Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Synthese 18 F-markierter Liganden zur Visualisierung der GABA-Bindungsstelle des GABA A-Rezeptors mittels PET

γ-Aminobuttersäure (GABA) ist der wichtigste inhibitorische Neurotransmitter im zentralen Nervensystem und bindet vorrangig an ionotrope GABAA-Rezeptoren. Diese sind an fast allen neuronalen Prozessen beteiligt und werden darüber hinaus mit neurologischen Erkrankungen wie Epilepsie, Angstzuständen, Schlafstörungen und Schizophrenie in Verbindung gebracht. Die PET bietet als molekulares bildgebendes Verfahren die Möglichkeit einzelne Stoffwechselvorgänge des GABAergen Systems zu visualisieren und zu quantifizieren. Durch den Einsatz eines 18F-markierten Radioliganden an die GABA-Bindungsstelle könnten so die Rezeptorverfügbarkeit des GABAA-Rezeptors gemessen und die Ausschüttung des Neurotransmitters GABA quantifiziert werden.rn4-(2-Naphthylmethyl)-5-(piperidin-4-yl)isothiazolole und -isoxazolole stellen aufgrund ihrer hohen Affinität gegenüber der GABA-Bindungsstelle und ihrer lipophilen Struktur vielversprechende Leitstrukturen für die Entwicklung eines PET-Tracers zur Visualisierung der GABA-Bindungsstelle dar. Daher wurden zunächst 19F-substituierte Referenzverbindungen synthetisiert, um diese hinsichtlich ihrer Eignung als Radioligand in in vitro-Studien zu evaluieren. Dazu wurde Fluor direkt sowie über eine Fluorethoxygruppe an Position 1 des Naphthalinrings eingeführt. Zusätzlich wurde ein Fluorethylether eines Isothiazolols als Referenz-verbindung synthetisiert. In anschließenden Verdrängungsstudien wurden die Affinitäten der synthetisierten Verbindungen mit [3H]Muscimol an Membranpräparaten aus Rattenhirnen, sowie transfizierten HEK293-Zellen bestimmt. Zusätzlich wurden die entsprechenden Log D-Werte bestimmt. Die Verbindung 5-(piperidin-4-yl)-4-(1-fluornaphth-2-ylmethyl)-isothiazol-3-ol VK5 zeigte in den in vitro-Studien die vielversprechendsten Ergebnisse (IC50 = 10 nM; Log D = 1,7) und wurde im Folgenden in einer dreistufigen Radiosynthese als 18F-Verbindung synthetisiert.rnZu diesem Zweck wurde ein geeigneter Markierungsvorläufer dargestellt und über eine n.c.a. SNAr-Markierung mit [18F]F- umgesetzt. Die Reaktionsparameter wurden hinsichtlich Reaktionszeit, -temperatur, Basenkonzentration und Lösungsmittel optimiert. Die zur Aktivierung einer SNAr ein-geführte Carbonylfunktion wurde in einem zweiten Schritt mit Triethylsilan/Trifluoressigsäure reduziert. Im finalen Schritt wurden zwei Schutzgruppen mit Bortrichlorid in DCM abgespaltet und [18F]VK5 als injektionsfertige Lösung in isotoner NaCl-Lösung erhalten. Es wurden radiochemische Ausbeuten von 0,7-1 % (EOS) nach einer durchschnittlichen Synthesedauer von 275 Minuten erhalten.rnDer Radioligand [18F]VK5 wurde anschließend in Autoradiographie-Versuchen an Hirnschnitten der Ratte hinsichtlich seiner Spezifität für die GABA-Bindungsstelle untersucht. Die unspezifische Bindung wurde durch die Zugabe von GABA bestimmt wonach kein signifikanter Unterschied festgestellt werden konnte. Die hohe unspezifische Bindung kann möglicherweise auf die niedrigen spezifischen Aktivitäten zurückgeführt werden. Diese lagen, bedingt durch die drei Schritte der Radiosynthese, in einem Bereich von 0,1-0,6 GBq/μmol. Die erhaltenen Ergebnisse lassen für zukünftige Versuche noch einige Optimierungsmöglichkeiten offen. Aufgrund der bisher erhaltenen Daten lässt sich daher keine definitive Aussage über die Eignung des Liganden [18F]VK5 als PET-Tracer treffen.rnγ-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the mammalian central nervous system and plays an important role in neuronal functions like cognition, learning and memory. Moreover, dysfunctions of the GABAergic system are involved in anxiety, schizophrenia, sleep disorders and epilepsy. Therefore PET ligands are valuable tools to study the pathophysiology of neuronal disorders, effects of pharmacotherapies and normal brain functions. Most radiotracers for PET and SPECT target the benzodiazepine binding site but till today there is no radioligand available for the imaging of the GABA recognition site of GABAA receptors. Those would provide the possibility to measure receptor availabilities as well as the release of GABA from presynaptic neurons.rn4-(2-Naphthylmethyl)-5-(piperidin-4-yl)isothiazololes and -isoxazololes seem to be promising lead structures for the development of PET-tracers for the GABA binding site, due to their high affinities and their lipophilic structures. Therefore new 19F-substituted reference compounds were synthesized to verify their suitability as PET-Tracers in in vitro assays. The fluorine label was introduced at position one of the naphthalene ring either directly or by a fluoroethoxy group. Additionally an isothiazolole was alkylated using fluoroethyltosylate. The affinities towards the GABAA receptor were determined using a radioligand competition binding assay using rat brain membrane preparations and [3H]muscimol. Some of the reference compounds showed high affinity towards the GABA binding site in low nanomolar range. Furthermore log D values were determined by HPLC. The most promising candidate of this series 5-(piperidin-4-yl)-4-(1-fluoronaphth-2-ylmethyl)-isothiazol-3-ol VK5 showed excellent binding properties (IC50 = 10 nM) and was selected for subsequent radiolabeling.rnTherefore a convenient labeling precursor was synthesized and labeled with 18F by direct n.c.a. nucleophilic aromatic substitution. The reaction conditions were optimized due to temperature, duration, solvent and amount of base. The carbonyl group, necessary for the activation of the naphthalene, ring was reduced with triethylsilane/trifluorofolic acid afterwards. During the final step, two protective groups were removed by borontrichloride whereby [18F]VK5 was obtained as injectable solution. Radiochemical yields of 0,7-1 % (EOS) were obtained after a synthesis time of about 275 minutes.rnAutoradiographic studies of [18F]VK5 on brain slices of rats were conducted to determine the specific binding to the GABA binding site. Unspecific binding was determined by addition of GABA but in any case no specific binding was observed. A possible explanation could be the low specific activity, resulting from the three radioactive steps. The obtained results indicate some possibilities for further improvements. Based upon the given results, there could be no definitive conclusion whether [18F]VK5 would be a suitable PET-Tracer or not.r

