A full understanding of Parkinson's Disease etiopathogenesis and of the causes of the preferential vulnerability of nigrostriatal dopaminergic neurons is still an unsolved puzzle. A multiple-hit ...hypothesis has been proposed, which may explain the convergence of familial, environmental and idiopathic forms of the disease. Among the various determinants of the degeneration of the neurons in Substantia Nigra pars compacta, in this review we will focus on the endotoxicity associated to dopamine dyshomeostasis. In particular, we will discuss the relevance of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) in the catechol-induced neurotoxicity. Indeed, the synergy between the catechol and the aldehyde moieties of DOPAL exacerbates its reactivity, resulting in modification of functional protein residues, protein aggregation, oxidative stress and cell death. Interestingly, αSynuclein, whose altered proteostasis is a recurrent element in Parkinson's Disease pathology, is considered a preferential target of DOPAL modification. DOPAL triggers αSynuclein oligomerization leading to synapse physiology impairment. Several factors can be responsible for DOPAL accumulation at the pre-synaptic terminals, i.e. dopamine leakage from synaptic vesicles, increased rate of dopamine conversion to DOPAL by upregulated monoamine oxidase and decreased DOPAL degradation by aldehyde dehydrogenases. Various studies report the decreased expression and activity of aldehyde dehydrogenases in parkinsonian brains, as well as genetic variants associated to increased risk in developing the pathology. Thus, we discuss how the deregulation of these enzymes might be considered a contributing element in the pathogenesis of Parkinson's Disease or a down-stream effect. Finally, we propose that a better understanding of the impaired dopamine metabolism in Parkinson's Disease would allow a more refined patients stratification and the design of more targeted and successful therapeutic strategies.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson’s disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light ...and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.
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•Cryo-electron tomography and integrative modeling reveal LRRK2’s structure in situ•Parkinson’s disease-linked LRRK2 forms double-helical filaments around microtubules•Homotypic interactions between the WD40 and COR domains form the filaments•The GTPase and kinase domains face the microtubule and cytoplasm, respectively
Cryo-electron tomography combined with integrative modeling yields the structure of LRKK2 in a cellular context and shows how filaments of the protein embrace microtubules.
Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are ...marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes.
Transcellular degradation of axonal mitochondria Davis, Chung-ha O.; Kim, Keun-Young; Bushong, Eric A. ...
Proceedings of the National Academy of Sciences - PNAS,
07/2014, Letnik:
111, Številka:
26
Journal Article
Recenzirano
Odprti dostop
It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these ...mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.
In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution ...and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins.
Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection. It produces an unusual intracellular infection in which a vegetative form, called the reticulate body (RB), ...replicates and then converts into an elementary body (EB), which is the infectious form. Here we use quantitative three-dimensional electron microscopy (3D EM) to show that C. trachomatis RBs divide by binary fission and undergo a sixfold reduction in size as the population expands. Conversion only occurs after at least six rounds of replication, and correlates with smaller RB size. These results suggest that RBs only convert into EBs below a size threshold, reached by repeatedly dividing before doubling in size. A stochastic mathematical model shows how replication-dependent RB size reduction produces delayed and asynchronous conversion, which are hallmarks of the Chlamydia developmental cycle. Our findings support a model in which RB size controls the timing of RB-to-EB conversion without the need for an external signal.
Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures ...cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast
staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells,
olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications.
Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary ...structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues.
The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such ...temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.
Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic causes of Parkinson disease (PD) and genome-wide association studies identify LRRK2 sequence variants ...as risk factors for sporadic PD. Intact kinase function appears critical for the toxicity of LRRK2 PD mutants, yet our understanding of how LRRK2 causes neurodegeneration remains limited. We find that most LRRK2 PD mutants abnormally enhance LRRK2 oligomerization, causing it to form filamentous structures in transfections of cell lines or primary neuronal cultures. Strikingly, ultrastructural analyses, including immuno-electron microscopy and electron microscopic tomography, demonstrate that these filaments consist of LRRK2 recruited onto part of the cellular microtubule network in a well-ordered, periodic fashion. Like LRRK2-related neurodegeneration, microtubule association requires intact kinase function and the WD40 domain, potentially linking microtubule binding and neurodegeneration. Our observations identify a novel effect of LRRK2 PD mutations and highlight a potential role for microtubules in the pathogenesis of LRRK2-related neurodegeneration.
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