Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.TDP-43 | ribonucleoprotein granules | liquid droplets | amyotrophic lateral sclerosis | neurons C ellular organelles allow eukaryotic cells to organize biochemical processes and concentrate specific cellular reactions in space and time. Although the role of membrane-bound organelles in cytoplasmic compartmentalization has long been recognized, the distinct biophysical properties and functions of non-membrane-bound organelles enriched in RNA and proteins have been recognized only recently (1-5). Ribonucleoprotein (RNP) granules, such as P granules in Caenorhabditis elegans (1), nucleoli in Xenopus laevis oocytes (2), yeast P bodies (3), and mammalian stress granules (6), show liquid droplet properties (reviewed in refs. 5, 7, 8), including fusion with rapid relaxation to a spherical shape, dynamic internal rearrangements, and rapid dissolution and assembly. Proteins comprising RNP granules share a common structure containing both RNA recognition motifs (RRMs) and low-complexity sequences (LCSs), intrinsically disordered regions that mediate protein-protein interactions (7, 9). In vitro characterization of human RNP granule proteins, RNA-binding protein FUS and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which are mutated in rare inherited forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (10-12), has revealed the LCS drives self-assembly of RNP granules through a process termed liquid-liquid phase separation (LLPS) (6, 13-18).Among eukaryotic cells, neurons face unique challenges in spatiotemporal cytoplasmic organization related to their com...

Autophagy is essential for maintaining cellular homeostasis in neurons, where autophagosomes undergo robust unidirectional retrograde transport along axons. We find that the motor scaffolding protein JIP1 binds directly to the autophagosome adaptor LC3 via a conserved LIR motif. This interaction is required for the initial exit of autophagosomes from the distal axon, for sustained retrograde transport along the mid-axon, and for autophagosomal maturation in the proximal axon. JIP1 binds directly to the dynein activator dynactin, but also binds to and activates kinesin-1 in a phosphorylation-dependent manner. Following JIP1 depletion, phosphodeficient JIP1-S421A rescues retrograde transport, while phosphomimetic JIP1-S421D aberrantly activates anterograde transport. During normal autophagosome transport, residue S421 of JIP1 may be maintained in a dephosphorylated state by autophagosome-associated MKP1 phosphatase. Moreover, binding of LC3 to JIP1 competitively disrupts JIP1-mediated activation of kinesin. Thus, dual mechanisms prevent aberrant activation of kinesin to ensure robust retrograde transport of autophagosomes along the axon.

Summary Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single molecule resolution, live-cell transport assays in primary neurons, computational image analysis and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron.

Graphical Abstract Highlights d Delivery of synaptic vesicle precursors occurs with high precision d Presynaptic sites are hotspots of dynamic GTP-rich microtubule plus ends d KIF1A binds more weakly to the GTP lattice, rapidly detaching from plus ends d A human KIF1A mutation perturbs lattice sensing and reduces synaptic strength SUMMARYNeurons in the CNS establish thousands of en passant synapses along their axons. Robust neurotransmission depends on the replenishment of synaptic components in a spatially precise manner.Using live-cell microscopy and single-molecule reconstitution assays, we find that the delivery of synaptic vesicle precursors (SVPs) to en passant synapses in hippocampal neurons is specified by an interplay between the kinesin-3 KIF1A motor and presynaptic microtubules. Presynaptic sites are hotspots of dynamic microtubules rich in GTPtubulin. KIF1A binds more weakly to GTP-tubulin than GDP-tubulin and competes with end-binding (EB) proteins for binding to the microtubule plus end. A disease-causing mutation within KIF1A that reduces preferential binding to GDP-versus GTPrich microtubules disrupts SVP delivery and reduces presynaptic release upon neuronal stimulation. Thus, the localized enrichment of dynamic microtubules along the axon specifies a localized unloading zone that ensures the accurate delivery of SVPs, controlling presynaptic strength in hippocampal neurons.

Cerebral amyloid angiopathy (CAA), limbic-predominant age-related TDP-43 encephalopathy neuropathological change (LATE-NC) and Lewy bodies occur in the absence of clinical and neuropathological Alzheimer’s disease, but their prevalence and severity dramatically increase in Alzheimer’s disease. To investigate how plaques, tangles, age and apolipoprotein E ε4 (APOE ε4) interact with co-pathologies in Alzheimer’s disease, we analysed 522 participants ≥50 years of age with and without dementia from the Center for Neurodegenerative Disease Research (CNDR) autopsy program and 1340 participants in the National Alzheimer's Coordinating Center (NACC) database. Consensus criteria were applied for Alzheimer’s disease using amyloid phase and Braak stage. Co-pathology was staged for CAA (neocortical, allocortical, and subcortical), LATE-NC (amygdala, hippocampal, and cortical), and Lewy bodies (brainstem, limbic, neocortical, and amygdala predominant). APOE genotype was determined for all CNDR participants. Ordinal logistic regression was performed to quantify the effect of independent variables on the odds of having a higher stage after checking the proportional odds assumption. We found that without dementia, increasing age associated with all pathologies including CAA (odds ratio 1.63, 95% confidence interval 1.38–1.94, P < 0.01), LATE-NC (1.48, 1.16–1.88, P < 0.01), and Lewy bodies (1.45, 1.15–1.83, P < 0.01), but APOE ε4 only associated with CAA (4.80, 2.16–10.68, P < 0.01). With dementia, increasing age associated with LATE-NC (1.30, 1.15–1.46, P < 0.01), while Lewy bodies associated with younger ages (0.90, 0.81–1.00, P = 0.04), and APOE ε4 only associated with CAA (2.36, 1.52–3.65, P < 0.01). A longer disease course only associated with LATE-NC (1.06, 1.01–1.11, P = 0.01). Dementia in the NACC cohort associated with the second and third stages of CAA (2.23, 1.50–3.30, P < 0.01), LATE-NC (5.24, 3.11–8.83, P < 0.01), and Lewy bodies (2.41, 1.51–3.84, P < 0.01). Pathologically, increased Braak stage associated with CAA (5.07, 2.77–9.28, P < 0.01), LATE-NC (5.54, 2.33–13.15, P < 0.01), and Lewy bodies (4.76, 2.07–10.95, P < 0.01). Increased amyloid phase associated with CAA (2.27, 1.07–4.80, P = 0.03) and Lewy bodies (6.09, 1.66–22.33, P = 0.01). In summary, we describe widespread distributions of CAA, LATE-NC and Lewy bodies that progressively accumulate alongside plaques and tangles in Alzheimer’s disease dementia. CAA interacted with plaques and tangles especially in APOE ε4 positive individuals; LATE-NC associated with tangles later in the disease course; most Lewy bodies associated with moderate to severe plaques and tangles.