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Age-dependent progression of apoptotic cell death. Representative examples show TUNEL-positive nuclei in neurons (Elav positive; A-A") and glia (Repo positive; B-B") in p35 mutant brains. Note the weaker Elav signal in the apoptotic neuron in comparison with surrounding cells (white arrow in A-A") and the weaker Repo signal in dying glial cell (white arrowhead in B-B"). Single confocal plan, optic slice: 1.6m. Scale bar: 10m. (C,D)The absolute number of TUNEL-positive cells per hemisphere is presented on the y axis (n≥6; mean ± s.e.m.). (C)Apoptosis in neurons. The pan-neuronal marker Elav was used to identify neurons (n≥6; mean ± s.e.m.) in three independent experiments. c, control. (D)Apoptosis in glia. The pan-glial marker Repo was used to identify glia (n≥6; mean ± s.e.m.). Time points under the chart show absolute age in days and refer to the position on the mortality curve; results are grouped according to the chronological points. For instance: 45-day-old p35 mutant flies reached 80% of mortality in population and were analyzed simultaneously with 45-day-old wild-type controls that reached only 20% in mortality.
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Altered function of Cdk5 kinase is associated with many forms of neurodegenerative disease in humans. We show here that inactivating the Drosophila Cdk5 ortholog, by mutation of its activating subunit, p35, causes adult-onset neurodegeneration in the fly. In the mutants, a vacuolar neuropathology is observed in a specific structure of the central b...
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... wild type and p35 mutants One simple hypothesis to explain the tissue loss in p35 mutants would be an increase in cell death. We therefore examined apoptotic cell death by TUNEL staining in whole-mount wild-type and mutant brains. In both controls and p35 mutants, we found an age-dependent progression in the frequency of apoptotic cell death ( Fig. 2A,B). A low, and approximately equal, number of TUNEL-positive cells was observed in the brains of young animals of both genotypes, ...
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... both neurons and glia (as assessed by molecular markers) as well as a small proportion of cells whose identity could not be ascertained. In controls the rate of neuronal cell death increased twofold by the middle of the lifespan and threefold in old adults over young animals (Fig. 2C). Glial cell apoptosis increased by 50% over the initial level in old controls (Fig. 2D). The portion of non- identifiable apoptotic cells also increased progressively, showing dynamics similar to neurons (not shown). TUNEL-positive cells were observed in all compartments of the central brain and did not reveal any compartment-specific ...
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... glia (as assessed by molecular markers) as well as a small proportion of cells whose identity could not be ascertained. In controls the rate of neuronal cell death increased twofold by the middle of the lifespan and threefold in old adults over young animals (Fig. 2C). Glial cell apoptosis increased by 50% over the initial level in old controls (Fig. 2D). The portion of non- identifiable apoptotic cells also increased progressively, showing dynamics similar to neurons (not shown). TUNEL-positive cells were observed in all compartments of the central brain and did not reveal any compartment-specific ...
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... analyzed). On average, MLBs and APGs were smaller and less mature then organelles in old flies of both genotypes (supplementary material Fig. S1). Organelles of the lysosomal- autophagosomal degradation pathway were detected in the soma as well as neurites in old neurons, and these structures were also detected in glia (supplementary material Fig. S2). We noted that cells containing two or more autophagosomal organelles were often clustered (Fig. 4D). Accumulation of large MLBs and APGs is consistent with previously published aging phenotypes in various cell types of 85-to 100-day-old flies ( Miquel et al., 1974) and in a fly model of spinobulbar muscular atrophy ( Pandey et al., ...
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... is a highly specialized portion of the axon where action potentials initiate, and we have shown that the structure and maintenance of the AIS are exquisitely sensitive to Cdk5 activity. Disrupting the structure of this portion of the axon would be expected to have profound effects on the physiological properties of the neuron and its subcellular organization, and perhaps, therefore, contribute to instability of the structure of the neuron (Trunova et al., 2011). ...
Citations
... Using fly genetics, researchers show that deregulation of cyclin-dependent kinase 5 (Cdk5) activity disrupts autophagy, leads to an overactive innate immune response, and results in dopamine neurodegeneration in Drosophila [51]. It was demonstrated that an overactive innate immune response was sufficient to trigger neuronal cell death. ...
Autophagy is an essential cellular process that involves the transport of cytoplasmic content in double-membraned vesicles to lysosomes for degradation. Neurons do not undergo cytokinesis, and thus, the cell division process cannot reduce levels of unnecessary proteins. The primary cause of neurodegenerative disorders (NDs) is the abnormal deposition of proteins inside neuronal cells, and this could be averted by autophagic degradation. Thus, autophagy is an important consideration when considering means of developing treatments for NDs. Various pharmacological studies have reported that the active components in herbal medicines exhibit therapeutic benefits in NDs, for example, by inhibiting cholinesterase activity and modulating amyloid beta levels, and α-synuclein metabolism. A variety of bioactive constituents from medicinal plants are viewed as promising autophagy controllers and are revealed to recover the NDs by targeting the autophagic pathway. In the present review, we discuss the role of autophagy in the therapeutic management of several NDs. The molecular process responsible for autophagy and its importance in various NDs and the beneficial effects of medicinal plants in NDs by targeting autophagy are also discussed.
