SYT6: a newly identified protein involved in ER - trans-Golgi network Membrane Contact Sites

Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.SYT6 is a newly identified lipid transport protein from ER - trans-Golgi network Membrane Contact Sites. Our results show that: • SYT6 contacts trans-Golgi network vesicles through its coiled-coil domain. • SYT6 can efectively respond to Ca2+ using its terminal C2C domain. • SYT6-C2 domains preferentially bind to negatively charged membranes (with PI4P and PS) in presence of Ca2+.This research has been funded by AEI (PID2021-127649OB-I00 and PGC-2018-098789-B-I00) and FEDER-Junta de Andalucía (UMA18-FEDERJA-154). The attendance to this meeting was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech

NTMC2T5: lipid transfer proteins at ER-chloroplast contact sites involved in plant stress.

Chloroplasts are the site of fatty acid synthesis in plants; however, these fatty acids are assembled into glycerolipids at the ER. Later on, some of these ER-assembled glycerolipids will be transferred back to the chloroplasts to be further modified and to form part of the chloroplastic membranes. Previous reports have shown that under some abiotic stresses, these plastid membranes suffer a large lipid remodelling and new precursors massively need to be transported from the ER to the chloroplast or vice versa. It has been suggested that the newly synthetized ER lipids are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in membrane contact sites (MCS). Some LTP at MCS contain particular domains, as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum. By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum. They are anchored to the chloroplast outer membrane, and they interact in trans with the ER (ER-chloroplast MCS). We have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins and Arabidopsis double knock-out mutant for these proteins showed less chloroplasts attached to nuclei at control conditions. And, we have investigated the NTMC2T5 protein domains involved in this clustering. Moreover, our analysis has demonstrated that Arabidopsis simple mutants show lower germination rates in media supplemented with NaCl and lower rates of expanded cotyledons in media supplemented with ABA. We have also performed biotinylation-based proximity labelling proteomics experiments in order to identify interactors of these proteins. Finally, we have performed lipidomic analysis to understand the role of these proteins.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Loss of Mecp2 causes atypical synaptic and molecular plasticity of parvalbumin-expressing interneurons reflecting rett syndrome–like sensorimotor defects

Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MECP2). Understanding the pathological processes impacting sensory-motor control represents a major challenge for clinical management of individuals affected by RTT, but the underlying molecular and neuronal modifications remain unclear. We find that symptomatic male Mecp2 knockout (KO) mice show atypically elevated parvalbumin (PV) expression in both somatosensory (S1) and motor (M1) cortices together with excessive excitatory inputs converging onto PV-expressing interneurons (INs). In accordance, high-speed voltage-sensitive dye imaging shows reduced amplitude and spatial spread of synaptically induced neuronal depolarizations in S1 of Mecp2 KO mice. Moreover, motor learning-dependent changes of PV expression and structural synaptic plasticity typically occurring on PV+ INs in M1 are impaired in symptomatic Mecp2 KO mice. Finally, we find similar abnormalities of PV networks plasticity in symptomatic female Mecp2 heterozygous mice. These results indicate that in Mecp2 mutant mice the configuration of PV+ INs network is shifted toward an atypical plasticity state in relevant cortical areas compatible with the sensory-motor dysfunctions characteristics of RTT.Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MECP2). Understanding the pathological processes impacting sensory-motor control represents a major challenge for clinical management of individuals affected by RTT, but the underlying molecular and neuronal modifications remain unclear. We find that symptomatic male Mecp2 knockout (KO) mice show atypically elevated parvalbumin (PV) expression in both somatosensory (S1) and motor (M1) cortices together with excessive excitatory inputs converging onto PV-expressing interneurons (INs). In accordance, high-speed voltage-sensitive dye imaging shows reduced amplitude and spatial spread of synaptically induced neuronal depolarizations in S1 of Mecp2 KO mice. Moreover, motor learning-dependent changes of PV expression and structural synaptic plasticity typically occurring on PV + INs in M1 are impaired in symptomatic Mecp2 KO mice. Finally, we find similar abnormalities of PV networks plasticity in symptomatic female Mecp2 heterozygous mice. These results indicate that in Mecp2 mutant mice the configuration of PV + INs network is shifted toward an atypical plasticity state in relevant cortical areas compatible with the sensory-motor dysfunctions characteristics of RTT

SMP-CONTAINING PROTEINS AT MEMBRANE CONTACT SITES: SUBCELLULAR LOCALIZATION AND CHARACTERIZATION.

