Cerebral Semaphorin3D is a novel risk factor for age-associated cognitive impairment.

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 < 0.001). Over-expression of homolog Sema3D gene in the nervous system of Drosophila reduced locomotor activity and lifespan by 25%. Mechanistically, Sema3D might reduce stemness and number of neural stem cells and potentially disrupt neuronal autophagy. Rapamycin restored density of dendritic spines in the hippocampus from mice injected with Sema3D lentivirus. Our novel small molecule increased viability of Sema3D-treated neurons and might improve autophagy efficiency, which suggested Sema3D could be a potential drug target. Video Abstract CONCLUSION: Our results highlight the importance of Sema3D in age-associated dementia. Sema3D could be a novel drug target for dementia treatment.

FTD-associated mutations in Tau result in a combination of dominant and recessive phenotypes.

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.

Disease-Associated Mutant Tau Prevents Circadian Changes in the Cytoskeleton of Central Pacemaker Neurons.

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.

Neuroprotective effects of PACAP against paraquat-induced oxidative stress in the Drosophila central nervous system.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that can arise after long-term exposure to environmental oxidative stressors, such as the herbicide paraquat (PQ). Here we investigated the potential neuroprotective action of vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) against PQ in Drosophila. We found that pre-treatment with this neuropeptide applied to the ventral nerve cord (VNC) at low doses markedly extended the survival of wild-type decapitated flies exposed to neurotoxic levels of PQ or dopamine (DA). In contrast and interestingly, application of a PACAP receptor antagonist, PACAP-6-38, had opposite effects, significantly decreasing the resistance of flies to PQ. PACAP also reduced PQ-induced caspase activation and reactive oxygen species (ROS) accumulation in the VNC. We then searched for the endogenous neuropeptide receptor potentially involved in PACAP-mediated neuroprotection in Drosophila. Knocking down the gene encoding the receptor Han/PDFR of the neuropeptide pigment-dispersing factor (PDF) in all neurons conferred to flies higher resistance to PQ, whereas PDFR downregulation restricted to PDF or DA neurons did not increase PQ resistance, but remarkably suppressed the neuroprotective action of PACAP. Further experiments performed with Pdf and Pdfr-deficient mutant strains confirmed that PDF and its receptor are required for PACAP-mediated neuroprotection in flies. We also provide evidence using split-green fluorescent protein (split-GFP) reconstitution that PDF neurons make synaptic contacts onto DA neurons in the abdominal VNC. Our results therefore suggest that the protective action of PACAP against PQ-induced defects in the Drosophila nervous system involves the modulation of PDFR signaling in a small number of interconnected neurons.

The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila.

Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. We now show that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation.

Reduced Expression of Foxp1 as a Contributing Factor in Huntington's Disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine expansion in the huntington protein (htt). The neuropathological hallmark of HD is the loss of neurons in the striatum and, to a lesser extent, in the cortex. Foxp1 is a member of the Forkhead family of transcription factors expressed selectively in the striatum and the cortex. In the brain, three major Foxp1 isoforms are expressed: isoform-A (∼90 kDa), isoform-D (∼70 kDa), and isoform-C (∼50 kDa). We find that expression of Foxp1 isoform-A and -D is selectively reduced in the striatum and cortex of R6/2 HD mice as well as in the striatum of HD patients. Furthermore, expression of mutant htt in neurons results in the downregulation of Foxp1 Elevating expression of isoform-A or -D protects cortical neurons from death caused by the expression of mutant htt On the other hand, knockdown of Foxp1 promotes death in otherwise healthy neurons. Neuroprotection by Foxp1 is likely to be mediated by the transcriptional stimulation of the cell-cycle inhibitory protein p21Waf1/Cip1 Consistently, Foxp1 activates transcription of the p21Waf1/Cip1 gene promoter, and overexpression of Foxp1 in neurons results in the elevation of p21 expression. Moreover, knocking down of p21Waf1/Cip1 blocks the ability of Foxp1 to protect neurons from mut-Htt-induced neurotoxicity. We propose that the selective vulnerability of neurons of the striatum and cortex in HD is related to the loss of expression of Foxp1, a protein that is highly expressed in these neurons and required for their survival.SIGNIFICANCE STATEMENT Although the mutant huntingtin gene is expressed widely, neurons of the striatum and cortex are selectively affected in Huntington's disease (HD). Our results suggest that this selectivity is attributable to the reduced expression of Foxp1, a protein expressed selectively in striatal and cortical neurons that plays a neuroprotective role in these cells. We show that protection by Foxp1 involves stimulation of the p21Waf1/Cip1 (Cdkn1a) gene. Although three major Foxp1 isoforms (A, C, and D) are expressed in the brain, only isoform-A has been studied in the nervous system. We show that isoform-D is also expressed selectively, neuroprotective and downregulated in HD mice and patients. Our results suggest that Foxp1 might be an attractive therapeutic target for HD.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development.

Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aß) 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 Aß-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.

Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster.

The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer's Disease (AD) due to its cleavage resulting in the production of the Aß peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including Drosophila, suggesting an important physiological function. Besides Aß, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aß, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation.

A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila.

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. Here, we show that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, we found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca(2+), also strongly enhanced PQ resistance. Finally, we found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans.

Organophosphate-induced changes in the PKA regulatory function of Swiss Cheese/NTE lead to behavioral deficits and neurodegeneration.

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.

A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease.

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 ß and ß' 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 ß' lobe, but not the ß or γ lobes, significantly inhibits negative geotaxis behavior. This suggests that modulation of the mushroom body ß' lobes by this dopaminergic pathway is specifically required for an efficient control of startle-induced locomotion in flies.

Lack of miRNA Misregulation at Early Pathological Stages in Drosophila Neurodegenerative Disease Models.

Late onset neurodegenerative diseases represent a major public health concern as the population in many countries ages. Both frequent diseases such as Alzheimer disease (AD, 14% incidence for 80-84 year-old Europeans) or Parkinson disease (PD, 1.4% prevalence for >55 years old) share, with other low-incidence neurodegenerative pathologies such as spinocerebellar ataxias (SCAs, 0.01% prevalence) and frontotemporal lobar degeneration (FTLD, 0.02% prevalence), a lack of efficient treatment in spite of important research efforts. Besides significant progress, studies with animal models have revealed unexpected complexities in the degenerative process, emphasizing a need to better understand the underlying pathological mechanisms. Recently, microRNAs (miRNAs), a class of small regulatory non-coding RNAs, have been implicated in some neurodegenerative diseases. The current data supporting a role of miRNAs in PD, tauopathies, dominant ataxias, and FTLD will first be discussed to emphasize the different levels of the pathological processes which may be affected by miRNAs. To investigate a potential involvement of miRNA dysregulation in the early stages of these neurodegenerative diseases we have used Drosophila models for seven diseases (PD, 3 FTLD, 3 dominant ataxias) that recapitulate many features of the human diseases. We performed deep sequencing of head small RNAs after 3 days of pathological protein expression in the fly head neurons. We found no evidence for a statistically significant difference in miRNA expression in this early stage of the pathological process. In addition, we could not identify small non-coding CAG repeat RNAs (sCAG) in polyQ disease models. Thus our data suggest that transcriptional deregulation of miRNAs or sCAG is unlikely to play a significant role in the initial stages of neurodegenerative diseases.

Behavioral consequences of dopamine deficiency in the Drosophila central nervous system.

The neuromodulatory function of dopamine (DA) is an inherent feature of nervous systems of all animals. To learn more about the function of neural DA in Drosophila, we generated mutant flies that lack tyrosine hydroxylase, and thus DA biosynthesis, selectively in the nervous system. We found that DA is absent or below detection limits in the adult brain of these flies. Despite this, they have a lifespan similar to WT flies. These mutants show reduced activity, extended sleep time, locomotor deficits that increase with age, and they are hypophagic. Whereas odor and electrical shock avoidance are not affected, aversive olfactory learning is abolished. Instead, DA-deficient flies have an apparently "masochistic" tendency to prefer the shock-associated odor 2 h after conditioning. Similarly, sugar preference is absent, whereas sugar stimulation of foreleg taste neurons induces normal proboscis extension. Feeding the DA precursor L-DOPA to adults substantially rescues the learning deficit as well as other impaired behaviors that were tested. DA-deficient flies are also defective in positive phototaxis, without alteration in visual perception and optomotor response. Surprisingly, visual tracking is largely maintained, and these mutants still possess an efficient spatial orientation memory. Our findings show that flies can perform complex brain functions in the absence of neural DA, whereas specific behaviors involving, in particular, arousal and choice require normal levels of this neuromodulator.