... Similar to the mammalian AIS, the AIS in flies is also linked to neurodegenerative stimuli. Deletion of Cdk5α (also called D-p35), encoding the activating subunit for cyclin dependent kinase 5 (Cdk5), has been shown to induce multiple degenerative phenotypes in Drosophila central brain neurons, including impaired autophagy, swelling of proximal axons, histologically-evident tissue loss (i.e., formation of Bvacuoles^in the brain), and age-dependent loss of neurons [26,27]. Cdk5α-null flies also exhibit an AIS that is severely shortened and indeed nearly absent: though a barrier remains that separates the somatodendritic and axonal compartments, the domain is so short that the characteristic pattern of accumulation of molecular markers cannot be detected [20]. ...
... Cdk5α-null flies also exhibit an AIS that is severely shortened and indeed nearly absent: though a barrier remains that separates the somatodendritic and axonal compartments, the domain is so short that the characteristic pattern of accumulation of molecular markers cannot be detected [20]. Notably, the position of the missing AIS correlates with the location where axonal swellings form in affected neurons and where histological tissue loss is observed [26]. However, it remains unclear if disruption of the AIS merely correlates with neurodegeneration or plays a causative role. ...
... Additional stocks for assessing the AIS included UAS-Act-RFP (BDSC) and UAS-Syt-HA (provided by Andreas Prokop, University of Manchester, Manchester, UK). Cdk5α overexpression conditions (UAS-Cdk5α, also called UAS-p35) have been previously described [20,[26][27][28][29][30]. In all experiments, MB-specific expression was accomplished with the gamma-neuron specific GAL4 driver 201Y-GAL4. ...
Neurodegenerative stimuli are often associated with perturbation of the axon initial segment (AIS), but it remains unclear whether AIS disruption is causative for neurodegeneration or is a downstream step in disease progression. Here, we demonstrate that either of two separate, genetically parallel pathways that disrupt the AIS induce axonal degeneration and loss of neurons in the central brain of Drosophila. Expression of a portion of the C-terminal tail of the Ank2-L isoform of Ankyrin severely shortens the AIS in Drosophila mushroom body (MB) neurons, and this shortening occurs through a mechanism that is genetically separate from the previously described Cdk5α-dependent pathway of AIS regulation. Further, either manipulation triggers morphological degeneration of MB axons and is accompanied by neuron loss. Taken together, our results are consistent with the hypothesis that disruption of the AIS is causally related to degeneration of fly central brain neurons, and we suggest that similar mechanisms may contribute to neurodegeneration in mammals.
... We have shown previously that increased or decreased activity of cyclin-dependent kinase 5 (Cdk5), achieved by altered expression of its essential activating subunit, Cdk5α (also called D-p35), causes a neurodegenerative syndrome in Drosophila that has extensive similarities to human NDs, including adult-onset degeneration and the death of neurons that are associated with learning and memory (mushroom body [MB] neurons), impaired autophagy, sensitivity to oxidative stress, and progressive loss of motor function, along with an accelerated rate of aging Trunova and Giniger, 2012). Cdk5 is a divergent member of the cyclin-dependent kinase family that does not associate with a classical cyclin for its activation and is not required for cell-cycle progression. ...
... If altering Cdk5α does not intensify microbial challenge, why does it activate the innate immune response? Defective autophagy can stimulate innate immunity, including AMP expression (Tusco et al., 2017;Wu et al., 2007), and data from us and others show that altering Cdk5α levels or inactivating Cdk5 kinase activity can disrupt autophagy (Nandi et al., 2017;Spurrier et al., 2018;Trunova and Giniger, 2012). Thus, we hypothesized that reduced autophagy in flies having an altered level of Cdk5α may be responsible for overactivated innate immunity. ...
... Previous experiments demonstrated that Cdk5α-altered flies have distorted autophagy in the brain at an older age Trunova and Giniger, 2012). To quantify autophagic flux specifically in DA neurons, we assayed the p62 ortholog, Ref(2)P (refractory to sigma P), and a tandem tagged Atg8 (GFP-mCherry-Atg8a). ...
