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Abstract Background We previously reported that miR-195 exerts neuroprotection by inhibiting Sema3A and cerebral miR-195 levels decreased with age, both of which urged us to explore the role of miR-195 and miR-195-regulated Sema3 family members in age-associated dementia. Methods miR-195a KO mice were used to assess the effect of miR-195 on aging and cognitive functions. Sema3D was predicted as a miR-195 target by TargetScan and then verified by luciferase reporter assay, while effects of Sema3D and miR-195 on neural senescence were assessed by beta-galactosidase and dendritic spine density. Cerebral Sema3D was over-expressed by lentivirus and suppressed by si-RNA, and effects of over-expression of Sema3D and knockdown of miR-195 on cognitive functions were assessed by Morris Water Maze, Y-maze, and open field test. The effect of Sema3D on lifespan was assessed in Drosophila. Sema3D inhibitor was developed using homology modeling and virtual screening. One-way and two-way repeated measures ANOVA were applied to assess longitudinal data on mouse cognitive tests. Results Cognitive impairment and reduced density of dendritic spine were observed in miR-195a knockout mice. Sema3D was identified to be a direct target of miR-195 and a possible contributor to age-associated neurodegeneration as Sema3D levels showed age-dependent increase in rodent brains. Injection of Sema3D-expressing lentivirus caused significant memory deficits while silencing hippocampal Sema3D improved cognition. Repeated injections of Sema3D-expressing lentivirus to elevate cerebral Sema3D for 10 weeks revealed a time-dependent decline of working memory. More importantly, analysis of the data on the Gene Expression Omnibus database showed that Sema3D levels were significantly higher in dementia patients than normal controls (p

Although mutations in the microtubules-associated protein Tau have long been connected with several neurodegenerative diseases, the underlying molecular mechanisms causing these tauopathies are still not fully understood. Studies in various models suggested that dominant gain-of-function effects underlie the pathogenicity of these mutants; however, there is also evidence that the loss of normal physiological functions of Tau plays a role in tauopathies. Previous studies on Tau in Drosophila involved expressing the human Tau protein in the background of the endogenous Tau gene in addition to inducing high expression levels. To study Tau pathology in more physiological conditions, we recently created Drosophila knock-in models that express either wildtype human Tau (hTauWT) or disease-associated mutant hTau (hTauV337M and hTauK369I) in place of the endogenous Drosophila Tau (dTau). Analyzing these flies as homozygotes, we could therefore detect recessive effects of the mutations while identifying dominant effects in heterozygotes. Using memory, locomotion and sleep assays, we found that homozygous mutant hTau flies showed deficits already when quite young whereas in heterozygous flies, disease phenotypes developed with aging. Homozygotes also revealed an increase in microtubule diameter, suggesting that changes in the cytoskeleton underlie the axonal degeneration we observed in these flies. In contrast, heterozygous mutant hTau flies showed abnormal axonal targeting and no detectable changes in microtubules. However, we previously showed that heterozygosity for hTauV337M interfered with synaptic homeostasis in central pacemaker neurons and we now show that heterozygous hTauK369I flies have decreased levels of proteins involved in the release of synaptic vesicles. Taken together, our results demonstrate that both mutations induce a combination of dominant and recessive disease-related phenotypes that provide behavioral and molecular insights into the etiology of Tauopathies.

A hallmark feature of Alzheimer’s disease (AD) and other Tauopathies, like Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17), is the accumulation of neurofibrillary tangles composed of the microtubule-associated protein Tau. As in AD, symptoms of FTDP-17 include cognitive decline, neuronal degeneration, and disruptions of sleep patterns. However, mechanisms by which Tau may lead to these disturbances in sleep and activity patterns are unknown. To identify such mechanisms, we have generated novel Drosophila Tauopathy models by replacing endogenous fly dTau with normal human Tau (hTau) or the FTDP-17 causing hTauV337M mutation. This mutation is localized in one of the microtubule-binding domains of hTau and has a dominant effect. Analyzing heterozygous flies, we found that aged hTauV337M flies show neuronal degeneration and locomotion deficits when compared to wild type or hTauWT flies. Furthermore, hTauV337M flies are hyperactive and they show a fragmented sleep pattern. These changes in the sleep/activity pattern are accompanied by morphological changes in the projection pattern of the central pacemaker neurons. These neurons show daily fluctuations in their connectivity, whereby synapses are increased during the day and reduced during sleep. Synapse formation requires cytoskeletal changes that can be detected by the accumulation of the end-binding protein 1 (EB1) at the site of synapse formation. Whereas, hTauWT flies show the normal day/night changes in EB1 accumulation, hTauV337M flies do not show this fluctuation. This suggests that hTauV337M disrupts sleep patterns by interfering with the cytoskeletal changes that are required for the synaptic homeostasis of central pacemaker neurons.

Expression of the human Parkinson-disease-associated protein α-synuclein in all Drosophila neurons induces progressive locomotor deficits. Here, we identify a group of 15 dopaminergic neurons per hemisphere in the anterior medial region of the brain whose disruption correlates with climbing impairments in this model. These neurons selectively innervate the horizontal β and β′ lobes of the mushroom bodies, and their connections to the Kenyon cells are markedly reduced when they express α-synuclein. Using selective mushroom body drivers, we show that blocking or overstimulating neuronal activity in the β′ lobe, but not the β or γ lobes, significantly inhibits negative geotaxis behavior. This suggests that modulation of the mushroom body β′ lobes by this dopaminergic pathway is specifically required for an efficient control of startle-induced locomotion in flies.

Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aβ) peptide fragments that accumulate in Alzheimer’s Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2) that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta), we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of our double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, our results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.

Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used Drosophila, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing the delayed symptoms of OPIDN but not in the acute toxicity.