Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling.

Dystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.

Endosomal sorting drives the formation of axonal prion protein endoggresomes.

The pathogenic aggregation of misfolded prion protein (PrP) in axons underlies prion disease pathologies. The molecular mechanisms driving axonal misfolded PrP aggregate formation leading to neurotoxicity are unknown. We found that the small endolysosomal guanosine triphosphatase (GTPase) Arl8b recruits kinesin-1 and Vps41 (HOPS) onto endosomes carrying misfolded mutant PrP to promote their axonal entry and homotypic fusion toward aggregation inside enlarged endomembranes that we call endoggresomes. This axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) mechanism forms pathologic PrP endoggresomes that impair calcium dynamics and reduce neuronal viability. Inhibiting ARESTA diminishes endoggresome formation, rescues calcium influx, and prevents neuronal death. Our results identify ARESTA as a key pathway for the regulation of endoggresome formation and a new actionable antiaggregation target to ameliorate neuronal dysfunction in the prionopathies.

LC3B phosphorylation regulates FYCO1 binding and directional transport of autophagosomes.

Macroautophagy (hereafter referred to as autophagy) is a conserved process that promotes cellular homeostasis through the degradation of cytosolic components, also known as cargo. During autophagy, cargo is sequestered into double-membrane vesicles called autophagosomes, which are predominantly transported in the retrograde direction to the perinuclear region to fuse with lysosomes, thus ensuring cargo degradation.1 The mechanisms regulating directional autophagosomal transport remain unclear. The ATG8 family of proteins associates with autophagosome membranes2 and plays key roles in autophagy, including the movement of autophagosomes. This is achieved via the association of ATG8 with adaptor proteins like FYCO1, involved in the anterograde transport of autophagosomes toward the cell periphery.1,3-5 We previously reported that phosphorylation of LC3B/ATG8 on threonine 50 (LC3B-T50) by the Hippo kinase STK4/MST1 is required for autophagy through unknown mechanisms.6 Here, we show that STK4-mediated phosphorylation of LC3B-T50 reduces the binding of FYCO1 to LC3B. In turn, impairment of LC3B-T50 phosphorylation decreases starvation-induced perinuclear positioning of autophagosomes as well as their colocalization with lysosomes. Moreover, a significantly higher number of LC3B-T50A-positive autophagosomes undergo aberrant anterograde movement to axonal tips in mammalian neurons and toward the periphery of mammalian cells. Our data support a role of a nutrient-sensitive STK4-LC3B-FYCO1 axis in the regulation of the directional transport of autophagosomes, a key step of the autophagy process, via the post-translational modification of LC3B.

The role of ESCRT during development and functioning of the nervous system.

The endosomal sorting complex required for transport (ESCRT) is made of subcomplexes (ESCRT 0-III), crucial to membrane remodelling at endosomes, nuclear envelope and cell surface. ESCRT-III shapes membranes and in most cases cooperates with the ATPase VPS4 to mediate fission of membrane necks from the inside. The first ESCRT complexes mainly serve to catalyse the formation of ESCRT-III but can be bypassed by accessory proteins like the Alg-2 interacting protein-X (ALIX). In the nervous system, ALIX/ESCRT controls the survival of embryonic neural progenitors and later on the outgrowth and pruning of axons and dendrites, all necessary steps to establish a functional brain. In the adult brain, ESCRTs allow the endosomal turn over of synaptic vesicle proteins while stable ESCRT complexes might serve as scaffolds for the postsynaptic parts. The necessity of ESCRT for the harmonious function of the brain has its pathological counterpart, the mutations in CHMP2B of ESCRT-III giving rise to several neurodegenerative diseases.

KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons.

In axons, proper localization of proteins, vesicles, organelles, and other cargoes is accomplished by the highly regulated coordination of kinesins and dyneins, molecular motors that bind to cargoes and translocate them along microtubule (MT) tracks. Impairment of axonal transport is implicated in the pathogenesis of multiple neurodegenerative disorders including Alzheimer's and Huntington's diseases. To understand how MT-based cargo motility is regulated and to delineate its role in neurodegeneration, it is critical to analyze the detailed dynamics of moving cargoes inside axons. Here, we present KymoAnalyzer, a software tool that facilitates the robust analysis of axonal transport from time-lapse live-imaging sequences. KymoAnalyzer is an open-source software that automatically classifies particle trajectories and systematically calculates velocities, run lengths, pauses, and a wealth of other parameters that are characteristic of motor-based transport. We anticipate that laboratories will easily use this package to unveil previously uncovered intracellular transport details of individually-moving cargoes inside neurons.

Regulation of postsynaptic function by the dementia-related ESCRT-III subunit CHMP2B.

The charged multivesicular body proteins (Chmp1-7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines.

Charged multivesicular body protein 2B (CHMP2B) of the endosomal sorting complex required for transport-III (ESCRT-III) polymerizes into helical structures deforming the plasma membrane.

The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.