Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants. DOI: https://doi.org/10. 1038/nm.3214 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-79905 Accepted Version Originally published at: Gulbins, E; Palmada, M; Reichel, M; Lüth, A; Böhmer, C; Amato, D; Müller, C P; Tischbirek, C H; Groemer, T W; Tabatabai, G; Becker, K A; Tripal, P; Staedtler, S; Ackermann, T F; van Brederode, J; Alzheimer, C; Weller, M; Lang, U E; Kleuser, B; Grassme, H; Kornhuber, J (2013). Acid sphingomyelinaseceramide system mediates effects of antidepressant drugs. Nature Medicine, 19 (7) Major depression may be triggered by psychological stress, inflammatory cytokines, and dysfunction of the hypothalamic-pituitary-adrenal axis, etc. 1-4 .The previously held monoamine hypothesis for the action of antidepressants has been questioned because the antidepressant effect of these drugs is not clearly associated with their monoaminergic effect; in fact, the antidepressant tianeptine is even a serotonin reuptake enhancer 5 . Furthermore, the direct effect on monoamines contrasts with the delay of antidepressant effects in patients. Recent concepts of the pathogenesis of major depression suggest a change of cellular plasticity predominantly in the hippocampus and a shift in the balance between neurogenic and antiapoptotic events that leads to neurodegeneration and hippocampal atrophy [6][7][8][9] . Antidepressants increase neurogenesis and reverse hippocampal atrophy associated with major depression 9 .Here, we tested the role of the acid sphingomyelinase (EC 3.1.4.12, sphingomyelin phosphodiesterase, human protein: ASM, murine protein: Asm, gene symbol: Smpd1) and ceramide system as a target for antidepressants. Asm is ubiquitously expressed and releases ceramide from sphingomyelin, predominantly in lysosomes but also in secretory lysosomes and on the plasma membrane 10-13 .The antidepres...

Acid sphingomyelinase (ASM) is an important lipid-metabolizing enzyme cleaving sphingomyelin to ceramide, mainly within lysosomes. Acid ceramidase (AC) further degrades ceramide to sphingosine which can then be phosphorylated to sphingosine-1-phosphate. Ceramide and its metabolite sphingosine-1-phosphate have been shown to antagonistically regulate apoptosis, cellular differentiation, proliferation and cell migration. Inhibitors of ASM or AC therefore hold promise for a number of new clinical therapies, e.g. for Alzheimer’s disease and major depression on the one hand and cancer on the other. Inhibitors of ASM have been known for a long time. Cationic amphiphilic substances induce the detachment of ASM protein from inner lysosomal membranes with its consecutive inactivation, thereby working as functional inhibitors of ASM. We recently experimentally identified a large number of hitherto unknown functional inhibitors of ASM and determined specific physicochemical properties of such cationic amphiphilic substances that functionally inhibit ASM. We propose the acronym “FIASMA” (Functional Inhibitor of Acid SphingoMyelinAse) for members of this large group of compounds with a broad range of new clinical indications. FIASMAs differ markedly with respect to molecular structure and current clinical indication. Most of the available FIASMAs are licensed for medical use in humans, are minimally toxic and may therefore be applied for disease states associated with increased activity of ASM.

Significanceα-Synuclein (α-Syn) aggregation underlies neurodegeneration in synucleinopathies. However, the nature of α-Syn aggregates and their toxic mechanisms in human pathology remains elusive. Here, we delineate a role of α-Syn oligomeric aggregates for axonal integrity in human neuronal models of synucleinopathies. α-Syn oligomers disrupt anterograde axonal transport of mitochondria by causing subcellular changes in transport-regulating proteins and energy deficits. An increase of α-Syn oligomers in human neurons finally results in synaptic degeneration. Together, our data provide mechanistic insights of α-Syn oligomeric toxicity in human neurons. Taking into account that α-Syn oligomers and axonal dysfunction are characteristic for early neurodegeneration in synucleinopathies, our data might deliver targets for therapeutic interference with early disease pathology.

We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimer's disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 µM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans.

Recently, it has been claimed that vesicles recycling spontaneously and during activity belong to different pools. Here we simultaneously measured, using spectrally separable styryl dyes, the release kinetics of vesicles recycled spontaneously or upon stimulation and the effects of the v-ATPase blocker folimycin on the frequency of miniature postsynaptic currents in rat hippocampal neurons. Our results provide evidence as to the identities of the vesicle pools recycling at rest and during stimulation.

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.

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Hereditary spastic paraplegias are a group of inherited motor neuron diseases characterized by progressive paraparesis and spasticity. Mutations in the spastic paraplegia gene SPG11, encoding spatacsin, cause an autosomal-recessive disease trait; however, the precise knowledge about the role of spatacsin in neurons is very limited. We for the first time analyzed the expression and function of spatacsin in human forebrain neurons derived from human pluripotent stem cells including lines from two SPG11 patients and two controls. SPG11 patients'-derived neurons exhibited downregulation of specific axonal-related genes, decreased neurite complexity and accumulation of membranous bodies within axonal processes. Altogether, these data point towards axonal pathologies in human neurons with SPG11 mutations. To further corroborate spatacsin function, we investigated human pluripotent stem cell-derived neurons and mouse cortical neurons. In these cells, spatacsin was located in axons and dendrites. It colocalized with cytoskeletal and synaptic vesicle (SV) markers and was present in synaptosomes. Knockdown of spatacsin in mouse cortical neurons evidenced that the loss of function of spatacsin leads to axonal instability by downregulation of acetylated tubulin. Finally, time-lapse assays performed in SPG11 patients'-derived neurons and spatacsin-silenced mouse neurons highlighted a reduction in the anterograde vesicle trafficking indicative of impaired axonal transport. By employing SPG11 patient-derived forebrain neurons and mouse cortical neurons, this study provides the first evidence that SPG11 is implicated in axonal maintenance and cargo trafficking. Understanding the cellular functions of spatacsin will allow deciphering mechanisms of motor cortex dysfunction in autosomal-recessive hereditary spastic paraplegia.