Innate immunity is central to the pathophysiology of neurodegenerative disorders, but it remains unclear why immunity is altered in the disease state and whether changes in immunity are a cause or a consequence of neuronal dysfunction. Here, we identify a molecular pathway that links innate immunity to age-dependent loss of dopaminergic neurons in Drosophila. We find, first, that altering the expression of the activating subunit of the Cdk5 protein kinase (Cdk5α) causes severe disruption of autophagy. Second, this disruption of autophagy is both necessary and sufficient to cause the hyperactivation of innate immunity, particularly expression of anti-microbial peptides. Finally, it is the upregulation of immunity that induces the age-dependent death of dopaminergic neurons. Given the dysregulation of Cdk5 and innate immunity in human neurodegeneration and the conserved role of the kinase in the regulation of autophagy, this sequence is likely to have direct application to the chain of events in human neurodegenerative disease.
... Consistent with its links to multiple NDDs, both gain and loss of Cdk5 function cause neuronal death in culture, and cause neurodegeneration in mouse models ( Cruz et al., 2003;Qu et al., 2007;Takahashi et al., 2010;Zheng et al., 2007). Similarly, we have shown previously that Cdk5α loss of function in the fly produces degeneration-like phenotypes that mimic cellular phenotypes observed in human disease ( Trunova and Giniger, 2012). This is consistent with a large and growing literature documenting that the fly is a valuable model for dissecting the molecular mechanisms underlying the cascade of events in human NDDs ( Feany and Bender, 2000;Iijima et al., 2004;Watson et al., 2008). ...
... This is consistent with a large and growing literature documenting that the fly is a valuable model for dissecting the molecular mechanisms underlying the cascade of events in human NDDs ( Feany and Bender, 2000;Iijima et al., 2004;Watson et al., 2008). While investigating Cdk5α-associated neurodegeneration in the fly, we noted that many of its central phenotypes resemble early onset of normal aging phenotypes ( Trunova and Giniger, 2012). As in human disease, this underscored the need to distinguish degeneration from aging, both analytically and mechanistically. ...
... Histology of Cdk5α-null flies has previously demonstrated age-dependent formation of 'vacuoles' in the brain, particularly in the mushroom bodies (MB) ( Trunova and Giniger, 2012). While these vacuoles are suggestive of neurodegeneration, this assay was not definitive as it did not directly demonstrate neuron loss, as opposed to alternative explanations, such as expansion of inter-neuronal spaces or reduced dendritic arborization. ...
Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. Here we develop a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. We applied this metric to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5, the ortholog of mammalian p35. Cdk5- mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected, age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, our results suggest that Cdk5α-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.
... Flies mutant for the Cdk5 co-activator p35 display adult onset neurodegeneration and reduced lifespan (Connell-Crowley et al., 2007;Trunova and Giniger, 2012). To test whether altered basal autophagy contributes to these phenotypes, we first examined the distribution of Atg8a in eye discs from fed 96-hr old larvae. ...
... Although the pathological activation of Cdk5 activity appears to be a contributing factor to the progression of some neurodegenerative diseases, we show that wild-type levels of Cdk5/p35 activity are necessary to support basal autophagy in the clearance of protein aggregates. We show that this effect, at least in part, is due to Cdk5-mediated phosphorylation of Acn as phospho-mimetic Acn S437D reverses the reduced basal autophagy and the shortened life span observed in p35 mutants due their adult-onset, progressive neurodegeneration (Connell-Crowley et al., 2007;Trunova and Giniger, 2012). ...
Cdk5 is a post-mitotic kinase with complex roles in maintaining neuronal health. The various mechanisms by which Cdk5 inhibits and promotes neurodegeneration are still poorly understood. Here, we show that in Drosophila melanogaster Cdk5 regulates basal autophagy, a key mechanism suppressing neurodegeneration. In a targeted screen, Cdk5 genetically interacted with Acinus (Acn), a primarily nuclear protein, which promotes starvation-independent, basal autophagy. Loss of Cdk5, or its required cofactor p35, reduces S437-Acn phosphorylation, whereas Cdk5 gain-of-function increases pS437-Acn levels. The phospho-mimetic S437D mutation stabilizes Acn and promotes basal autophagy. In p35 mutants, basal autophagy and lifespan are reduced, but restored to near wild-type levels in the presence of stabilized AcnS437D. Expression of aggregation-prone polyQ-containing proteins or the Amyloid-β42 peptide, but not alpha-Synuclein, enhances Cdk5-dependent phosphorylation of S437-Acn. Our data indicate that Cdk5 is required to maintain the protective role of basal autophagy in the initial responses to a subset of neurodegenerative challenges.
... An alternative mechanism for the role of p35 in survival is seen in a study of a mutant p35 in Drosophila [37]. The mutation causes an increase in apoptotic and necrotic cell death, axonal fragmentation, accumulation of autophagosomes packed with crystalline-like depositions, and finally leads to neurodegeneration. ...
