Aging Differentially Affects Axonal Autophagosome Formation and Maturation.

Misregulation of neuronal macroautophagy/autophagy has been implicated in age-related neurodegenerative diseases. We compared autophagosome formation and maturation in primary murine neurons during development and through aging to elucidate how aging affects neuronal autophagy. We observed an age-related decrease in the rate of autophagosome formation leading to a significant decrease in the density of autophagosomes along the axon. Next, we identified a surprising increase in the maturation of autophagic vesicles in neurons from aged mice. While we did not detect notable changes in endolysosomal content in the distal axon during early aging, we did observe a significant loss of acidified vesicles in the distal axon during late aging. Interestingly, we found that autophagic vesicles were transported more efficiently in neurons from adult mice than in neurons from young mice. This efficient transport of autophagic vesicles in both the distal and proximal axon is maintained in neurons during early aging, but is lost during late aging. Our data indicate that early aging does not negatively impact autophagic vesicle transport nor the later stages of autophagy. However, alterations in autophagic vesicle transport efficiency during late aging reveal that aging differentially impacts distinct aspects of neuronal autophagy.Abbreviations: ACAP3: ArfGAP with coiled-coil, ankyrin repeat and PH domains 3; ARF6: ADP-ribosylation factor 6; ATG: autophagy related; AVs: autophagic vesicles; DCTN1/p150Glued: dynactin 1; DRG: dorsal root ganglia; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; LAMP2: lysosomal-associated protein 2; LysoT: LysoTracker; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK8IP1/JIP1: mitogen-activated protein kinase 8 interacting protein 1; MAPK8IP3/JIP3: mitogen-activated protein kinase 8 interacting protein 3; mCh: mCherry; PE: phosphatidylethanolamine.

Structural basis for membrane recruitment of ATG16L1 by WIPI2 in autophagy.

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.

TUBB4A mutations result in both glial and neuronal degeneration in an H-ABC leukodystrophy mouse model.

Mutations in TUBB4A result in a spectrum of leukodystrophy including Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (H-ABC), a rare hypomyelinating leukodystrophy, often associated with a recurring variant p.Asp249Asn (D249N). We have developed a novel knock-in mouse model harboring heterozygous (Tubb4aD249N/+) and the homozygous (Tubb4aD249N/D249N) mutation that recapitulate the progressive motor dysfunction with tremor, dystonia and ataxia seen in H-ABC. Tubb4aD249N/D249N mice have myelination deficits along with dramatic decrease in mature oligodendrocytes and their progenitor cells. Additionally, a significant loss occurs in the cerebellar granular neurons and striatal neurons in Tubb4aD249N/D249N mice. In vitro studies show decreased survival and dysfunction in microtubule dynamics in neurons from Tubb4aD249N/D249N mice. Thus Tubb4aD249N/D249N mice demonstrate the complex cellular physiology of H-ABC, likely due to independent effects on oligodendrocytes, striatal neurons, and cerebellar granule cells in the context of altered microtubule dynamics, with profound neurodevelopmental deficits.

Inside human and other animal cells, filaments known as microtubules help support the shape of the cell and move proteins to where they need to be. Defects in microtubules may lead to disease. For example, genetic mutations affecting a microtubule component called TUBB4A cause a rare brain disease in humans known as H-ABC. Individuals with H-ABC display many symptoms including abnormal walking, speech defects, impaired swallowing, and several cognitive defects. Abnormalities in several areas of the brain, including the cerebellum and striatum contribute to these defects. . In these structures, the neurons that carry messages around the brain and their supporting cells, known as oligodendrocytes, die, which causes these parts of the brain to gradually waste away. At this time, there are no therapies available to treat H-ABC. Furthermore, research into the disease has been hampered by the lack of a suitable "model" in mice or other laboratory animals. To address this issue, Sase, Almad et al. generated mice carrying a mutation in a gene which codes for the mouse equivalent of the human protein TUBB4A. Experiments showed that the mutant mice had similar physical symptoms to humans with H-ABC, including an abnormal walking gait, poor coordination and involuntary movements such as twitching and reduced reflexes. H-ABC mice had smaller cerebellums than normal mice, which was consistent with the wasting away of the cerebellum observed in individuals with H-ABC. The mice also lost neurons in the striatum and cerebellum, and oligodendrocytes in the brain and spinal cord. Furthermore, the mutant TUBB4A protein affected the behavior and formation of microtubules in H-ABC mice. The findings of Sase, Almad et al. provide the first mouse model that shares many features of H-ABC disease in humans. This model provides a useful tool to study the disease and develop potential new therapies.

Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons.

Mitophagy, the selective removal of damaged mitochondria, is thought to be critical to maintain neuronal homeostasis. Mutations of proteins in the pathway cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration. In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage. Mitophagy-associated proteins were sequentially recruited to depolarized mitochondria followed by sequestration into autophagosomes. The localization of these mitophagy events had a robust somal bias. In basal and induced conditions, engulfed mitochondria remained in non-acidified organelles for hours to days, illustrating efficient autophagosome sequestration but delayed lysosomal fusion or acidification. Furthermore, expression of an ALS-linked mutation in the pathway disrupted mitochondrial network integrity and this effect was exacerbated by oxidative stress. Thus, age-related decline in neuronal health or expression of disease-associated mutations in the pathway may exacerbate the slow kinetics of neuronal mitophagy, leading to neurodegeneration.

Expression of WIPI2B counteracts age-related decline in autophagosome biogenesis in neurons.

Autophagy defects are implicated in multiple late-onset neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's, Huntington's, and Parkinson's diseases. Since aging is the most common shared risk factor in neurodegeneration, we assessed rates of autophagy in mammalian neurons during aging. We identified a significant decrease in the rate of constitutive autophagosome biogenesis during aging and observed pronounced morphological defects in autophagosomes in neurons from aged mice. While early stages of autophagosome formation were unaffected, we detected the frequent production of stalled LC3B-negative isolation membranes in neurons from aged mice. These stalled structures recruited the majority of the autophagy machinery, but failed to develop into LC3B-positive autophagosomes. Importantly, ectopically expressing WIPI2B effectively restored autophagosome biogenesis in aged neurons. This rescue is dependent on the phosphorylation state of WIPI2B at the isolation membrane, suggesting a novel therapeutic target in age-associated neurodegeneration.

Vesicular degradation pathways in neurons: at the crossroads of autophagy and endo-lysosomal degradation.

Autophagy and endo-lysosomal degradation are two parallel degradation pathways essential for maintaining neuronal health and function. Autophagosomes and endosomes sequester cellular cargo through different mechanisms, but these pathways converge upon fusion with lysosomes. Both pathways are spatially regulated, with distinct features evident in the soma, axons, and dendrites, possibly as an adaptation to the unique morphology of neurons and the specific demands of each compartment. Relatively little is known about how autophagy and endo-lysosomal degradation interact and how their activities may be coordinated. We review our current understanding of autophagy and endo-lysosomal degradation in neurons, highlighting common features and differences as well as the intersection of these two essential cellular pathways.

Long-distance axonal transport of AAV9 is driven by dynein and kinesin-2 and is trafficked in a highly motile Rab7-positive compartment.

Adeno-associated virus (AAV) vectors can move along axonal pathways after brain injection, resulting in transduction of distal brain regions. This can enhance the spread of therapeutic gene transfer and improve treatment of neurogenetic disorders that require global correction. To better understand the underlying cellular mechanisms that drive AAV trafficking in neurons, we investigated the axonal transport of dye-conjugated AAV9, utilizing microfluidic primary neuron cultures that isolate cell bodies from axon termini and permit independent analysis of retrograde and anterograde axonal transport. After entry, AAV was trafficked into nonmotile early and recycling endosomes, exocytic vesicles, and a retrograde-directed late endosome/lysosome compartment. Rab7-positive late endosomes/lysosomes that contained AAV were highly motile, exhibiting faster retrograde velocities and less pausing than Rab7-positive endosomes without virus. Inhibitor experiments indicated that the retrograde transport of AAV within these endosomes is driven by cytoplasmic dynein and requires Rab7 function, whereas anterograde transport of AAV is driven by kinesin-2 and exhibits unusually rapid velocities. Furthermore, increasing AAV9 uptake by neuraminidase treatment significantly enhanced virus transport in both directions. These findings provide novel insights into AAV trafficking within neurons, which should enhance progress toward the utilization of AAV for improved distribution of transgene delivery within the brain.