α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, includingParkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Current understanding of amyloid-β (Aβ) metabolism and toxicity provides an extensive list of potential targets for developing drugs for treating Alzheimer's disease. We took two independent approaches, including synaptic-plasticity-based analysis and behavioral screening of synthetic compounds, for identifying single compounds that are capable of rescuing the Aβ-induced memory loss in both transgenic fruit fly and transgenic mouse models. Two clinically available drugs and three synthetic compounds not only showed positive effects in behavioral tests but also antagonized the Aβ oligomers-induced activation of the epidermal growth factor receptor (EGFR). Such surprising converging outcomes from two parallel approaches lead us to conclude that EGFR is a preferred target for treating Aβ-induced memory loss.A myloid-β (Aβ) oligomers-induced memory loss is thought to be a hallmark of Alzheimer's disease (AD) progression (1-3). Aβ peptides are cleaved from a membrane protein APP via β-and γ-secretases' activities (4). They can be removed through activities of neprilysin, insulin-degradation enzyme, and possibly other mechanisms (5-7). Aβ is also able to bind with a large array of target proteins, such as EphB2, TNF-R1, RAGE1, and NMDA receptor and prion (8)(9)(10)(11)(12) to exert a wide range of effects, including synaptic transmission, protein transportation, mitochondrial functions, and others (13-15). Thus, there are a large number of potential targets for developing AD treatment based on the Aβ hypothesis, for example, the mechanisms either reducing the production of Aβ peptides or enhancing the degradation process. It remains, however, an open question as to whether some of these targets are, at the organism level, better suited for drug development than others. One important reason is that manipulating activities of such production and degradation enzymes may affect many other physiological proteins. Thus, reported failures in a number of Aβ-based drug efforts (16) stress the necessity of identifying such preferred targets.To evaluate the possibility of finding preferred targets, we looked for overlapping and converging effects of identified targets with multiple independent approaches. First, following the direction of a synaptic-plasticity-based, mechanism-guided study, we continued to work on the signal transduction pathway of PI3-kinase that has been shown to mediate an Aβ-induced change in long-term synaptic depression as well as the Aβ-induced memory loss in Aβ42-expressing Drosophila, which recapitulates several ADlike symptoms (17). These efforts led us to find Aβ42 oligomersinduced activation of the epidermal growth factor receptor (EGFR) and the rescue of Aβ-induced memory loss in transgenic Drosophila and APP/PS1 double transgenic mouse models through treatments with clinically available EGFR inhibitors. Second, we worked to identify single compounds capable of rescuing Aβ-induced memory loss through large-scale behavioral screening with Aβ42-expressing transgenic fruit flies, followed by a confirma...

Kainic acid (KA) treatment is a well-established model of hippocampal neuron death mediated in large part by KA receptor-induced excitotoxicity. KA-induced, delayed neuron death has been shown previously to follow the induction of seizures and exhibit characteristics of both apoptosis and necrosis. Growing evidence supports a role of autophagic stress-induced death of neurons in several in vitro and in vivo models of neuron death and neurodegeneration. However, whether autophagic stress also plays a role in KA-induced excitotoxicity has not been previously investigated. To examine whether KA alters the levels of proteins associated with or known to regulate the formation of autophagic vacuoles, we isolated hippocampal extracts from control mice and in mice following 2-16h KA injection. KA induced a significant increase in the amount of LC3-II, a specific marker of autophagic vacuoles, at 4-6h following KA, which indicates a transient induction of autophagic stress. Levels of autophagy-associated proteins including ATG5 (conjugated to ATG12), ATG6 and ATG7 did not change significantly after treatment with KA. However, ratios of phospho-mTOR/ mTOR were elevated from 6-16h, and ratios of phospho-Akt/Akt were elevated at 16h following KA treatment, suggesting a potential negative feedback loop to inhibit further stimulation of autophagic stress. Together these data indicate the transient induction of autophagic stress by KA which may serve to regulate excitotoxic death in mouse hippocampus. Keywords kainic acid; hippocampus; autophagy; ATG5; ATG7; Akt; mTOR; LC3The excitatory amino acid neurotransmitter glutamate is known to play an important role in a vast array of neuronal activities as well as in the induction of excitotoxic neurodegeneration through massive activation of its receptors [1;2]. Kainic acid (KA) is a potent glutamate receptor agonist with selectivity towards non-N-methyl-D-aspartate (NMDA)-type glutamate receptors [3;4]. KA is well known for its ability to induce seizures within minutes of its administration and is followed by a delayed excitotoxic neuron death in the hippocampus several hours later, in part through an increase in intracellular calcium and activation of calcium-dependent neuron death pathways [5][6][7]. Both apoptotic and necrotic death of neurons Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Autophagic stress results from alterations in autophagy, a lysosomal degradation pathway that is responsible for the homeostatically regulated turnover of macronutrients and organelles [10]. Macroautophagy, the most prominent form of autophagy ...

Multiple intracellular signals are altered in Alzheimer's disease brain tissues, including the PI3K/Akt pathway. However, the pathological relevance of such alterations is poorly understood. In vitro studies yield results that seem to be consistent with the conventional perception in which an up-regulation of the cell survival pathway, PI3K pathway, is protective in Alzheimer's disease pathogenesis. The current in vivo genetic approach, however, reveals that inhibition of the PI3K pathway leads to rescuing of the β-amyloid peptide (Aβ)-induced memory loss in the Drosophila brain. We began our inquiry into the molecular basis of this memory loss by studying Aβ42-induced enhancement of long-term depression. We found that long-term depression is restored to a normal level through inhibition of PI3K activity. Aβ42-induced PI3K hyperactivity is directly confirmed by immunostaining of the PI3K phosphorylation targets, phospholipids. Such observations lead to the following demonstration that Aβ42-induced memory loss can be rescued through genetic silencing or pharmacological inhibition of PI3K functions. Our data suggest that Aβ42 stimulates PI3K, which in turn causes memory loss in association with an increase in accumulation of Aβ42 aggregates.

