9 research outputs found
Early Changes in Exo- and Endocytosis in the EAE Mouse Model of Multiple Sclerosis Correlate with Decreased Synaptic Ribbon Size and Reduced Ribbon-Associated Vesicle Pools in Rod Photoreceptor Synapses
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally
leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies
also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported
alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis
(EAE) mouse model of MS. In the present study, we found that the previously observed decreased
imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size,
whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller
photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even
as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis
was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished
synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting
non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated
with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse
pathology in early EAE
ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis
Biogenesis of large dense core vesicles and molecular mechanisms of dead-end docking in mouse chromaffin cells
SUMMARY
Ca2+-dependent regulated exocytosis is major form of exocytosis in neurons and neuroendocrine cells. These cells release neurotransmitters and hormones through exocytosis of secretory vesicles upon external stimuli. Regulated exocytosis occurs in different stages: biogenesis of the secretory vesicles, translocation of these vesicles, that are targeted and docked to the plasma membrane (PM). Ultimately when the Ca2+ concentration strongly increases, fusion of secretory vesicles with the PM occurs inducing release of the neurotransmitter cargo outside the cells. In the last 30 years, adrenal medullary chromaffin cells have emerged as a model system to study regulated secretory pathway because they are round, rendering them easy to patch and because they can be used for membrane capacitance measurements. Furthermore, due to their common origin with the sympathetic neurons their molecular machinery for regulated secretion is very similar to that found in neurons. Even though chromaffin cells are extensively used to study the regulated secretory pathway, biogenesis and recycling of the chromaffin secretory granules which are called large dense core vesicles (LDCVs) still remains unresolved.
In order to get more insights into these processes, I used adrenal chromaffin cells from newborn mice and successfully electroporated them with Neuropeptide Y (NPY)-mCherry fusion protein construct to specifically label the newly generated LDCVs. I followed them by fixing the chromaffin cells at different time intervals from 2 h to 24 h post transfection. Immunostaining of the cis-Golgi compartment and the cortical actin network was used to analyze the distribution of the newly formed vesicles over time. I could show that initial expression of NPY-mCherry was already visible after 2 h post transfection but it was localized near Golgi and that few vesicles were formed. Over the time LDCVs moved away from the Golgi and distributed in the cytoplasm. LDCVs accumulated in the cortical actin ring but not directly adjacent to the PM. Furthermore, there was only partial colocalization between NPY and the cis- or trans-Golgi markers irrespective of the time used to study colocalization. With the help of correlative fluorescence electron microscopy (CLEM), we could show that the non-overlapping area near the Golgi, which was neither cis- nor trans-Golgi, was indeed another Golgi compartment. The identification of this compartment remains to be elucidated. I was also able to show that the vesicular proteins Synaptobrevin-2 (Syb-2), Cellubrevin (Ceb), and Synaptotagmin1 (Syt1) gets associated with the newly generated LDCVs at a later stage in the biogenesis because their colocalization with NPY-mCherry increased over time and was highest at 24 h post transfection. Moreover, I also studied the endocytosis of Syt1 in NPY-mCherry transfected chromaffin cells by inducing exocytosis followed by endocytosis with application of 60mM KCl, allowing the uptake of lumenal domain anti-Syt1 antibody, and fixing the cells at increasing time of recovery. I also used anti-TGN38 and anti-Rab11A antibodies to study the fate of the endocytosed Syt1. I counted the number of Syt1 punctae overlapping with NPY-mCherry after stimulation and the number of Syt1 punctae remaining at the cell periphery. We observed that Syt1 required a minimum of 2 h for recycling, and this recycling was not mediated by Rab11A positive endosomes because the number of punctae positive for endocytosed Syt1 and Rab11A were scarce. The three major conclusions from this study were:
1. Generation of vesicles needs at least 2 h and a new Golgi compartment devoid of GM130 and TGN38 retains the expression of NPY-mCherry probably for loading of vesicles with neuropeptides and Granins.
2. Association of vesicular membrane proteins with the newly formed vesicles happens at late stage in biogenesis.
3. Recycling of Syt1 needs a minimum of 2 h after first round of exo/endocytosis and it is not dependent on Rab11A positive endosomes.
