in particular astrocytes, also play critical roles in both modes by releasing different kinds of molecules (e.g., D-serine secreted by astrocytes). On the other hand, neurons produce factors that can regulate the activity of glial cells, including their ability to release regulatory molecules. In the last fifteen years it has been demonstrated that both neurons and glial cells release extracellular vesicles (EVs) of different kinds, both in physiologic and pathological conditions. Here we discuss the possible involvement of EVs in the events underlying learning and memory, in both physiologic and pathological condition