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Abstract Background Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer’s disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations. Methods Here, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs. Results ENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq. Conclusions Our work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer’s disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

IntroductionThe neurotrophin system plays a pivotal role in the development, morphology, and survival of the nervous system, and its dysregulation has been manifested in numerous neurodegenerative and neuroinflammatory diseases. Neurotrophins NGF and BDNF are major growth factors that prevent neuronal death and synaptic loss through binding with high affinity to their specific tropomyosin-related kinase receptors namely, TrkA and TrkB, respectively. The poor pharmacokinetic properties prohibit the use of neurotrophins as therapeutic agents. Our group has previously synthesized BNN27, a prototype small molecule based on dehydroepiandrosterone, mimicking NGF through the activation of the TrkA receptor.MethodsTo obtain a better understanding of the stereo-electronic requirements for selective activation of TrkA and TrkB receptors, 27 new dehydroepiandrosterone derivatives bearing a C17-spiro-dihydropyran or cyclobutyl moiety were synthesized. The new compounds were evaluated for their ability (a) to selectively activate the TrkA receptor and its downstream signaling kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death, and (b) to induce phosphorylation of TrkB and to promote cell survival under serum deprivation conditions in NIH3T3 cells stable transfected with the TrkB receptor and primary cortical astrocytes. In addition the metabolic stability and CYP-mediated reaction was assessed.ResultsAmong the novel derivatives, six were able to selectively protect PC12 cells through interaction with the TrkA receptor and five more to selectively protect TrkB-expressing cells via interaction with the TrkB receptor. In particular, compound ENT-A025 strongly induces TrkA and Erk1/2 phosphorylation, comparable to NGF, and can protect PC12 cells against serum deprivation-induced cell death. Furthermore, ENT-A065, ENT-A066, ENT-A068, ENT-A069, and ENT-A070 showed promising pro-survival effects in the PC12 cell line. Concerning TrkB agonists, ENT-A009 and ENT-A055 were able to induce phosphorylation of TrkB and reduce cell death levels in NIH3T3-TrkB cells. In addition, ENT-A076, ENT-A087, and ENT-A088 possessed antiapoptotic activity in NIH-3T3-TrkB cells exclusively mediated through the TrkB receptor. The metabolic stability and CYP-mediated reaction phenotyping of the potent analogs did not reveal any major liabilities.DiscussionWe have identified small molecule selective agonists of TrkA and TrkB receptors as promising lead neurotrophin mimetics for the development of potential therapeutics against neurodegenerative conditions.

One common event that is the most detrimental in neurodegenerative disorders, even though they have a complex pathogenesis, is the increased rate of neuronal death. Endogenous neurotrophins consist of the major neuroprotective factors, while brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor TrkB are described in a number of studies for their important neuronal effects. Normal function of this receptor is crucial for neuronal survival, differentiation, and synaptic function. However, studies have shown that besides direct activation, the TrkB receptor can be transactivated via GPCRs. It has been proven that activation of the 5-HT4 receptor and transactivation of the TrkB receptor have a positive influence on neuronal differentiation (total dendritic length, number of primary dendrites, and branching index). Because of that and based on the main structural characteristics of LM22A-4, a known activator of the TrkB receptor, and RS67333, a partial 5-HT4 receptor agonist, we have designed and synthesized a small data set of novel compounds with potential dual activities in order to not only prevent neuronal death, but also to induce neuronal differentiation in neurodegenerative disorders.

Neurodegenerative diseases are incurable and debilitating conditions, characterized by progressive loss and degeneration of vulnerable neuronal populations. Currently, there are no effective therapies available for the treatment of most neurodegenerative disorders. A panel of extracts exhibiting interesting chemical profiles among a high number of bacterial strains isolated from East Mediterranean marine sediments and macroorganisms were evaluated for their activity on TrkB-expressing cells. Among them, the actinobacterial strain Streptomyces sp. BI0788, exhibiting neuroprotective activity in vitro, was selected and cultivated in large-scale. The chemical analysis of its organic extract resulted in the isolation of four new butanolides (1, 4–6), along with two previously reported butanolides (2 and 3) and eight previously reported butenolides (7–14). Compounds 2–4 and 7–14 were evaluated for their neuroprotective effects on TrkB-expressing NIH-3T3 cells. Among them, metabolites 3, 4, 7, 10, 11, 13 and 14 exhibited significant protective activity on the aforementioned cells through the activation of TrkB, the high-affinity receptor for the Brain-Derived Neurotrophic Factor (BDNF), which is well known to play a crucial role in neuronal cell survival and maintenance.

Neurotrophins are growth factors that exert important neuroprotective effects by preventing neuronal death and synaptic loss. Nerve Growth Factor (NGF) acts through the activation of its high-affinity, pro-survival TrkA and low-affinity, pro-apoptotic p75NTR receptors. NGF has been shown to slow or prevent neurodegenerative signals in Alzheimer’s Disease (AD) progression. However, its low bioavailability and its blood–brain-barrier impermeability limit the use of NGF as a potential therapeutic agent against AD. Based on our previous findings on synthetic dehydroepiandrosterone derivatives, we identified a novel NGF mimetic, named ENT-A013, which selectively activates TrkA and exerts neuroprotective, anti-amyloid-β actions. We now report the chemical synthesis, in silico modelling, metabolic stability, CYP-mediated reaction phenotyping and biological characterization of ENT-A013 under physiological and neurodegenerative conditions. We show that ENT-A013 selectively activates the TrkA receptor and its downstream kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death. Moreover, ENT-A013 promotes survival of primary Dorsal Root Ganglion (DRG) neurons upon NGF withdrawal and protects hippocampal neurons against Amyloid β-induced apoptosis and synaptic loss. Furthermore, this neurotrophin mimetic partially restores LTP impairment. In conclusion, ENT-A013 represents a promising new lead molecule for developing therapeutics against neurodegenerative disorders, such as Alzheimer’s Disease, selectively targeting TrkA-mediated pro-survival signals.

Hydrobioid freshwater gastropods were collected from mainland and insular Greece. Several threatened taxa, such as Graecoanatolica vegorriticola, Pseudamnicola negropontina, Pseudamnicola pieperi, Pseudobithynia eubooensis and Pseudoislamia balcanica, were recorded from new localities. Trichonia trichonica, which has been considered extinct from its type locality for the last twenty eight years, was re-discovered, whereas the presence of Daphniola exigua, G. vegorriticola, Marstoniopsis graeca, P. pieperi and Pseudobithynia trichonis in their type localities was verified. The taxonomic status of P. negropontina and the newly discovered populations of G. vegorriticola was elucidated using COI sequence data. The new data recorded during this survey indicate that the IUCN status of some Greek endemic hydrobioids needs to be updated.