To clarify the role of microglia in brain homeostasis and disease, an understanding of their maintenance, proliferation and turnover is essential. The lifespan of brain microglia, however, remains uncertain, and reflects confounding factors in earlier assessments that were largely indirect. We genetically labeled single resident microglia in living mice and then used multiphoton microscopy to monitor these cells over time. Under homeostatic conditions, we found that neocortical resident microglia were long-lived, with a median lifetime of well over 15 months; thus, approximately half of these cells survive the entire mouse lifespan. While proliferation of resident neocortical microglia under homeostatic conditions was low, microglial proliferation in a mouse model of Alzheimer's β-amyloidosis was increased threefold. The persistence of individual microglia throughout the mouse lifespan provides an explanation for how microglial priming early in life can induce lasting functional changes and how microglial senescence may contribute to age-related neurodegenerative diseases.
Deposition of aggregated amyloid- (A) peptide in brain is
Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.
Here we describe how to anesthetize and image Drosophila larvae as to follow 'the life history' of identified synapses and synaptic components. This protocol is sensitive, for example, the distribution of glutamate receptors expressed at physiological levels can be monitored. Typically, 2-20 time points can be recorded in the intact organism. Finally, we discuss how to extract the kinetic information on protein dynamics from two-color fluorescence recovery after photo-bleaching (FRAP) measurements and give advice how to keep the in vivo imager's five arch enemies--limited temporal and spatial resolution, injury of the animal, inactivation of proteins and movement artifacts--in check. While we focus on synapses, as model structure, the protocol can easily be adapted to study other developmental processes such as muscle growth, gut development or tracheal branching.
The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. Here, we show that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3 completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3 function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site.
Abstract. Cholesterol-lowering drugs such as statins influence the proteolytic processing of the amyloid-β protein precursor (AβPP) and are reported to stimulate the activity of α-secretase, the major preventive secretase of Alzheimer's disease. Statins can increase the α-secretase activity by their cholesterol-lowering properties as well as by impairment of isoprenoids synthesis. In the present study, we elucidate the contribution of these pathways in α-secretase activation. We demonstrate that zaragozic acid, a potent inhibitor of squalene synthase which blocks cholesterol synthesis but allows synthesis of isoprenoids, also stimulates α-secretase activity. Treatment of human neuroblastoma cells with 50 µM zaragozic acid resulted in a ∼3 fold increase of α-secretase activity and reduced cellular cholesterol by ∼30%. These effects were comparable to results obtained from cells treated with a low lovastatin concentration (2 µM). Zaragozic acid-stimulated secretion of α-secretase cleaved soluble AβPP was dose dependent and saturable. Lovastatin-or zaragozic acid-stimulated increase of α-secretase activity was completely abolished by a selective ADAM10 inhibitor. By targeting the α-secretase ADAM10 to lipid raft domains via a glycosylphosphatidylinositol anchor, we demonstrate that ADAM10 is unable to cleave AβPP in a cholesterol-rich environment. Our results indicate that inhibition of cholesterol biosynthesis by a low lovastatin concentration is sufficient for α-secretase activation.
Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution [1][2][3] . While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter 1 in stable transgenic lines and the exon trap line FasII-GFP , the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.
No abstract
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