Notch (N) is a cell surface receptor that mediates an evolutionarily ancient signaling pathway to control an extraordinarily broad spectrum of cell fates and developmental processes. To gain insights into the functions of N signaling in the adult brain, we examined the involvement of N in Drosophila olfactory learning and memory. Long-term memory (LTM) was disrupted by blocking N signaling in conditional mutants or by acutely induced expression of a dominant-negative N transgene. In contrast, neither learning nor early memory were affected. Furthermore, induced overexpression of a wild-type (normal) N transgene specifically enhanced LTM formation. These experiments demonstrate that N signaling contributes to LTM formation in the Drosophila adult brain.A ctivation of Notch (N) receptors has been linked to the specification of many cell types in both vertebrates and invertebrates (1). Binding of ligands such as Delta (2) causes cleavage of the intracellular domain of the N protein (3-5). The cleaved cytoplasmic domain of N (Nintra) enters the nucleus, in which it regulates expression of target genes (1). Intercellular communication mediated by N signaling consists of two different modes: lateral inhibitory signaling and inductive signaling. A prototypic example of lateral inhibitory signaling is that loss of N function causes a cluster of equivalent proneural cells to all assume the default neuronal fate rather than an epidermal fate (6). Inductive N signaling, on the other hand, mediates interactions between cells that are nonequivalent before the signal initiates, such as induction of cone cells by photoreceptor cells and specification of midline cells in the embryo (7-9).In contrast to the extensive understanding of the vital role of N in development, the significance of N signaling in adult brains has yet to be revealed, although there is continuous presence of N protein and its ligands in the adult vertebrate nervous system (10). It has been reported that N signaling regulates neurite outgrowth in mammals (11), and chronic reduction of N activity in adult fruit flies also leads to progressive neurological syndromes (12). Processing of N requires ␥-secretase activity, whereas presenilin (PS) is a critical component of the ␥-secretase complex (13). Mutations in the PS genes are associated with the early onset of Alzheimer's disease (14). Conditional knockout of the PS1 gene in mice is also associated with reduced clearance of hippocampal memory traces (15). Although involvement of N in these PS mutant phenotypes remains to be determined, it has been shown that N plays a role in PS-mediated formation of neural projections in postmitotic neurons necessary for learned thermotaxis in Caenorhabditis elegans (16). In the present study, we examined the role of N signaling in learning and memory in Drosophila.A Pavlovian procedure that pairs odors with foot-shock (17) was used in this study to assess the effects of N signaling on adult behavioral plasticity. Genetic analyses have demonstrated that memory formation afte...

Accelerated forgetting has been identified as a feature of Alzheimer’s disease (AD), but the therapeutic efficacy of the manipulation of biological mechanisms of forgetting has not been assessed in AD animal models. Ras-related C3 botulinum toxin substrate 1 (Rac1), a small GTPase, has been shown to regulate active forgetting in Drosophila and mice. Here, we showed that Rac1 activity is aberrantly elevated in the hippocampal tissues of AD patients and AD animal models. Moreover, amyloid-beta 42 could induce Rac1 activation in cultured cells. The elevation of Rac1 activity not only accelerated 6-hour spatial memory decay in 3-month-old APP/PS1 mice, but also significantly contributed to severe memory loss in aged APP/PS1 mice. A similar age-dependent Rac1 activity-based memory loss was also observed in an AD fly model. Moreover, inhibition of Rac1 activity could ameliorate cognitive defects and synaptic plasticity in AD animal models. Finally, two novel compounds, identified through behavioral screening of a randomly selected pool of brain permeable small molecules for their positive effect in rescuing memory loss in both fly and mouse models, were found to be capable of inhibiting Rac1 activity. Thus, multiple lines of evidence corroborate in supporting the idea that inhibition of Rac1 activity is effective for treating AD-related memory loss.Electronic supplementary materialThe online version of this article (10.1007/s13238-019-0641-0) contains supplementary material, which is available to authorized users.

Seven new iridoid glucosides, 6''-O-trans-sinapoylgenipin gentiobioside (1), 6''-O-trans-p-coumaroylgenipin gentiobioside (2), 6''-O-trans-cinnamoylgenipin gentiobioside (3), 6'-O-trans-p-coumaroylgeniposide (4), 6'-O-trans-p-coumaroylgeniposidic acid (5), 10-O-succinoylgeniposide (6), and 6'-O-acetylgeniposide (7), two new monoterpenoids, 11-(6-O-trans-sinapoylglucopyranosyl)gardendiol (8) and 10-(6-O-trans-sinapoylglucopyranosyl)gardendiol (9), and three known ones, 6'-O-trans-sinapoylgeniposide (10), geniposide (11), and 10-O-acetylgeniposide (12), were isolated from the fruit of Gardenia jasminoides. The structures of these compounds were elucidated on the basis of 1D and 2D NMR spectra analyses. Furthermore, short-term memory assays on an Abeta transgenic drosophila model showed that compounds 4 and 6-12 can improve the short-term memory capacity to varying degrees, with compounds 4 and 7 being the most active ones, suggesting that these compounds may have a potential antagonism effect against Alzheimer's disease.

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