Our group previously showed that dead-end vesicles which remain attached at the PM for a long time without fusing exist in bovine chromaffin cells. We demonstrated that target-Soluble NSF Attachment Protein Receptors (t-SNAREs) mediates dead-end docking via formation of unproductive t-SNARE acceptor complexes that hinders the fusion of docked vesicles with the PM. However, the vesicular component interacting with the unproductive t-SNARE acceptor complexes remained unknown. We used knock out mouse models for different vesicular proteins like Syb-2, Ceb, Syt1, and Syt7. I perfused the mouse chromaffin cells with high free Ca2+ and used the combination of membrane capacitance measurement through patch-clamp technique and total internal reflection fluorescence microscopy (TIRFM) allowing the analysis of single vesicle fusion and of their behavior during the recording period. First I was able to show that dead-end vesicles also existed in mouse chromaffin cells. Then I analyzed the effect of the absence of the above cited vesicular proteins on dead-end docking. Syb-2 KO induced significant reduction in global secretion but no effect on the number of dead-end vesicles. Also, there was no docking defect in Syb-2 KO cells. Ceb KO showed no effect on any of the TIRFM parameters and no effect on overall secretion. Syt1 KO cells showed strong docking defect and a high reduction in single vesicle secretion in comparison to control. However, no influence on overall exocytosis or number of dead-end vesicles was found. Syt7 KO cells also showed mild but significant docking defect and strong decrease in number of vesicles that were secreted in TIRF in comparison to WT control. Surprisingly, there was also a massive decline in the number of dead-end vesicles. The specificity of the effect of Syt7 on dead-end docking was further tested by overexpressing Syt7 in Syt7 KO, which rescued the number of dead-end vesicles. From this study, I concluded that:
1. Dead-end vesicles also exist in mouse chromaffin cells.
2. v-SNAREs have no role in dead-end docking.
3. Syt1 has an important role in productive docking but not in dead-end docking.
4. Syt7 is the only vesicular protein to be involved in unproductive docking.ZUSAMMENFASSUNG
Ca2+-abhĂ€ngige regulierte Exozytose ist die Hauptform der Exozytose in Neuronen und neuroendokrinen Zellen. Diese Zellen setzen Neurotransmitter und Hormone durch Exozytose von sekretorischen Vesikeln auf entsprechende Stimuli hin frei. Die regulierte Exozytose erfolgt in verschiedenen Stadien: Biogenese der sekretorischen Vesikel, Translokation dieser Vesikel, zur Plasmamembran (PM) und âAndockenâ dieser Vesikel mit der PM. Nach einem Ansteig der intrazellulĂ€ren Ca2+-Konzentration erfolgt die Fusion von sekretorischen Vesikeln mit der PM ein, die zur Freisetzung der Neurotransmitter-Ladung fĂŒhrt. In den letzten 30 Jahren sind Chromaffin-Zellen des Nebennierenmarks als Modellsystem etabliert worden, um den regulierten sekretorischen Weg zu untersuchen. Dies hat verschiedene GrĂŒnde: zum einen sind Chromaffine Zellen rund, was sie leicht zu patchen und was fĂŒr MembrankapazitĂ€tsmessungen sehr nĂŒtzlich macht. DarĂŒber hinaus ist aufgrund ihrer gemeinsamen Herkunft mit den sympathischen Neuronen aus der Neuralleiste ihre molekulare Maschinerie fĂŒr regulierte Sekretion sehr Ă€hnlich wie bei Neuronen. Obwohl Chromaffin-Zellen weitgehend verwendet werden, um den regulierten sekretorischen Weg zu untersuchen, bleibt die Biogenese und das Recycling der chromaffinen Sekretionsgranula, die hier als groĂe dichte Kern-Vesikel (large dense core vesicles, LDCVs) bezeichnet werden, noch ungelöst.