... The mutation causes an increase in apoptotic and necrotic cell death, axonal fragmentation, accumulation of autophagosomes packed with crystalline-like depositions, and finally leads to neurodegeneration. The study revealed aggregated depositions of endogenous material as well as enhanced accumulation of autophagic organelles from ultrastructural analysis of the p35 mutant, which may contribute to neuronal death [37]. Whether it occurs in a podocyte p35KO and initiates apoptosis will be an important subject for future investigation. ...
Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases.
... To begin to test our hypothesis, we examined glial and neuronal cell death in GFAP R79H flies with altered levels of Nos. We and others have previously observed loss of endogenous cell-type-specific markers in apoptotic cells 8,32 . We therefore introduced b-gal, expressed using UAS-lacZ and repo-GAL4, as a transgenic marker and performed double labelling with TUNEL to identify dying glia specifically and distinguish neuronal from glial cell death ( Supplementary Fig. 2k). ...
Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human disease states remains largely undefined. Here we use Alexander disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander disease, and possibly other neurodegenerative disorders with glial dysfunction.
... Furthermore, p35 is required for ERK½ activation, which induces up-regulation of Bcl2, a known anti-apoptotic factor (Wang et al. 2005, Zheng et al. 2007. The p35 deletion is associated with vacuolar pathology, progressive cell death and increased formation of autophago-lysosomes in Drosophila brains, which supports a neurodegenerative phenotype (Trunova & Giniger 2012). The close relationship between survival and active Rac as cell death and active RhoA, which were up-regulated or inhibited by CDK5 silencing, respectively, were reversed by the reduction of p35 using p35 shRNA; thus, these findings support the critical role of p35 for survival via the modulation of Rho GTPase. ...
P35 AND RAC1 CONTRIBUTE TO NEUROPROTECTION FOLLOWING CDK5 RNAI: CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate-mediated excitotoxicity using primary hippocampal neurons transduced with AAV2.5 viral vector eGFP-tagged SCR or CDK5 shRNA-miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down-stream Rho GTPase activity. Furthermore, p35 overexpression and constitutively active Rac1 mimicked CDK5 silencing-induced neuroprotection. In addition, 3xTg-AD mice (24 months old) were injected in the hippocampus with SCR or CDK5 shRNA-miR, and spatial learning and memory were performed three weeks post injection using "Morris" water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Worms lacking UNC-51 display axonal membrane defects, indicating a role of autophagy in synaptic plasticity, which indirectly interferes with learning and memory (Sigmond et al., 2008;Ragagnin et al., 2013). In Drosophila, inhibition of the cyclin-dependent kinase 5 (cdk5) kinase ortholog decreases autophagy, shortens lifespan and causes structural defects in central brain regions associated with olfactory learning and memory (Trunova and Giniger, 2012). In both flies and worms, autophagy deficiency leads to abnormal accumulation of protein aggregates thus promoting pathological mechanisms associated with neurodegenerative disorders, such as HD and AD (Ling et al., 2009;Low et al., 2013). ...
The aging process has been associated with numerous pathologies at the cellular, tissue, and organ level. Decline or loss of brain functions, including learning and memory, is one of the most devastating and feared aspects of aging. Learning and memory are fundamental processes by which animals adjust to environmental changes, evaluate various sensory signals based on context and experience, and make decisions to generate adaptive behaviors. Age-related memory impairment is an important phenotype of brain aging. Understanding the molecular mechanisms underlying age-related memory impairment is crucial for the development of therapeutic strategies that may eventually lead to the development of drugs to combat memory loss. Studies in invertebrate animal models have taught us much about the physiology of aging and its effects on learning and memory. In this review we survey recent progress relevant to conserved molecular pathways implicated in both aging and memory formation and consolidation.
... In addition, CDK5 gene knock-out mice showed significant nervous system disorders and embryonic defects [22] , all of which indicate the significance of CDK5 in central nervous system development. May previous studies have indicated a role for CDK5 in the developing nervous system [23][24][25][26] . ...
In the field of developmental neurobiology, accurate and ordered regulation of the cell cycle and apoptosis are crucial factors contributing to the normal formation of the neural tube. Preliminary studies identified several genes involved in the development of neural tube defects. In this study, we established a model of developmental neural tube defects by administration of retinoic acid to pregnant rats. Gene chip hybridization analysis showed that genes related to the cell cycle and apoptosis, signal transduction, transcription and translation regulation, energy and metabolism, heat shock, and matrix and cytoskeletal proteins were all involved in the formation of developmental neural tube defects. Among these, cell cycle-related genes were predominant. Retinoic acid ment caused differential expression of three cell cycle-related genes p57kip2, Cdk5 and Spin, the expression levels of which were downregulated by retinoic acid and upregulated during normal neural tube formation. The results of this study indicate that cell cycle-related genes play an important role in the formation of neural tube defects. P57kip2, Cdk5 and Spin may be critical genes in the pathogenesis of neural tube defects.