Membrane contact sites (MCS) are microdomains where two membranes of two different organelles are in close apposition, but they do not fuse. MCS are essential for non-vesicular transport of lipids. This lipid transport is mediated by several families of proteins which all of them contain a lipid transport domain, as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. The most studied SMP protein is Arabidopsis SYT1 which is known to be involved in tolerance to multiple abiotic stresses. Later studies in other SMP proteins of the same family have shown that SYT1 and homologous such as SYT3 or SYT5 gave similar results. However, little information is available about the role other SMP proteins in plants. We have studied the occurrence of additional SMP proteins in A. thaliana and S. lycopersicum. In order to identify these proteins, SMP sequences from human and yeast were used to identify their remote orthologues in A. thaliana and S. lycopersicum, allowing the identification of several putative encoding SMP domains. We have found that some of the identified proteins are exclusive of plants as they do not have direct orthologs in yeast nor human. Transient expression in N. benthamiana leaves followed by confocal microscopy was used to study the subcellular localization of these proteins. Our results show that some of these proteins are localized at ER-Golgi contact sites and two other proteins at ER-Chloroplast sites. Finally, to determine whether these proteins are involved in abiotic stress tolerance, we have analysed the root growth and seed germination rates of Arabidopsis mutants for these genes under different conditions. Some of these mutants have shown different germination rates in media supplemented with NaCl and different rates of expanded cotyledons in media supplemented with ABA. These results suggest that some these proteins may be implicated in abiotic stress signalling through an ABA-dependent pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación (BIO2017-82609-R)

NTMC2T5 protein family: newly identified ER-chloroplast contact site proteins involved in abiotic stress.

Plants are sessile organisms and therefore they have perfected a complex molecular signalling network to detect and respond to the different environmental stresses such as high temperatures, salinity, or drought. In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the endoplasmic reticulum (ER). Then, the newly synthetized lipids in the ER are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in ER-chloroplast membrane contact sites (MCS). Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved LTP in eukaryotes that localize at MCS. They are involved in tethering of these MCS through interaction with other proteins/membrane lipids and in transferring of glycerolipids between these two membranes. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum by searching remote orthologs of human E-Syt1 (SMP protein). By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum that are anchored to the chloroplast outer membrane and are interacting with the ER (at ER-chloroplast MCS). Our preliminary data have unequivocally demonstrated that NTMC2T5 proteins are anchored to the chloroplast, and they bind in trans the ER. Additionally, it is predicted that these proteins contain a SMP domain which is a lipid-transfer domain, indicating that these proteins could be responsible for some of the lipid transferring events at ER-chloroplast MCS that are still unknown. Our preliminary phenotypic analyses have shown that these proteins are involved in salt tolerance. Finally, we have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins in Nicotiana benthamiana leaves.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Stabilizing Immature Dendritic Spines in the Auditory Cortex: A Key Mechanism for mTORC1-mediated Enhancement of Long-term Fear Memories