Um mehr Einblicke in diese Prozesse zu erhalten, benutzte ich adrenale Chromaffin-Zellen der Nebenniere von neugeborenen MĂ€usen und transfizierte sie erfolgreich mit einem Neuropeptid Y (NPY)-mCherry-Fusionsprotein-Konstrukt, um die neu erzeugten LDCVs spezifisch zu markieren. Ich untersuchte die Biogenese der chromaffinen Granula, indem ich die Chromaffinzellen in verschiedenen Zeitintervallen von 2 h bis 24 h nach der Transfektion fixierte. Die ImmunfĂ€rbung des cis-Golgi-Kompartiments und des kortikalen Aktin-Netzwerks wurde verwendet, um die Verteilung der neu gebildeten Vesikel im Laufe der Zeit zu analysieren. Ich konnte zeigen, dass eine erste Expression von NPY-mCherry bereits 2 h nach der Transfektion sichtbar war. Die Expression 2 h nach der Transfektion war aber in der NĂ€he von Golgi beschrĂ€nkt und es wurden nur wenige Vesikel zu diesem Zeitpunkt gebildet. Im Laufe der Zeit entfernten sich die LDCVs vom Golgi und verteilten sich im Zytoplasma. LDCVs sammelten sich im kortikalen Aktinring an, aber nicht direkt neben der PM. DarĂŒber hinaus gab es nur eine partielle Kolokalisierung zwischen NPY und den cis- oder trans-Golgi-Markern. Mit Hilfe der korrelativen Fluoreszenz-Elektronenmikroskopie (CLEM) konnten wir zeigen, dass die nicht ĂŒberlappende FlĂ€che in der NĂ€he des Golgi, die weder cis- noch trans-Golgi war, tatsĂ€chlich ein weiteres Golgi-Kompartiment darstellte. Die Identifizierung dieses unbekannten Golgi-Kompartimentes bleibt abzuklĂ€ren. Ich konnte auch zeigen, dass die vesikulĂ€ren Proteine Synaptobrevin-2 (Syb-2), Cellubrevin (Ceb) und Synaptotagmin1 (Syt1) mit den neu erzeugten LDCVs in einem spĂ€teren Stadium der Biogenese assoziiert werden, weil ihre Kolokalisation mit NPY-mCherry erste im Laufe der Zeit sich erhöhte und am höchsten 24 h nach der Transfektion war. DarĂŒber hinaus habe ich auch die Endozytose von Syt1 in NPY-mCherry-transfizierten Chromaffin-Zellen untersucht. Dazu habe ich zunĂ€chst mittels einer Applikation von 60 mM KCl eine Exozytose induziert, gefolgt von einer Endozytose, was die Aufnahme von luminalen DomĂ€nen-Anti-Syt1-Antikörpern ermöglicht und die Zellen nach unterschiedlichen Zeitpunkten fixiert. Ich habe u.a. Anti-TGN38 und Anti-Rab11A-Antikörper verwendet, um das Schicksal des endozytosierten Syt1 zu untersuchen. Ich zĂ€hlte die Anzahl der Syt1 Punkte, die mit NPY-mCherry nach der Stimulation ĂŒberlappten, und die Anzahl der Syt1 Punkte, die an der Zellperipherie verbleiben. Wir beobachteten, dass Syt1 mindestens 2 h fĂŒr das Recycling benötigte, und dieses Recycling nicht durch Rab11A positive Endosomen vermittelt wurde, da die Anzahl der Punkte, die sowohl fĂŒr endozytierte Syt1 als auch fĂŒr Rab11A positive waren, gering war. Die drei wichtigsten Schlussfolgerungen aus dieser Studie waren:
1. Die Erzeugung von Vesikeln (LDCVs) benötigt mindestens 2 h und benötigt ein neues Golgi-Kompartiment, das weder GM130 noch TGN38 aufweist, und wahrscheinlich fĂŒr die Beladung von Vesikeln mit Neuropeptiden und Graninen zustĂ€ndig ist.
2. Die VerknĂŒpfung von vesikulĂ€ren Membranproteinen mit den neu gebildeten Vesikeln erfolgt im spĂ€ten Stadium der Biogenese.
3. Recycling von Syt1 braucht mindestens 2 Stunden nach der ersten Runde der Exo / Endozytose und ist nicht abhÀngig von Rab11A positiven Endosomen.
Unsere Gruppe zeigte zuvor, dass sogenannte âdead-endâ Vesikel, die an der PM lange haften bleiben, ohne zu fusionieren, auch in Rinder-Chromaffin-Zellen vorhanden sind. Wir haben gezeigt, dass sogenannte target-Soluble NSF Attachment Protein Receptors (t-SNAREs) das dead-end Docking durch Bildung von unproduktiven t-SNARE-Akzeptorkomplexen vermitteln, die die Verschmelzung von angedockten Vesikeln mit der PM behindern. Die vesikulĂ€re Komponente, die mit den unproduktiven t-SNARE-Akzeptorkomplexen interagiert, blieb jedoch unbekannt. Wir verwendeten Knock out Maus Modelle fĂŒr verschiedene vesikulĂ€re Proteine wie Syb-2, Ceb, Syt1 und Syt7. Ich habe die Maus-Chromaffin-Zellen mit hohen Konzentrationen von freiem Ca2+ perfundiert und die Kombination der MembrankapazitĂ€tsmessung durch Patch-Clamp-Technik und Total-Reflexions-Fluoreszenzmikroskopie (TIRFM) verwendet, um die Analyse der Einzelvesikelfusion und ihres Verhaltens wĂ€hrend der Aufzeichnungsperiode zu ermöglichen. Zuerst konnte ich zeigen, dass dead-end Vesikel auch in Maus-Chromaffin-Zellen existierten. Dann analysierte ich die Wirkung der Abwesenheit der oben genannten vesikulĂ€ren Proteine auf dead-end Docking. Syb-2 KO induzierte eine signifikante Reduktion der globalen Sekretion, aber keine Wirkung auf die Anzahl der dead-end Vesikel. Auch gab es keinen Docking-Defekt in Syb-2 KO-Zellen. Ceb KO zeigte keine Wirkung auf einen der TIRFM-Parameter und keine Auswirkung auf die Gesamtsekretion. Syt1-KO-Zellen zeigten einen starken Docking-Defekt und eine hohe Reduktion der Einzel-Vesikel-Sekretion im Vergleich zur Kontrolle. Es wurde jedoch kein Einfluss auf die Gesamt-Exozytose oder die Anzahl der dead-end Vesikel festgestellt. Syt7-KO-Zellen zeigten auch einen leichten, aber signifikanten Docking-Defekt und eine starke Abnahme der Anzahl von Vesikeln, die im Vergleich zur WT-Kontrolle in TIRF sezerniert wurden. Ăberraschenderweise gab es auch einen massiven RĂŒckgang der Anzahl der dead-end Vesikel. Die SpezifitĂ€t der Wirkung von Syt7 auf dead-end Docking wurde weiter durch Ăberexpression von Syt7 in Syt7 KO, die die Anzahl der dead-end Vesikel wieder herstellt getestet. Aus dieser Studie kam ich zu folgenden Schlussfolgerungen:
1. Dead-End-Vesikel gibt es auch in Maus-Chromaffin-Zellen.
2. v-SNAREs haben keine Rolle im dead-end Docking.
3. Syt1 hat eine wichtige Rolle bei der produktiven Andockung, aber nicht bei dead-end Docking.
4. Syt7 ist das einzige vesikulÀre Protein, das sowohl in unproduktives Docking involviert ist
ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis
Disturbed Presynaptic Ca2+ Signaling in Photoreceptors in the EAE Mouse Model of Multiple Sclerosis
Multiple sclerosis (MS) is a demyelinating disease caused by an auto-reactive immune system. Recent studies also demonstrated synapse dysfunctions in MS patients and MS mouse models. We previously observed decreased synaptic vesicle exocytosis in photoreceptor synapses in the EAE mouse model of MS at an early, preclinical stage. In the present study, we analyzed whether synaptic defects are associated with altered presynaptic Ca2+ signaling. Using high-resolution immunolabeling, we found a reduced signal intensity of Cav-channels and RIM2 at active zones in early, preclinical EAE. In line with these morphological alterations, depolarization-evoked increases of presynaptic Ca2+ were significantly smaller. In contrast, basal presynaptic Ca2+ was elevated. We observed a decreased expression of Na+/K+-ATPase and plasma membrane Ca2+ ATPase 2 (PMCA2), but not PMCA1, in photoreceptor terminals of EAE mice that could contribute to elevated basal Ca2+. Thus, complex Ca2+ signaling alterations contribute to synaptic dysfunctions in photoreceptors in early EAE
Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses
The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor
outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3
as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also
enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons.
In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-
dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor
synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron
microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was
predominantly found on vesicles distributed within the entire terminal. These findings indicate that
the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins.
In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-
dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active
zone. Mutations in Nphp3 are known to be associated with SeniorâLĂžken Syndrome 3 (SLS3). Visual
impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from
malfunctions of the photoreceptor synapse
The Calcineurin-Binding, Activity-Dependent Splice Variant Dynamin1xb Is Highly Enriched in Synapses in Various Regions of the Central Nervous System
In the present study, we generated and characterized a splice site-specific monoclonal antibody that selectively detects the calcineurin-binding dynamin1 splice variant dynamin1xb. Calcineurin is a Ca2+-regulated phosphatase that enhances dynamin1 activity and is an important Ca2+-sensing mediator of homeostatic synaptic plasticity in neurons. Using this dynamin1xb-specific antibody, we found dynamin1xb highly enriched in synapses of all analyzed brain regions. In photoreceptor ribbon synapses, dynamin1xb was enriched in close vicinity to the synaptic ribbon in a manner indicative of a peri-active zone immunolabeling. Interestingly, in dark-adapted mice we observed an enhanced and selective enrichment of dynamin1xb in both synaptic layers of the retina in comparison to light-adapted mice. This could be due to an illumination-dependent recruitment of dynamin1xb to retinal synapses and/or due to a darkness-induced increase of dynamin1xb biosynthesis. These latter findings indicate that dynamin1xb is part of a versatile and highly adjustable, activity-regulated endocytic synaptic machinery
Early autoâimmune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis
Abstract Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an autoâimmune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and wellâvalidated mouse models of optic neuritis that ribbon synapses in the myelinâfree retina are targeted by an autoâreactive immune system even before alterations in the optic nerve have developed. The autoâimmune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis