Carlos G. Dotti

Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation

The presynaptic protein α-synuclein has been implicated in the pathogenesis of Parkinson’s disease. First, two missense mutations A30P and A53T cause inheritable early onset Parkinson’s disease in some families. Secondly, α-synuclein is present in Lewy bodies of affected nerve cells in the predominant sporadic type of Parkinson’s disease as well as in dementia with Lewy bodies. We demonstrate in the rat optic system that a portion of α-synuclein is carried by the vesicle-moving fast component of axonal transport and that it binds to rat brain vesicles through its amino-terminal repeat region. We find α-synuclein with the A30P mutation of familial Parkinson’s disease devoid of vesicle-binding activity and propose that mutant α-synuclein may accumulate, leading to assembly into Lewy body filaments.

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.

Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis

The sprouting of neurites, which will later become axons and dendrites, is an important event in early neuronal differentiation. Studies in living neurons indicate that neuritogenesis begins immediately after neuronal commitment, with the activation of membrane receptors by extracellular cues. These receptors activate intracellular cascades that trigger changes in the actin cytoskeleton, which promote the initial breakdown of symmetry. Then, through the regulation of gene transcription, and of microtubule and membrane dynamics, the newly formed neurite becomes stabilized. A key challenge is to define the molecular machinery that regulates the actin cytoskeleton during initial neurite sprouting. We propose that analysing the molecules involved in actin-dependent mechanisms in non-neuronal systems, such as budding yeast and migrating fibroblasts, could help to uncover the secrets of neuritogenesis.

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho‐associated coiled‐coil kinase, the Rac‐associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co‐localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Neuronal membrane cholesterol loss enhances amyloid peptide generation

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimers disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its β-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1–APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Centrosome localization determines neuronal polarity.

Neuronal polarization occurs shortly after mitosis. In neurons differentiating in vitro, axon formation follows the segregation of growth-promoting activities to only one of the multiple neurites that form after mitosis. It is unresolved whether such spatial restriction makes use of an intrinsic program, like during C. elegans embryo polarization, or is extrinsic and cue-mediated, as in migratory cells. Here we show that in hippocampal neurons in vitro, the axon consistently arises from the neurite that develops first after mitosis. Centrosomes, the Golgi apparatus and endosomes cluster together close to the area where the first neurite will form, which is in turn opposite from the plane of the last mitotic division. We show that the polarized activities of these organelles are necessary and sufficient for neuronal polarization: (1) polarized microtubule polymerization and membrane transport precedes first neurite formation, (2) neurons with more than one centrosome sprout more than one axon and (3) suppression of centrosome-mediated functions precludes polarization. We conclude that asymmetric centrosome-mediated dynamics in the early post-mitotic stage instruct neuronal polarity, implying that pre-mitotic mechanisms with a role in division orientation may in turn participate in this event.

Neuronal Polarity: Vectorial Cytoplasmic Flow Precedes Axon Formation

Axon formation in multipolar neurons is believed to depend on the existence of precise sorting mechanisms for axonal membrane and membrane-associated proteins. Conclusive evidence in living neurons, however, is lacking. In the present study, we use light and video microscopy to address this issue directly. We show that axon formation is preceded by the appearance in one of the multiple neurites of (1) a larger growth cone, (2) a higher amount and greater transport of membrane organelles, (3) polarized delivery of TGN-derived vesicles, (4) a higher concentration of mitochondria and peroxisomes, (5) a higher concentration of a cytosolic protein, and (6) a higher concentration of ribosomes. These results provide evidence for the involvement of bulk cytoplasmic flow as an early determinant of neuronal morphological polarization. Molecular sorting events would later trigger the establishment of functional polarity.

Asymmetric membrane ganglioside sialidase activity specifies axonal fate

Axon specification triggers the polarization of neurons and requires the localized destabilization of filamentous actin. Here we show that plasma membrane ganglioside sialidase (PMGS) asymmetrically accumulates at the tip of one neurite of the unpolarized rat neuron, inducing actin instability. Suppressing PMGS activity blocks axonal generation, whereas stimulating it accelerates the formation of a single (not several) axon. PMGS induces axon specification by enhancing TrkA activity locally, which triggers phosphatidylinositol-3-kinase (PI3K)- and Rac1-dependent inhibition of RhoA signaling and the consequent actin depolymerization in one neurite only. Thus, spatial restriction of an actin-regulating molecular machinery, in this case a membrane enzymatic activity, before polarization is enough to determine axonal fate.

Establishment of neuronal polarity: lessons from cultured hippocampal neurons

In recent years we have learned a great deal about the molecular mechanisms underlying axonal elongation and navigation and the manner in which extracellular signals modify a growth cones course of action. Yet, the mechanisms responsible for the earlier events of axonal and dendritic generation are just beginning to be understood. The recent advances in this exciting field highlight the importance of studies of cell migration and axonal elongation for our current understanding of the establishment of neuronal polarity.

RhoA/ROCK regulation of neuritogenesis via profilin IIa–mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.