Mammalian target of rapamycin (mTOR) pathway has emerged as a key molecular mechanism underlying memory processes. Although mTOR inhibition is known to block memory processes, it remains elusive whether and how an enhancement of mTOR signaling may improve memory processes. Here we found in male mice that the administration of VO-OHpic, an inhibitor of the phosphatase and tensin homolog (PTEN) that negatively modulates AKT-mTOR pathway, enhanced auditory fear memory for days and weeks, while it left short-term memory unchanged. Memory enhancement was associated with a long-lasting increase in immature-type dendritic spines of pyramidal neurons into the auditory cortex. The persistence of spine remodeling over time arose by the interplay between PTEN inhibition and memory processes, as VO-OHpic induced only a transient immature spines growth in the somatosensory cortex, a region not involved in long-term auditory memory. Both the potentiation of fear memories and increase in immature spines were hampered by rapamycin, a selective inhibitor of mTORC1.These data revealed that memory can be potentiated over time by the administration of a selective PTEN inhibitor. Besides disclosing new information on the cellular mechanisms underlying long-term memory maintenance, our study provides new insights on the cellular mechanisms that aid enhancing memories over time.Significance StatementThe neuronal mechanisms that may help improve the maintenance of long-term memories are still elusive. The inhibition of mammalian-target of rapamycin (mTOR) signaling shows that this pathway plays a crucial role in synaptic plasticity and memory formation. However, if its activation may strengthen long-term memory storage is unclear. We assessed the consequences of positive modulation of AKT-mTOR pathway obtained by VO-OHpic administration, a phosphatase and tensin homolog inhibitor, on memory retention and underlying synaptic modifications. We found that mTOR activation greatly enhanced memory maintenance for weeks by producing a long-lasting increase of immature-type dendritic spines in pyramidal neurons of the auditory cortex. These results offer new insights on the cellular and molecular mechanisms that can aid enhancing memories over time

Effects of forced swimming stress on ERK and histone H3 phosphorylation in limbic areas of Roman high-and low-avoidance rats

Stressful events evoke molecular adaptations of neural circuits through chromatin remodeling and regulation of gene expression. However, the identity of the molecular pathways activated by stress in experimental models of depression is not fully understood. We investigated the effect of acute forced swimming (FS) on the phosphorylation of the extracellular signal-regulated kinase (ERK)1/2 (pERK) and histone H3 (pH3) in limbic brain areas of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Roman high-avoidance rats) to stress-induced depression-like behavior. We demonstrate that FS markedly increased the density of pERK-positive neurons in the infralimbic (ILCx) and the prelimbic area (PrLCx) of the prefrontal cortex (PFCx), the nucleus accumbens, and the dorsal blade of the hippocampal dentate gyrus to the same extent in RLA and RHA rats. In addition, FS induced a significant increase in the intensity of pERK immunoreactivity (IR) in neurons of the PFCx in both rat lines. However, RHA rats showed stronger pERK-IR than RLA rats in the ILCx both under basal and stressed conditions. Moreover, the density of pH3-positive neurons was equally increased by FS in the PFCx of both rat lines. Interestingly, pH3-IR was higher in RHA than RLA rats in PrLCx and ILCx, either under basal conditions or upon FS. Finally, colocalization analysis showed that in the PFCx of both rat lines, almost all pERK-positive cells express pH3, whereas only 50% of the pH3-positive neurons is also pERK-positive. Moreover, FS increased the percentage of neurons that express exclusively pH3, but reduced the percentage of cells expressing exclusively pERK. These results suggest that (i) the distinctive patterns of FS-induced ERK and H3 phosphorylation in the PFCx of RHA and RLA rats may represent molecular signatures of the behavioural traits that distinguish the two lines and (ii) FS-induced H3 phosphorylation is, at least in part, ERK-independent

A role for hemopexin in oligodendrocyte differentiation and myelin formation.

