Expression of a Fragment of Ankyrin 2 Disrupts the Structure of the Axon Initial Segment and Causes Axonal Degeneration in Drosophila.

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.

Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila.

BACKGROUND: During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors. RESULTS: We show here that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. We further show that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, we show that Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and provide evidence that it also acts at additional steps of MB pruning. CONCLUSIONS: These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5, we suggest that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, we speculate that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it uses during developmental remodeling.

Ex vivo culturing of whole, developing Drosophila brains.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab(1) has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)(2), was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine(3) causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically. A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly(4), and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern(5). Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches(6), by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B1(7), and that is dependent on the activity of the ubiquitin-proteasome system(6). Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Absence of the Cdk5 activator p35 causes adult-onset neurodegeneration in the central brain of Drosophila.

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 brain, the 'mushroom body', which is the seat of olfactory learning and memory. Analysis of cellular phenotypes in the mutant brains reveals some phenotypes that resemble natural aging in control flies, including an increase in apoptotic and necrotic cell death, axonal fragmentation, and accumulation of autophagosomes packed with crystalline-like depositions. Other phenotypes are unique to the mutants, notably age-dependent swellings of the proximal axon of mushroom body neurons. Many of these phenotypes are also characteristic of mammalian neurodegenerative disease, suggesting a close relationship between the mechanisms of Cdk5-associated neurodegeneration in fly and human. Together, these results identify the cellular processes that are unleashed in the absence of Cdk5 to initiate the neurodegenerative program, and they provide a model that can be used to determine what part each process plays in the progression to ultimate degeneration.

At the Fulcrum in Health and Disease: Cdk5 and the Balancing Acts of Neuronal Structure and Physiology.

Cdk5 has been implicated in a multitude of processes in neuronal development, cell biology and physiology. These influence many neurological disorders, but the very breadth of Cdk5 effects has made it difficult to synthesize a coherent picture of the part played by this protein in health and disease. In this review, we focus on the roles of Cdk5 in neuronal function, particularly synaptic homeostasis, plasticity, neurotransmission, subcellular organization, and trafficking. We then discuss how disruption of these Cdk5 activities may initiate or exacerbate neural disorders. A recurring theme will be the sensitivity of Cdk5 sequelae to the precise biological context under consideration.

Cdk5 regulates the size of an axon initial segment-like compartment in mushroom body neurons of the Drosophila central brain.

The axon initial segment (AIS) is the specialized compartment of vertebrate axons where action potentials are initiated. Despite longtime attention to the unique functions of this compartment, the mechanisms that regulate AIS formation and maintenance are not known. Here, we identify a novel compartment in Drosophila mushroom body neurons that mirrors the molecular hallmarks of the vertebrate AIS as judged by accumulation of the anchoring protein Ankyrin1, presence of a specialized actin cytoskeleton, exclusion of both axon-specific and somatodendritic-specific cell surface proteins, and accumulation of a unique combination of voltage-gated ion channels. Using pharmacological treatments, we show that, similar to the vertebrate AIS, the integrity of this region of γ-neurons and its ability to tether membrane proteins depends on an intact actin cytoskeleton. We further show that Cdk5/p35 kinase regulates the formation and maintenance of the putative AIS by controlling the position of its distal boundary. Thus, boosting Cdk5 activity in γ-neurons extends the AIS by as much as 100%, while eliminating Cdk5 activity causes the domain to shrink proximally or disappear altogether. These data demonstrate that Cdk5/p35 kinase is a key regulator of the development and maintenance of the AIS in Drosophila.

Drosophila brain tumor metastases express both neuronal and glial cell type markers.

Loss of either lgl or brat gene activity in Drosophila larvae causes neoplastic brain tumors. Fragments of tumorous brains from either mutant transplanted into adult hosts over-proliferate, and kill their hosts within 2 weeks. We developed an in vivo assay for the metastatic potential of tumor cells by quantifying micrometastasis formation within the ovarioles of adult hosts after transplantation and determined that specific metastatic properties of lgl and brat tumor cells are different. We detected micrometastases in 15.8% of ovarioles from wild type host females 12 days after transplanting lgl tumor cells into their abdominal cavities. This frequency increased significantly with increased proliferation time. We detected micrometastases in 15% of ovarioles from wild type host females 10 days after transplanting brat tumor cells into their abdominal cavities. By contrast, this frequency did not change significantly with increased proliferation time. We found that nearly all lgl micrometastases co-express the neuronal cell marker, ELAV, and the glial cell marker, REPO. These markers are not co-expressed in normal brain cells nor in tumorous brain cells. This indicates deregulated gene expression in these metastatic cells. By contrast, most of the brat micrometastases expressed neither marker. While mutations in both lgl and brat cause neoplastic brain tumors, our results reveal that metastatic cells arising from these tumors have quite different properties. These data may have important implications for the treatment of tumor metastasis.