Chilean experience using “Theranostics” for treating metastatic neuroendocrine tumors with [177Lu]Lu DOTA-TATE

Introduction: Well-differentiated, Neuroendocrine Tumors (NET) are highly heterogenic and slow-growing pathologies, characterized by unspecific symptomatology and elevated expression of somatostatin receptors (SSTR). Despite the high incidence of NETs, several patients are diagnosed in advance stages of the disease when surgery is insufficient to treat the pathology. Peptide receptor radionuclide therapy (PRRT) has emerged as a new state-of-the-art treatment for NET-patients in advanced stages.&nbsp;Results: In this retrospective study, between 2004 and 2018 a total of 66 patients with advanced-stage-NETs, refractory to other therapies, were treated with [177Lu]Lu DOTA TATE.&nbsp; At the end of the study, 56.1% of the patients were alive and the median overall survival for all patients in the study was 86.3 months. Patients that received doses ≥ 22.2 GBq showed increased overall survival (OS) in comparison with patients that received doses &lt; 22.2 GBq (HR, 0.168; 95%CI 0.12- 0.99; p&lt;0.001), adjusted by gender. Likewise, patients that received doses ≥29.6 GBq had an increased OS (HR, 0.42; 95%CI 0.19-0.94, p&lt;0.05).&nbsp;Conclusion: Although several studies have shown that PRRT is an effective alternative for advanced NET, patients in South America have no regular access to PRRT. Our study proves that [177Lu]Lu-DOTA-TATE effectively increases the survival of patients with metastatic NET and provides an excellent alternative in terms of cost-efficiency for South American countries.&nbsp;</p

Whole-body biodistribution and radiation dosimetry of [18F]PR04.MZ: a new PET radiotracer for clinical management of patients with movement disorders

Purpose [18F]PR04.MZ is a new PET imaging agent for dopamine transporters, providing excellent image quality and allowing for the evaluation of patients with movement disorders such as Parkinson’s disease. The objective of this study was to evaluate the biodistribution and radiation dosimetry of [18F]PR04.MZ by serial PET imaging. Methods Six healthy subjects (n = 3 males, n = 3 females) were enrolled in this study. A series of 14 whole-body PET/CT scans were acquired until 5.5 h post-injection of 200 ± 11 MBq of [18F]PR04.MZ. After rigid co-registration, volumes of interest were outlined either on CT or PET images. Time-integrated activity coefficients were calculated for selected source organs. Organ absorbed doses, and the effective dose were calculated using IDAC-Dose 2.1. Results Physiological uptake of [18F]PR04.MZ was mainly observed in the striatum, brain, liver, gall bladder, intestine, red marrow and cortical bone. [18F]PR04.MZ was primarily excreted via hepatobiliary clearance and, to a lower extent, via renal clearance. The normalized absorbed doses were highest in gall bladder wall (32.2 ± 6.4 µGy/MBq), urinary bladder wall (27.2 ± 4.5 µGy/MBq), red marrow (26.5 ± 1.4 µGy/MBq), cortical bone surface (26.3 ± 2.5 µGy/MBq), liver (22.5 ± 1.8 µGy/MBq) and kidneys (21.8 ± 1.1 µGy/MBq). The effective dose according to ICRP 60 and 103 was 16.3 ± 1.1 and 16.6 ± 1.5 µSv/MBq, respectively. Conclusion [18F]PR04.MZ has a favourable dosimetry profile, comparable to those of other 18F-labelled PET tracers, and is suitable for larger clinical applications. Trial registration CEC SSM Oriente, Santiago, Chile, permit 20140520