Myelin formation and maintenance are crucial for the proper function of the CNS and are orchestrated by a plethora of factors including growth factors, extracellular matrix components, metalloproteases and protease inhibitors. Hemopexin (Hx) is a plasma protein with high heme binding affinity, which is also locally produced in the CNS by ependymal cells, neurons and glial cells. We have recently reported that oligodendrocytes (OLs) are the type of cells in the brain that are most susceptible to lack of Hx, as the number of iron-overloaded OLs increases in Hx-null brain, leading to oxidative tissue damage. In the current study, we found that the expression of the Myelin Basic Protein along with the density of myelinated fibers in the basal ganglia and in the motor and somatosensory cortex of Hx-null mice were strongly reduced starting at 2 months and progressively decreased with age. Myelin abnormalities were confirmed by electron microscopy and, at the functional level, resulted in the inability of Hx-null mice to perform efficiently on the Rotarod. It is likely that the poor myelination in the brain of Hx-null mice was a consequence of defective maturation of OLs as we demonstrated that the number of mature OLs was significantly reduced in mutant mice whereas that of precursor cells was normal. Finally, in vitro experiments showed that Hx promotes OL differentiation. Thus, Hx may be considered a novel OL differentiation factor and the modulation of its expression in CNS may be an important factor in the pathogenesis of human neurodegenerative disorders

Identification of NTMC2T5, a new lipid transfer protein family at ER-chloroplast contact sites involved in stress response

Plants are sessile organisms and they have perfected a complex molecular signalling network to detect and respond to different environmental stresses. In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the ER. Then, the newly synthetized lipids in the ER are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins. These LTP would be localized in ER-chloroplast membrane contact sites (MCS), which are microdomains where membranes of these two different organelles are closely apposed but not fussing. SMP domain proteins are evolutionarily conserved LTP in eukaryotes that localize at MCS. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum. By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum that are anchored to the chloroplast outer membrane and are interacting with the ER (at ER-chloroplast MCS. Our preliminary data have demonstrated that NTMC2T5 proteins are anchored to the chloroplast, and they bind in trans the ER. Additionally, it is predicted that these proteins contain a SMP domain which is a lipid-transfer domain, indicating that these proteins could be responsible for some of the lipid transferring events at ER-chloroplast MCS that are still unknown. We show the results of the lipidomic analysis we have performed in order to understand the role of these proteins. And our phenotypic analyses have shown that these proteins are involved in salt tolerance. Additionally, we have observed that clustering of chloroplasts occurred when we overexpressed these proteins. And Arabidopsis double knock-out mutant for these proteins showed less chloroplasts attached to the nucleus in epidermal cells, suggesting that these proteins could be involved in these chloroplast signalling events after stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Localization and characterization of SMP-containing proteins in Membrane Contact Sites

Membrane contact sites (MCS) are discrete regions where two membranes from different organelles are closely apposed (10-30 nm). In those regions, non-vesicular transfer of lipids takes place to ensure proper organelle functioning. Arabidopsis SYT1 is one of the best characterized MCS protein, and it plays a relevant role in tolerance to abiotic stresses. SYT1 is a SMP (synaptotagmin-like mitocondrial lipid binding domain) containing protein localized at ER-PM contact sites. Recent studies suggest that this protein transfer glycerolipids between these two membranes. However, little is known about other SMP-containing proteins in plants, as their localization or their role in abiotic stress. We have focused on studying the rest of SMP-containing proteins in Arabidopsis thaliana and Solanum lycopersicum. To identify them, human E-Syt1 sequence was used to find the remote orthologues in plants. An interesting highlight of those results was that some SMP-containing proteins are exclusive from plants, there are no orthologues in human nor yeast. The subsequent step was the study of their subcellular location, that was carried out in Nicotiana benthamiana by transient expression of the SMP-containing proteins from Arabidopsis and Solanum, followed by confocal microscopy imaging. We have found that those proteins locate in different MCS across the cell: SYT6, NTMC2T6 and Tex2 localise in ER-Golgi contact sites, NTMC2T5 in ER-Chloroplast contact sites, and we have also confirmed that Solanum CLB1 and SYT5 localized at ER-PM contact sites as their Arabidopsis counterparts. Additionally, we have analysed the root growth, seed germination rates and fully expanded cotyledons of Arabidopsis mutants for these genes in media supplemented with salt or ABA, and our results suggest that some of these proteins might be implicated in abiotic stress signalling through an ABA pathway.This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación (BIO2017-82609-R), and meeting assistance was granted by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech